Rotaviral Enterotoxin Nonstructural Protein 4 Targets … · · 2012-08-102 Capsule: Background:...

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Rotaviral Enterotoxin Nonstructural Protein 4 Targets Mitochondria for

Activation of Apoptosis During Infection

Rahul Bhowmick1 Umesh Chandra Halder2Shiladitya Chattopadhyay3Shampa Chanda4Satabdi Nandi1 Parikshit Bagchi2Mukti Kant Nayaksect5Oishee Chakrabartiyen

NobumichiKobayashiФ MamtaChawla-Sarkar

Division of Virology National Institute of Cholera and Enteric Diseases P-33 CIT Road Scheme- XM Beliaghata Kolkata 700010 India

sect Department of Zoology University of Calcutta Ballygunge Kolkata 700019 India

yen Structural Genomics Section Saha Institute of Nuclear Physics Kolkata India

Ф Department of Hygeine Sapporo Medical University Sapporo Japan Corresponding Author Dr MamtaChawla-Sarkar Division of Virology National Institute of Cholera and Enteric Diseases P-33 CIT Road Scheme-XM Beliaghata Kolkata-700010 West Bengal India Tel +91-33-2353-7470 Fax + 91-33-2370-5066 Email chawlam70gmailcom sarkarmcicmrorgin Running title- Rotaviral NSP 4 translocates to mitochondria

httpwwwjbcorgcgidoi101074jbcM112369595The latest version is at JBC Papers in Press Published on August 10 2012 as Manuscript M112369595

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Capsule

Background Rotaviral Nonstructural protein 4 (NSP-4) disrupts Ca+2 ion homeostasis by translocating to endoplasmic reticulum

Result In this study we show translocation of NSP 4 to mitochondria dissipation of mitochondrial potential and initiation of apoptosis which NSP1 counteracts during early infection

Conclusion NSP 4 and NSP1 regulate apoptosis during infection

Significance Study signifies modulation of cellular survival and apoptotic machinery by rotavirus for their own benefit

Abstract

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins Rotavirus is known to express six nonstructural and six structural proteins Among them NSP 4 is the enterotoxin known to disrupt cellular Ca+2 homeostasis by translocating to endoplasmic reticulum In this study we have observed translocation of NSP 4 to mitochondria resulting in dissipation of mitochondrial membrane potential both during viral infection as well as in NSP 4 overexpression Furthermore transfection of N-terminal and C-terminal truncated NSP 4 mutants followed by analyzing NSP 4 localization by immunofluorescence microscopy identified 61-83 amino acid (aa) region as the shortest mitochondria targeting signal (MTS) NSP 4 exert its proapoptotic effect by interacting with mitochondrial proteins ANT and VDAC resulting in dissipation of mitochondrial potential release of cytochrome c (cyt c) from mitochondria and caspase activation During early infection apoptosis activation by NSP 4 is inhibited by the activation of cellular survival pathways (PI3KAKT) since PI3Kinase inhibitor results in early induction of apoptosis However in presence of both PI3Kinase inhibitor and NSP 4 siRNA apoptosis was delayed suggesting that early apoptotic signal is initiated by NSP 4 expression This proapoptotic function of NSP 4

is balanced by another viral encoded protein NSP1 which is implicated in PI3KAKT activation since over expression of both NSP 4 and NSP1 in cells resulted in reduced apoptosis compared to only NSP 4 expressing cells Overall the study reports mechanism by which enterotoxin NSP 4 exerts cytotoxicity and mechanism by which virus counteracts it at the early stage for efficient infection

Introduction

Rotavirus a nonenveloped double stranded RNA virus belonging to the family reoviridae is the major cause of severe gastroenteritis in children under age of 5 years and causes two million hospitalizations and four lakh fifty thousand deaths per year worldwide (1) The virus with its 11 segmented dsRNA genome encodes six structural proteins (VP1 to VP4 VP6 and VP7) which form the virion and six nonstructural proteins (NSP1 to NSP6) which are translated only in host cells after virus infection and do not form part of the mature infectious virus (2) Nonstructural proteins play a crucial role in infection by establishing host virus interaction by carrying out diverse function Among them NSP 4 is identified as viral enterotoxin (3)

NSP 4 is a 175 aa long protein having fundamental role in both viral morphogenesis and pathogenesis (3 4) It was reported that exogenous addition of NSP 4 can induce an age and dose dependent diarrhea in mouse model (5) NSP 4 localizes in endoplasmic reticulum and acts as an intracellular receptor of the double layered viral particle during the entry of sub viral particle into endoplasmic reticulum (6 7 8 and 9) NSP 4 has a membrane destabilizing activity due to its viroporin domain by which it can create transmembrane aqueous pore in endoplasmic reticulum and increase the cytoplasmic Ca+2 concentration (10) Increase in cytoplasmic Ca+2 concentration and activation of Bax have been shown to play a role in rotavirus induced cell death (11 12 and 13) It is natural for virus an obligatory parasite to modulate the cell death machinery for winning the struggle between the virus and host (14) During the early stage of infection virus inhibits apoptosis

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elicited as an innate immunity response either by modulating cellular signaling pathway or mimicking cellular antiapoptotic proteins In contrast to promote the viral dissemination virus stimulates apoptosis by either inducing cellular apoptotic signaling pathways or directly appointing viral proteins in proapoptotic function during late stages of infection

There are reports of viroporins encoded by several RNA viruses to interact with mitochondria and cause mitochondrial destabilization during infection (15) Since NSP 4 is also a viroporin and has an inherent property to interact and destabilize membrane (10 16) we analyzed whether NSP 4 also results in disruption of mitochondria Results suggested that NSP 4 localizes to mitochondria and destabilizes it by interacting with mitochondrial proteins VDAC and ANT leading to induction of proapoptotic stimuli which is counteracted by NSP1 induced survival pathway during early stages of infection to encourage viral replication

Experimental procedure

Ethics Statement This investigation was approved by the Institutional Animal Ethics Committee National Institute of Cholera amp Enteric Diseases Indian Council of Medical Research (Registration No- NICEDCPCSEAAW(215)2012-IAECSSO) and (Approval 6520082009) registered under ldquoCommittee for the Purpose of Control and Supervision of Experiments on Laboratory Animalsrdquo Ministry of Environment and Forests Government of India and conforms with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health Publication 85ndash23 revised 1996

Viruses cells and viral infection The monkey kidney cells (MA104) 293T cells HeLa cells were cultured in minimal essential medium (MEM) Dulbecco modified Eagle medium (DMEM) supplemented with 10 heat-inactivated fetal bovine serum (FBS) 2 mM l-glutamine 2 mM sodium pyruvate and 1X PSF (penicillin streptomycin and fungizone) respectively at 37degC humifiied incubator with 5 CO2 The simian rotavirus strain SA11 and

NSP1 mutant strain A5-16 were used in this study For infection viruses were activated with acetylated trypsin (10gml) at 37degC for 30 minutes (min) and added to the phosphate buffer saline (PBS) washed cells at indicated multiplicity of infection for 45 min at 37degC Unbound virus was removed by 3 washes with media and infection was continued in fresh DMEM or MEM The time of virus removal was taken as 0 hr post infection for all experiments At different time points cells were freeze thawed for cell lysis (17) Extracted and purified viral preparations were titrated by plaque assay (18) Construction of vectors Vectors were constructed using the primers having specific restriction sites (supplementary table 1) Anibodies reagents and inhibitor Rabbit polyclonal antibody against NSP 4 was raised against peptide fragment of NSP 4 according to standard protocols at the department of Virology and Parasitology Fujita Health University School of Medicine Aichi Japan Antibodies against cytochrome c (sc-13156) His probe (sc-803) VDAC (sc-8828) ANT (sc-11433) Bax (sc-493) were from SantaCruz Biotechnology Antibodies against caspase-9 (9501 9502) caspase-7 (9491 9491) caspase-3 (9662 9664) PARP (9541 9542) hexokinase (C35C4) Cox 4 (4844S) GAPDH (14C10) were from Cell Signaling Technology Antibody against FLAG epitope (SAB4200071) was from Sigma Antibody against Lamp2 was purchased from invitrogen Antibody against Trap alpha was donated by Dr RS Hegde (National Institutes of Health Bethesda MD 20892 USA) All antibodies were used at manufacture recommended dilution ATP (A9187) ADP (A2754) BAPTA-AM (A1076) TMRE (87917) FURA-2AM (F0888) broad spectrum caspase inhibitor Z-VAD-FMK (V116) iodixanol were from Sigma PI3K inhibitor (LY294002) (9901) was purchased from Cell Signaling Technology

Plasmid and siRNA transfection Plasmids were transfected in 293T and HeLa cells with Lipofectamine 2000 (Invitrogen) whereas siRNA were transfected in 293T and MA104 cells with siPORT-NeoFX (Ambion) according

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to manufacturerrsquos instructions Custom-Synthetic siRNA against NSP 4 was obtained from Dharmacon Bax siRNA (Flexi Tube Gene Solution for Bax GS581) was obtained from Qiagen

Western blot analysis Whole cell lysates [extracted with Totex buffer (20 mM Hepes at pH 79 035 M NaCl 20 glycerol 1 NP-40 1 mM MgCl2 05 mM EDTA 01 mM EGTA 50 mM NaF and 03 mM Na3VO4) containing a mixture of protease and phosphatase inhibitor (Sigma)] cytoplasmic or mitochondrial extract in vitro translated product or immunoprecipitated products were prepared and subjected to SDS-PAGE followed by immunoblotting according to standard protocols (19) using specific primary antibody with manufacturerrsquos recommended dilutions For anti NSP 4 antibody 13000 dilution was used Primary antibodies were identified with HRP conjugated secondary antibody (Pierce Rockford IL) and chemiluminescent substrate (Millipore Billerica MA) In vitro translated product containing biotinylated proteins were detected by immunoblotting using Pierce High Sensitivity streptavidin-HRP (Thermo scientific Rockford IL USA) Where necessary to confirm protein loading blots were reprobed with β-Actin GAPDH or Cox 4 The immunoblots shown are representative of three independent experiments Cell fractionation Mitochondria were isolated from either infected MA104 or transfected 293T cells by the differential centrifugation method Cells were washed with cold phosphate buffered saline (PBS) scrapped and resuspended in 1-2 (wv) Tritonx-100001-003 (wv) NP-40 04-06 (wv) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) in ice for 30 min for cell disruption followed by centrifugation at 1000g for 10 min Supernatants (supe) were collected and centrifuged at 7000g for 10 min to pellet the mitochondria supe were saved as cytoplasm Pellet was washed with buffer (025 M sucrose and 10 mM HEPES pH-75) and then centrifuged at 7000g for 10 min and saved as mitochondria For protein extraction the pellets were resuspended in buffer containing 7 M urea

2 M thiourea 4 CHAPS 120 mM dithiothreitol (DTT) 2 ampholytes (pH 3-10) and 40 mM Tris-HCl and further incubated in ice for 30 min Pure mitochondrial fractions from SA11 infected (8h) cells were isolated by ultracentrifugation using iodixanol as described previously (20) Endoplasmic reticulum fractions and mitochondrial fractions were isolated from SA11 infected (8h) cells by ultracentrifugation using sucrose gradient as described previously (21) Co-immunoprecipitation Infected or transfected cells were washed with cold PBS and then mitochondria were isolated as described before and mitochondrial lysate were clarified by incubation (2h) at 4oC followed by centrifugation with protein A-Sepharose beads and the supe were incubated with anti-Flag or anti-His anti NSP 4 antibodies overnight at 40C and with protein A-Sepharose for further 4h Beads were washed 5 times with 1ml wash buffer (200 mM Tris pH-80 100 mM NaCl and 05 NP-40) and bound proteins were eluted with SDS sample buffer before separation on 12 SDS-PAGE gels followed by immunoblotting with anti-Flag or anti-His or anti NSP 4 antibodies Invitro transcription translation and purification pcDNSP 4 pcDVDAC1 and pcDANT3 were subjected to invitro coupled transcription and translation (IVT) using TNT Quick Coupled TranscriptionTranslation system (Promega Medison USA) according to manufacturerrsquos protocol In presence of TranscendTM biotinylated-lysyl t-RNA 2 microg plasmid was added to TNT Quick Master Mix for 90 min at 30oC and the products were separated by SDS PAGE and immunoblotted using Pierce High sensitivity streptavidin-HRP (Thermo Scientifics Rockford USA) (Supplementary Fig-1A) Recombinant proteins were purified on Ni2+ -NTA magnetic agarose beads under native conditions and the purity was validated by immunoblot analysis using antibodies against NSP 4 VDAC and ANT (Supplementary Fig -1B) Invitro Mitochondria import assay and cytochrome crelease assay Overnight starved

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Balbc mouse livers were isolated and homogenized followed by fractionation as described previously (22) Purified functionally active mitochondria were resuspended in MRM-S buffer (250 mM sucrose 10 mM Hepes 1 mM ATP 5 mM succinate 008 mM ADP and 2 mM K2HPO4 [pH-74]) and 50 microg of mitochondria (by total protein) were incubated with different amount of purified IVT NSP 4 with appropriate control (pcDNA6 IVT product) for 1h at 30oC then mitochondria were pelleted (7000g X 10 min) and suspended in resuspension buffer (7 M urea 2 M thiourea 4 CHAPS 120 mM dithiothreitol (DTT) 2 ampholytes (pH 3-10) and 40 mM Tris-HCl) and extracted protein were immunoblotted with anti NSP 4 antibody The supe were analyzed for cytochrome c release by immunoblotting with anti-cytochrome c antibody Analysis of mitochondrial depolarization with TMRE fluorescence Functionally active purified mitochondria were incubated with different amount of IVT NSP 4 for 10 min at RT 1 ml of 50 nM TMRE dye dissolved in MRM-S buffer was added to IVT NSP 4 treated mitochondria After 10 min incubation at room temperature TMRE fluorescence was measured in a fluorometer (PTI Fluorescence spectrophotometer) with 490 nm excitation wavelength and 575 nm emission scanning Immune fluorescence microscopy To assess localization of NSP 4 to mitochondria HeLa cells were seeded in a four well chamber slide (BD Pharmingen SanDiego CA) and transfected with pAcGFP1-C2NSP 4 deletion mutants or fragments of NSP 4 cloned in pAcGFP1-C2 After 16 h cells were fixed with paraformaldehyde (4 [wtvol] in [PBS]) for 10 min at room temperature (RT) then permeabilized with 01 Triton X-100 for 20 min at 4oC Cell were then incubated with blocking solution (PBS supplemented with 5 [volvol] horse serum and 5 [volvol] goat serum) for 1 h at room temperature followed by cold PBS (3X) wash and incubation with primary antibodies (α Mn-SOD) for 2 h at RT Unbound primary antibodies were washed with PBS (3X) followed by 1 h incubation (RT) with RRX-conjugated anti-rabbit antibody After 5

times washing with PBS slides were mounted with Vectashield-DAPI and observed under fluorescence microscope Excitation and emission detection for each flour was performed sequentially to avoid cross talk Confocal microscopy and imaging To assess subcellular localization of NSP 4 in different organelles such as endoplasmic reticulum mitochondria or lysosome HeLa cells were processed as previously and incubated with primary antibodies against trap alpha (ER marker) Cox 4 (mitochondrial marker) Lamp 2 (lysosome marker) for 2h at RT Unbound primary antibodies were washed with PBS (3X) followed by 1h incubation (RT) with Alexa 546 conjugated secondary antibody After 5 time PBS wash confocal microscopy was performed utilizing LSM510-Meta confocal microscopy system (Zeiss) equipped with an Ar-ion laser (for GFP excitation with the 488 nm line) and a He-Ne laser (for TMRE and Alexa Flour 546 excitation with the 543 line) A 63x 14 NA oil immersion objective was used for all imaging For comparisons between multiple samples images were collected during a single session using identical excitation and detection settings The detector gain settings were chosen to allow imaging of the desired cells within the linear range of the photomultiplier tube without saturating pixels For imaging localization of different proteins randomly chosen fields of cells were imaged with the above laser lines The complete set of experiments was performed twice to eliminate artifacts arising from individual experiments FACS characterization of mitochondrial membrane potential At indicated time points transfected 293T cells and infected MA104 cells were trypsinized and resuspended in 1 ml PBS Suspension was centrifuged at 300g for 5 min and pellet were resuspended into 1 ml of 100 nM TMRE for 20 min at 37oC before direct analysis on flowcytometer (Aria II BD Biosciences San Jose California) using a 488 nm laser As a positive control for membrane depolarization cells were treated with CCCP and stained with TMRE (data not shown)

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TUNEL assay pcDNSP 4 transfected or control vector transfected 293T cells with or without different treatment were harvested at indicated time points and stained using a APO-BRDUtrade Kit (BD Pharmingen) for TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay according to the manufacturerrsquos protocol For flow cytometry data acquisition and analysis were performed on BD FACS Aria cytometer using a BD FACS Diva data management system Trypsin treatment of isolated mitochondria To identify proteins located peripherally on the outer mitochondrial membrane mitochondria fractionated from NSP 4 expressing 293T cells (24h post transfection) or mouse liver mitochondria incubated with IVT NSP 4 (2 microM) treated with trypsin on ice for 30 min followed by centrifugation at 6700g for 15 min at 4oC (23) The pellet was washed 2 times with MESH buffer (20 mMHepes-NaOH pH-74 220 mMmannitol 70 mM sucrose and 01 mM EDTA) and analyzed with 15 SDS-PAGE Western blot analysis was performed using hexokinase (24) and VDAC (25) specific antibody as markers of peripheral outer mitochondrial membrane protein and a protein located within the mitochondria respectively Alkaline treatment of isolated mitochondria To distinguish integral membrane protein from peripheral membrane and soluble proteins either mitochondria fractionated from pcDNSP 4 transfected 293T cells after 24h or mouse liver mitochondria incubated with IVT NSP 4 (2 microM) as before were treated with 01M sodium carbonate (Na2CO3) pH-113 for 30 min on ice followed by centrifugation at 13000g for 10 min at 4oC as described previously (26) The pellet fraction containing the inner and outer mitochondrial membrane were directly solubilized in SDS sample buffer and the supe containing peripheral membrane intramembrane space and matrix protein were first concentrated by trichloroacetate precipitation and then solubilized and separated by 15 SDS-PAGE Western blot analyses was done using hexokinase and VDAC as markers of peripheral outer mitochondrial membrane protein and a

protein located within the mitochondria respectively Potassium chloride (KCl) treatment of isolated mitochondria To separate the outer and inner mitochondrial membrane either mitochondria fractionated from pcDNSP 4 transfected 293T cells after 24h or mouse liver mitochondria incubated with IVT NSP 4 (2 microM) as before were treated with 10 mM KCl for 10 min on ice as previously described (27) followed by centrifugation at 2500g The pellet fraction contains the intact inner mitochondrial membrane and matrix leaving the outer mitochondrial membrane and intermembrane space in the supe The pellet fraction was washed and centrifuged at 300g before solubilization in SDS sample buffer The supe were first concentrated by trichloroacetate precipitation and then solubilized in SDS sample buffer followed by separation with SDS-PAGE and western blot analyses using VDAC and Cox 4 (28) as markers of outer mitochondrial membrane protein and inner mitochondrial membrane protein respectively Determination of intracellular Ca+2 293T cells were transfected with pCDNSP 4 either in presence or absence of BAPTA-AM (50 microM) (added 6h post transfection) and intracellular Ca+2 concentration was measured as described previously (29 30) with FURA 2AM at indicated time points

Fluorescence resonance energy transfer (FRET) To assess localization of NSP 4 to mitochondria HeLa cells were seeded in a four well chamber slide (BD Pharmingen San Diego CA) and transfected with pAcGFP1-C2NSP 4 After 16h cells were treated with 150 nM TMRE for 20 min at room temperature followed by PBS wash (3X) 5 min each Fluorescence microscopy was performed utilizing LSM710-NLO microscopy system (Zeiss) equipped with an Ar-ion laser (for GFP excitation with the 488 nm line) and a He-Ne laser (for TMRE excitation with the 543 nm line) A 63x 14 NA oil immersion objective was used for all imaging For comparisons between multiple samples images were collected during a single session using identical excitation and detection

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settings The detector gain settings were chosen to allow imaging of the desired cells within the linear range of the photo multiplier tube without saturating pixels For imaging proximity between mitochondria and NSP 4 randomly chosen fields of cells were imaged with the above laser lines 3 dishes were imaged for each set of transfections and the complete set of experiments was performed twice to eliminate artifacts arising from individual experiments For Fluorescence Resonance Energy Transfer (FRET) analyses defined regions of interest (ROI) was photobleached at full laser power (100 power 100 transmission) of the 543 nm laser beam change in fluorescence was monitored in both the channels by scanning the whole cell at low laser power (10 power 03 transmission) as previously described (31) Two images were recorded of the donor and acceptor before and after photo bleaching The fluorescence intensities of the two channels were plotted over time as described previously (32) Statistical analysis Data are expressed as meanplusmnSD of at least three independent experiments (nge3) In all tests P=005 was considered statistically significant by guest on June 4 2018

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Results

NSP 4 induces Ca+2 ion and Bax independent apoptosis Purified NSP 4 protein has been reported to cause diarrhea in mice (5) To characterize its cytotoxic effect we transiently transfected 293T cells with either pcDNSP 4 or empty vector and after 24h apoptosis was measured by measuring DNA fragmentation using TUNEL assay according to the manufacturerrsquos protocol Expression of glycosylated (26KD 28KD) and non glycosylated (20KD) form of NSP 4 was confirmed by immunoblotting with NSP 4 antisera (Fig-1A) Ectopic expression levels of NSP 4 protein in pcDNSP 4 expressing cells [24h] and cells infected with SA11 [11-14hpi] were assessed by western blotting followed by densitometry analysis by quantity one software version 463 (Bio-Rad) using GAPDH as normalization control (Fig-1B) Results revealed significant increase in TUNEL positive cells in pcDNSP 4 transfected cells (44) compared to empty vector (74) (Fig-1C [I II]) To know whether the apoptois induced by NSP 4 expression was by extrinsic or intrinsic pathway cleavage of caspase-8 caspase-9 caspase-7 caspase-3 and PARP was analyzed by immunoblotting the cell extracts of NSP 4 expressing 293T cells at indicated time points Cleavage of caspase-8 was not observed but caspase-9 caspase-3 and PARP were cleaved in pcDNSP 4 transfected cells compared to empty vector transfected cells suggesting activation of intrinsic apoptotic pathway (Fig-1D) Activation of Bax (13) and elevation of Ca+2 ion concentration (11 12) has been previously reported during rotavirus induced cell death but whether these were responsible for NSP 4 induced apoptosis is not known To assess this 293T cells transfected with Bax-siRNA (24h) were transfected with either pcDNSP 4 or pcDNA6 followed by treatment (6h post pcDNSP 4 transfection) with an intracellular Ca+2 chelator (50microM BAPTA-AM) for 24 h Apoptosis was measured by TUNEL assay which revealed 25-50 decrease in apoptosis in NSP 4 expressing cells treated with Bax-siRNA (303) (Fig-1C [VI]) or BAPTA-AM (244) (Fig-1C[IV]) alone or together (215) (Fig-1C[VIII]) compared to NSP 4 expressing cells

Modulation of Ca+2 ion concentration and Bax expression by BAPTA-AM and Bax-siRNA was measured by FURA-2 fluorescence and immnoblotting respectively As shown in Fig-1E NSP 4 mediated elevation of Ca+2 ion concentration was buffered in presence of BAPTA-AM Similarly cellular Bax was significantly reduced (more than 80) in Bax-siRNA transfected cells (Fig-1F) Since Bax-siRNA and Ca+2 chelator could not reverse NSP 4 induced apoptosis completely it suggested role of other factors in NSP 4 mediated apoptosis

NSP 4 depolarizes mitochondria and induces apoptosis through intrinsic pathway Caspase-9 cleavage without caspase-8 activation indicated involvement of intrinsic pathway (Fig-1D) thus effect of NSP 4 on mitochondrial membrane potential was measured 293T cells transfected with pcDNSP 4 or control vector (16h post transfection) were treated with TMRE (100 nM) for 20 min at 37oC followed by flowcytometric analysis Increase in mitochondrial depolarization in pcDNSP 4 transfected cells (41) was observed (Fig-2A[II]) compared to controls (55) (Fig-2A [I]) This suggested that NSP 4 alone can depolarize mitochondria independent of viral replication or expression of other viral proteins To nullify the effects of Bax or elevated Ca+2 ion concentration same experiment was repeated in presence of either Bax-siRNA (Fig-A[IIIIV]) or Ca+2 chelator (Fig-A [V VI]) or both (Fig-A[VIIVIII]) as described previously Concurrent with previous results presence of Ca+2 chelator (239) and Bax-siRNA (29) resulted in decreased depolarization compared to only NSP 4 expressing cells but still significant amount of mitochondrial membrane potential dissipation (20) was observed compared to 293T control cells in presence of both Bax-siRNA and Ca+2 chelator (Fig-2A) To further analyze whether this depolarizing effect of NSP 4 is direct or dependent on other cellular factors increasing amount of purified in vitro transcribed and translated (IVT) NSP 4 was incubated with purified functional mouse liver mitochondria Mitochondrial membrane potential was measured after incubation with TMRE (50nM) as described in materials and

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methods Results confirmed that purified IVT NSP 4 can depolarize mitochondria in a cell free in vitro system (Fig-2B) suggesting that NSP 4 can depolarize mitochondria both in cellular and in cell free condition independent of other viral proteins and cellular factors To see the downstream effect of mitochondria depolarization 293T cells transiently transfected with pcDNSP 4 or empty vector control were either treated with BAPTA-AM and Bax-siRNA or left untreated After 24h presence of cyt c in cytosol was assessed by immunoblotting As shown in Fig 2C cyt c release was observed in pcDNSP 4 transfected cells and in presence of BAPTA-AM and Bax-siRNA release of cyt c was attenuated but still significant amount of cyt c was found in cytosol To further verify cyt c release assay was done with purified mouse liver mitochondria and IVT NSP 4 protein Consistent with previous results concentration dependent cyt c release from mitochondria was observed in presence of purified NSP 4 in cell free system (Fig-2D) suggesting that NSP 4 induced apoptosis may be triggered by a direct effect on mitochondria To confirm this pcDNSP 4 or empty vector transfected 293T cells were treated with broad spectrum caspase inhibitor z vad-fmk (10 microM) and release of cyt c into cytosol from mitochondria was assessed by immunoblotting (Fig-2E) As shown in Fig-2E release of cyt c from mitochondria to cytosol was observed in presence or absence of caspase inhibitor suggesting that caspase activation by NSP 4 is downstream of cyt c release However caspases play vital role in NSP 4 induced cell death as apoptosis is significantly inhibited in presence of zvad-fmk (10 microM) (Fig-2F)

NSP 4 localizes to mitochondria Since previous results suggest direct effect of NSP 4 on mitochondria it was hypothesized that NSP 4 may translocate to mitochondria To examine this possibility MA104 cells were either infected with SA11 strain (2 moi) or mock infected before mitochondria enriched fractions were isolated at increasing time points Immunoblotting with NSP 4 antisera revealed presence of nonglycosylated NSP 4 (20KD) in mitochondrial fraction from 4hpi (Fig-3A) Similarly mitochondrial enriched fraction of

pcDNSP 4 transfected 293T cells for 24 h revealed presence of nonglycosylated NSP 4 (20KD) suggesting that mitochondrial translocation of NSP 4 was independent of other viral components (Fig-3B) Mitochondrial fraction was immunoblotted with Cox 4 specific antibody as mitochondrial protein control and anti calnexin anti Lysosomal-associated membrane protein 2 (lamp2) for detecting other organeller contamination As shown in Fig-3B lysosomal contamination was not observed though minimal ER contamination was found To eliminate this ER contamination subcellular fractionation by gradient centrifugation using iodixanol was carried out and copurification of NSP 4 with mitochondria was confirmed (supplymentary fig-4A) To confirm whether mitochondria translocating ability of NSP 4 is independent of other cellular factors purified functional mouse liver mitochondria were incubated with IVT NSP 4 Mitochondrial fraction was precipitated and subjected to immunoblotting Results revealed presence of NSP 4 with the mitochondrial proteins (Fig-3C) Proximity of mitochondria and NSP 4 was further confirmed by FRET There is a significant overlap between the emission spectra of GFP and excitation spectra of TMRE making it a well matched FRET pair When two fret partners remain closer than 10 nm the intensity of fluorescence emission of the donor (GFP) is quenched by the acceptor (TMRE) This can be detected by photobleaching of the acceptor flour which results in increase of donor emission intensity To visualize the spatial proximity we transfected pAcGFP1-C2NSP 4 in Hela cells and after 16h cells were labeled with TMRE (150 nm) To see whether TMRE quenches the fluorescence of NSP 4-GFP photobleaching of TMRE was done as described in materials and methods which showed an immediate increase in emission of NSP 4-GFP indicating close proximity of two flours (Fig-3D E) Overall results confirmed presence of NSP 4 protein in mitochondria Previously it was reported that NSP 4 translocates to endoplasmic reticulum To confirm its other subcellular localization we carried out confocal microscopy with mitochondria endoplasmic reticulum and lysosomal markers Results confirmed localization of NSP 4 in mitochondria and

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endoplasmic reticulum but not in lysosome (supplementary Fig-3B) The proportion of NSP 4 present in mitochondria and endoplasmic reticulum was also confirmed by subcellular fractionation of SA11 infected MA104 cells (8hpi) using sucrose gradient as described in materials and methods Results showed that at 8 hpi the amount of NSP 4 present in endoplasmic reticulumwas 3 fold higher than that in mitochondria (supplementary Fig-3C)

61 - 83 amino acid of NSP 4 comprises the MTS To map the MTS region we generated a panel of sequential amino and carboxyl terminal truncated mutants of NSP 4 cloned in pAcGFP1-C2 (Fig- 4A)These constructs were designed to encompass or omit potential α helices (Fig-4D) To know the sub-cellular localization of the mutant NSP 4 compared with the wild type HeLa cells were transfected with either pAcGFP1-C2 or wild type or NSP 4 mutant constructs and after 16h cells were fixed α MnSOD (mitochondrial marker) was stained using anti-α MnSOD antibody followed by anti-rabbit rhodamine labeled secondary antibody When pAcGFP1-C2 was expressed alone in HeLa cells a diffuse green fluorescence was observed in both cytoplasm and nucleus but in case of NSP 4-GFP fusion protein fluorescence was found to localize at filamentous cytoplasmic structures confirmed as mitochondria by indirect immunofluorescence with α MnSOD (red) (Fig- 4B) The N terminal truncated NSP 4 mutant NSP 4Δ1-83 only showed diffused fluorescence and no co-localization with the mitochondrial marker whereas other N terminal truncated mutants NSP 4Δ1-25 NSP 4Δ1-48 NSP 4Δ1-60 showed mitochondrial localization similar to wild type NSP 4 (Fig-4B) In case of C terminal truncated mutants except for NSP 4Δ74-175 which showed diffused fluorescence like control vector (pAcGFP1-C2) vector all other mutants NSP 4Δ143-175 NSP 4Δ92-175 showed mitochondrial localization (Fig-4B) Based on these results 61-91 amino acid stretch can be predicted to harbor MTS region Sequence analysis revealed presence of one amphiphatic α helix within this region (Fig-4C) Presence of amphiphatic α helices have been reported in other mitochondria translocating cellular and viral proteins (33 32 and 34) But

to confirm whether this helix alone comprises the MTS or flanking regions are also necessary different small fractions within 61-91 aa region were cloned and subcellular localization was observed as described earlier NSP 4 (61-91) NSP 4 (61-83) mutant showed mitochondrial localization like wild type NSP 4 but NSP 4 (74-91) showed diffused fluorescence like empty vector (Fig-4B) This result revealed that not only the helical region (74-83) within the 61-83 aa region but the flanking region (61-73) was also required for mitochondrial localization

NSP 4 integrates both to outer and inner mitochondrial membrane After confirming that NSP 4 localizes to mitochondria proper localization of NSP 4 within mitochondria was examined To determine whether NSP 4 remains exposed (completely or partially) on the surface of outer mitochondrial membrane or is integrated within the mitochondria we treated the mitochondrial fraction isolated from either pcDNSP 4 transiently transfected 293T cells or from IVT NSP 4 treated mouse liver mitochondria with increasing concentration of trypsin Western blot analysis was performed with antibodies against NSP 4 hexokinase as a peripheral protein marker and voltage dependent anion channel (VDAC) as an integral protein marker Results showed that trypsin completely cleaved hexokinase but not VDAC and NSP 4 suggesting that NSP 4 was not exposed to the surface of the mitochondria (Fig-5A top) To rule out the possibility that the procedure of mitochondria isolation may affect trypsin sensitivity of NSP 4 experiments were repeated in presence of 01 TritonX100 As expected both VDAC and NSP 4 were cleaved by trypsin in presence of TritonX100 confirming that NSP 4 is integrated within the mitochondria (Fig-5A bottom)

For further confirmation isolated mitochondria were treated to separate the integral membrane proteins from peripheral membrane and soluble proteins in the intermembrane space and matrix with sodium carbonate pH 115 followed by differential centrifugation Immunoblotting was performed with anti NSP 4 antibody and hexokinase and VDAC were used as peripheral and integral protein markers respectively As

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shown in Fig-5B NSP 4 was confirmed to be a membrane integral protein Furthermore isolated mitochondria were treated with 10mM KCl followed by differential centrifugation to separate inner membrane from outer membrane and inter membrane space as KCl treatment disrupts outer membrane while leaving the inner membrane intact Fractions were subjected to western blot analysis with anti NSP 4 antibody and Cox 4 and VDAC proteins were assessed as inner and outer membrane markers respectively Result revealed presence of NSP 4 in both outer and inner membrane fractions (Fig-5C)

NSP 4 interacts with VDAC and ANT Different mitochondrial proteins play an essential role in controlling apoptosis (35) Since NSP 4 was found to be a mitochondria integral protein it is possible that it interacts with VDAC and ANT which is targeted by several other viral mitochondria translocating proteins (36 37 38 and 39) Hence to know the functional involvement of VDAC and ANT these were cloned in pcDNA6 and transfected in 293T cells After 24h cells were lysed and subcellular fractions were separated as described previously Immunoblotting revealed that both overexpressed VDAC and ANT were present in mitochondria in transfected cells (Fig-6A) though small amount of VDAC was observed in cytoplasm too (Fig-6A) To detect interaction between NSP 4 and VDAC ANT we either cotransfected pcDVDAC1 pcDANT3 with pFLAG-CMV6-NSP 4 or these three constructs were transfected individually After 24h post transfection immunoprecipitation was done (with either anti Flag antibody or anti His antibody) as mentioned in materials and methods followed by immunoblotting with reciprocal antibody Irrespective of whether NSP 4 was pulled down using anti FLAG or VDAC1ANT3 by anti-His antibodies specific interaction between NSP 4 and VDAC1 ANT3 was confirmed (fig-6B) Input lysate were probed using Cox 4 anti FLAG anti His antibodies To confirm this interaction during virus infection 293T cells expressing either VDAC1 or ANT3 were either infected with SA11 (2 moi) or mock infected Co-immunoprecipitation was done with anti NSP 4 antibody after 10 hpi followed by

immunoblotting with anti-His and anti NSP 4 antibodies Results confirmed interaction between NSP 4 and VDAC1 and ANT3 protein during virus infection (Fig-6C) Thus it can be concluded that NSP 4 causes depolarization of mitochondria and release of cyt c probably through interacting with VDAC and ANT

During SA11 infection Ca+2 ion elevation and Bax activation independent mitochondrial depolarization occurs From the previous experiments it was confirmed that overexpression of NSP 4 leads to its translocation to mitochondria (Fig-3B) and dissipation of mitochondrial membrane potential (Fig-2B) but whether similar effects are observed during viral infection was not proved Therefore we either infected MA104 cells with SA11 at 2 moi or mock infected After 2 hpi-14 hpi cells were incubated with TMRE (100 nM) for 20 min and depolarization of mitochondria was measured by flowcytometry Results revealed significant increase in depolarization (46-664) in time dependent manner from 5h of post infection (Fig-7A) To confirm whether this depolarization was independent of Ca+2 ion flux and Bax activation cells were transiently transfected with Bax-siRNA After 24h cells were infected with SA11 (2 moi) and after 2hpi 50 microM BAPTA-AM was added and mitochondrial depolarization was measured at 2hpi-14hpi As shown in Fig-7B BAPTA-AM and Bax-siRNA together reduced the intensity of mitochondrial depolarization (324 at 14 hpi) compared to virus infected cells (664 at 14 hpi) (Fig-7A) though still significant mitochondrial depolarization is observed which is independent of Ca+2 ion or Bax activation This is consistent with our previous results following NSP 4 over expression (Fig-2A) To confirm this depolarization is due to NSP 4 we infected NSP 4 siRNA or scrambled NSP 4 siRNA transfected MA104 cells and measured the mitochondrial depolarization Compared to scrambled NSP 4 siRNA transfected cells significant decrease in mitochondrial depolarization was observed in NSP 4 siRNA transfected cells following SA11 infection (2-14 hpi) (Fig-7C)

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At 5 hpi mitochondrial depolarization was observed in SA11 infected MA104 cells suggesting release of cyt c from mitochondria into cytosol To assess this MA104 cells were either infected with SA11 (at 2 moi) or left untreated for given time points and western blot analysis was done for cytoplasmic cytc following subcellular fractionation As expected release of cytc from mitochondria was detected as early as 5 hpi whereas Bax was found to translocate to mitochondria from 11hpi onwards (Fig-8A) However when release of cyt c to cytosol was assessed in MA104 cells transfected with NSP 4 siRNA 24h prior to SA11 infection cyt c release to cytosol was not observed until 11-14 hpi compared to 5 hpi in only SA11 infected cells transfected with scrambled NSP 4 siRNA (Fig-8B) This delay in cyt c release was due to downregulation of NSP 4 since gt80 reduction in NSP 4 expression was observed in siRNA transfected cells (Fig-8C) suggesting that early cyt c release is due to NSP 4

In Rotavirus early infection intrinsic apoptotic stimuli initiated by NSP 4 is supressed by virus initiated survival pathways Cyt c release into cytoplasm has been shown to facilitate activation of caspase-9 by formation of functional apoptosome (40) Thus activation of caspases or other apoptotic marker proteins were analyzed in time dependent manner following SA11 infection Cell lysates were prepared from MA104 cells infected with either SA11 or mock infected (2hpi-14hpi) followed by immunoblotting with caspase-9 PARP pAKT AKT and XIAP specific antibodies Upregulation of antiapoptotic protein XIAP and AKT phosphorylation was observed as early as 2 hpi until 8 hpi (Fig-8D) which is consistent with our previous report (41) In contrast cleavage of caspase-9 and PARP protein was observed after 11 hpi (Fig-8D) previous studies with rotaviral strain A5-13 from our lab have reported induction of apoptosis during late hours of infection (after 12 hpi) which was attributed to induction of antiapoptotic pathway during early infection (41) Thus to confirm whether activation of PI3KAKT pathway counteracts effect of cyt c release MA104 cells were either infected with SA11 or mock infected and treated

with 10microM LY294002 (PI3K inhibitor) at post absorption Caspase-9 cleavage and expression of pAKT and XIAP was measured by immunoblotting in time dependent manner As shown in (Fig-8E) LY294002 treatment resulted in downregulationof XIAP protein and early activation of caspase-9 cleavage In addition downregulation of both NSP 4 (NSP 4 siRNA) and PI3KAKT (by LY294002) significantly delayed caspase-9 cleavage until 11-14hpi (Fig-8F) compared to only LY294002 alone treated cells (Fig-8E) For further confirming the role of NSP1 activated PI3KAKT pathway we infected MA104 cells with NSP1 mutant rotavirus strain A5-16 at 2 moi and analyzed the cyt c release from mitochondria and caspase activation at indicated time points (Fig-9A) Result revealed that compared to SA11 infection (Fig-8A) there is no change in cyt c release during A5-16 infection (Fig-9A) but caspase activation start at early time point (8hpi) (Fig-9A) This suggested the role of PI3KAKT pathway in preventing NSP 4 initiated apoptotic stimuli during early infection This counter acting role of NSP1 was further proved when release of cyt c from mitochondria (Fig-9C) and cell death was measured (Fig-9D) in NSP 4 and NSP1 co transfected cells Compared to only NSP 4 expressing cells significant decrease in apoptosis was observd in NSP 4 and NSP1 coexpressing cells Overall results confirmed role of NSP 4 in early release of cyt c from mitochondria to cytosol but delay in caspase-9 activation due to the activation of cellular survival pathway during rotavirus infection

Discussion

Rotaviral nonstructural protein NSP 4 is the first discovered viral enterotoxin (3 5 and 4) with subcelluar localization at ER (10) In this study we showed NSP 4 localizes to mitochondria and induces apoptosis Other viral proteins such as PB1F2 of influenza virus (36) HBX of hepatitis B virus (37) VPR of human immune deficiency virus (38) localize to mitochondria and trigger apoptotic pathways Induction of apoptosis during virus infection is important for viral release and dissemination of viral progeny (42) Unlike influenza virus (36) human immunodeficiency virus (38) htlv 1 (39) etc no

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rotavirus encoded protein has been shown to directly induce apoptosis Previous studies have analyzed increase in Ca+2 ion concentration (12) and activation of proapoptotic protein Bax (13) as probable cause of rotavirus induced apoptosis Ca+2 homeostasis is stringently maintained in cells because disruption in Ca+2 homeostasis predisposes cells towards apoptosis (43) Bax is a Bcl2 family cytoplasmic protein which after activation by diverse stimuli inserts into mitochondria leading to mitochondrial depolarization and release of pro-apoptotic proteins from mitochondria resulting in initiation of caspase activation and apoptosis (44 45 46 47 and 48)

In this study we propose that NSP 4 mediated apoptosis is not fully dependent on Ca+2 ion concentration or Bax activation since significant apoptosis was observed in NSP 4 expressing cells in presence of Bax-siRNA and Ca+2chelator (Fig-1C) This suggested that some other proapoptotic factor or pathways are activated by NSP 4 Apoptosis can be broadly divided into extrinsic and intrinsic pathway Extrinsic pathway is initiated by death receptor stimulation and caspase-8 activation whereas intrinsic pathway involves mitochondrial dysfunction resulting in release of proapoptotic proteins from mitochondria and activation of caspase-9 (49 50 and 51) Analysis of both pathways following NSP 4 over expression revealed depolarization of mitochondria release of cyt c into cytosol and activation of caspase-9 both in vitro (Fig-2A 2C 1D) in cell free condition (Fig-2B 2D) and during virus infection (Fig-7A 8A 8D) suggesting the involvement of intrinsic pathway Since pcDNSP 4 expression or IVT-NSP 4 treatment resulted in cyt c release from mitochondria and NSP 4 siRNA significantly delayed cyt c release in SA11 infection (Fig-8B) direct role of NSP 4 on mitochondria was postulated Close proximal presence of NSP 4 and mitochondria was observed by doing FRET analysis suggesting that NSP 4 may have to translocate to mitochondria for this function (Fig-3D) Presence of NSP 4 in mitochondria was further confirmed by immunoblotting subcellular fractionation of cells infected with SA11 or transfected with pcDNSP 4 (Fig-3A B)

Quantitation of NSP 4 in subcellular fractions confirmed 3 fold higher conc of NSP 4 in ER compared to mitochondrial fraction (supplementary -4b)

Although during NSP 4 over expression all forms of NSP 4 were evident (FIG-1A) but only non glycosylated form was copurified with mitochondrial fraction (Fig-3B) which is further confirmed by translocation of IVT NSP 4 to mitochondria (Fig-3C) as in vitro transcription and translation does not support glycosylation

To know the shortest region necessary for mitochondrial translocation of NSP 4 various deletion mutants were constructed Immunoflouresence microscopy following transfection identified a 23 amino acid long region (aa 61-83) to be sufficient for mitochondrial translocation (Fig-4B) May be there are other regions involved for more efficient translocation It was reported that full length NSP 4 is targeted to ER by an uncleaved signal sequence (aa 25-44) (52) and gets inserted into the membrane by a viroporin region (aa 47-90) (10) Identified MTS (61-83) is within the reported viroporin domain which is responsible for NSP 4 mediated membrane destabilization Similar to other mitochondrial proteins NSP 4 also has a putative amphipathic helix (aa 74-83) (Fig-4C) within the MTS region (33 34) Once the MTS region of NSP 4 was identified next question was to know the localization of NSP 4 within mitochondria No effect of trypsin on mitochondrial NSP 4 in absence of detergent (TritonX100) (Fig-5A upper panel) and degradation of NSP 4 by trypsin in presence of tritonX100 (Fig-5A lower panel) suggested that NSP 4 is not localized on peripheral mitochondrial membrane but is part of integral membrane Similarly alkaline [Sodium carbonate (pH-115)] and KCl treatment confirmed it to be an insoluble protein embedded both in outer and inner mitochondrial membranes (Fig-5B 5C) which is similar to the PB1F2 protein of influenza virus (32) This suggested that NSP 4 might be targeting VDAC and ANT To confirm this hypothesis NSP 4 was co-immunoprecipitated with VDAC1 and ANT3 in both SA11 infected and pcDNSP 4

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expressing cells (Fig-6B 6C) Isoforms VDAC1 and ANT3 were utilized because of their abundance and ubiquitousness (53 54) Overall it can be hypothesized that NSP 4 contributes to mitochondrial dysfunction by interacting with VDAC and ANT possibly through VDAC closure with impaired substrate supply to mitochondria and or inhibition of ANT with shortage of ATP production which leads to apoptosis But whether a single NSP 4 molecule binds with both VDAC and ANT forming a bridge or it binds separately cannot be defined from this study and it does not exclude possible interaction of NSP 4 with other mitochondrial proteins involved in mitochondrial permeability transition Further in-depth analysis is required to know the exact mechanism of permeability transition During infection NSP 4 was found to be present in mitochondria as early as 4hpi which increases with time (Fig-4A) This correlates with significant mitochondrial depolarization and release of cyt c from 5 hpi (Fig-7A 8A) In contrast Bax translocation was observed at11hpi (Fig-8A) suggesting this two processes to be independent Consistent to our observation previous report with RRV rotavirus strain had shown cyt c release at 9 hpi and Bax activation at 12 hpi (13) Cytosolic cyt c interacts with APAF-1 (57-44) triggering its oligomerisation which allows the recruitment of procaspase-9 leading to its autocatalytic cleavage and activation Activated caspase-9 then initiates the cascade of caspase activation leading to apoptosis (55 56 and 57) Surprisingly we observed caspase-9 cleavage after 10-11 hpi in spite of cyt c release at 5 hpi (Fig-8D) Thus some other regulatory pathway may be involved to prevent early activation of caspase-9 by cyt c Induction of antiapoptotic pathways such as PI3KAKT during early steps of viral infection have been reported previously (41) Both rotavirus NSP1 and influenza virus NS1 proteins were shown to interact with PI3K and induce AKT phosphorylation and upregulation of XIAP (41 58) which can bind with caspase-9 and caspase-3 and inhibit their autocatalytic activity (59 60) In SA11 infection both AKT phosphorylation and XIAP upregulation were observed as early as 2hpi but was downregulated

after 10-12 hpi (Fig-8D) Role of PI3KAKT activation in delaying apoptosis was confirmed when significant caspase-9 cleavage was observed as early as 8 hpi in presence of PI3K inhibitor (Fig-8E) However in presence of both PI3K inhibitor and NSP 4 siRNA caspase-9 activation was delayed further (14 hpi) (Fig-8F) suggesting that down regulation of NSP 4 results in inhibition of initiation of proapoptotic signaling resulting in delayed apoptosis Induction of apoptosis during late hours is due to both host innate immune responses and other viral induced pathways for efficient viral dissemination (41)

Overall the results from previous study and our study suggested dual role of NSP 4 during rotavirus infection It elevates the Ca+2 ion concentration during infection which results in disturbance of cellular homeostasis triggering stress response and plays a vital role in pathogenesis In addition we have shown that it translocates to mitochondria and disrupts mitochondrial membrane potential eliciting intrinsic apoptotic pathway But the activation of early apoptosis may not be beneficial for virus as it will abort its replication and formation of infectious progeny (61) To maintain this balance rotavirus counteracts the effect of NSP 4 by activating PI3KAKT pathway through rotaviral protein NSP1 We have previously shown early apoptosis induction and slower growth rate of NSP1 mutant strain compared to wild type rotavirus strain (41) NSP1 mutant strain A5-16 resulted in cyt c release (FIG-9A) and mitochondrial depolarization (Supplementary Fig-2) similar to SA11 (5 hpi) but unlike SA11 strain early caspase-9 activation (FIG-9A) was observed Counteracting role of NSP1 was further confirmed when only NSP 4 expressing cells induce much higher apoptosis compared to NSP 4 and NSP1 coexpressing cells (Fig-9D) inspite of comparable cyt c release from mitochondria (Fig-2C 9C) This type of cell death regulation by two virus encoded proteins has been also observed in influenza virus polio virus and adenovirus etc (58 41 62 and 63) During the course of evolution viruses have employed viral proteins host intrinsic apoptotic pathways for their own benefit

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20 Wood-Allum CA Barber SC Kirby J Heath P Holden H Mead R Higginbottom A Allen S Beaujeux T Alexson SE Ince PG Shaw PJ (2006) Impairment of mitochondrial anti-oxidant defence in SOD1-related motor neuron injury and amelioration by ebselenBrain129 1693-709

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22 Frezza C Cipolat S and Scorrano L (2007) Organelle isolation functional mitochondria from mouse liver muscle and cultured fibroblasts Nat Protoc2 287-295

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25 Chen J and Siddiqui A (2007) Hepatitis B virus X protein stimulates the mitochondrial translocation of Raf-1 via oxidative stress J Virol 81 6757ndash6760

26 Goping IS Gross A Lavoie JN Nguyen M and Jemmerson R (1998) Regulated targeting of Bax to mitochondria J Cell Biol 143 207ndash215

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28 Rahmani Z Huh KW Lasher R and Siddiqui A (2000) Hepatitis Bvirus X protein colocalizes to mitochondria with human voltage-dependentanion channel HVDAC3 and alters its transmembrane potential J Virol 74 2840ndash2846

29 Peacuterez JF Chemello ME Liprandi F Ruiz MC and Michelangeli F (1998)Oncosis in MA104 cells is induced by rotavirus infection through an increase in intracellular Ca+2concentrationVirology252 17-27

30 Grynkyewicz G Poenie MandTsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties J Biol Chem 260 3440-3450

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32 Gibbs JS Malide D Hornung F Bennink JR and Yewdell JW (2003) The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function J Virol 77 7214-7224

33 Endo T and Kohda D (2002)Functions of outer membrane receptors in mitochondrial protein import BiochemBiophysActa1592 3-14

34Gouttenoire J Montserret R Kennel A Penin F and Moradpour D (2009) An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association J Virol 83 11378-11384

35 Zamzami N and Kroemer G (2001) The mitochondrion in apoptosis how Pandoras box opens Nat Rev Mol Cell Biol2 67-71

36 Zamarin D Garciacutea-Sastre A Xiao X Wang R and Palese P (2005) Influenza virus PB1F2 protein induces cell death through mitochondrial ANT3 andVDAC1 PLoSPathog1 e4

37 Rahmani Z Huh KW Lasher R and Siddiqui A (2000) Hepatitis B virus X protein colocalizesto mitochondria with a human voltage-dependent anion channel HVDAC3 and alters its transmembrane potential J Virol 74 2840ndash2846

38 Jacotot E Ferri KF El Hamel C Brenner C Druillennec SHoebeke J Rustin P Meacutetivier D Lenoir C Geuskens M Vieira HL Loeffler M Belzacq AS Briand JP Zamzami N Edelman L Xie ZH Reed JC Roques BP and Kroemer G (2001) Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2 J Exp Med 193 509ndash519

39 D DAgostino L Ranzato G Arrigoni I Cavallari F Belleudi M Torrisi M Silic-Benussi T Ferro V Petronilli O Marin L Chieco-Bianchi P Bernardi V Ciminale (2002) Mitochondrial alterations induced by the p13II protein of human T-cell leukemia virus type 1 Critical role of arginine residuesJournal of Biological Chemistry277 pp 34424ndash34433

40 Srinivasula SM Ahmad M Fernandes-Alnemri T and Alnemri ES (1998)Autoactivation of procaspase-9 by Apaf-1-mediated oligomerizationMol Cell 1 949-957

41 Bagchi P Dutta D Chattopadhyay S Mukherjee A HalderUCSarkar S Kobayashi N Komoto S Taniguchi K and Chawla-Sarkar M (2010) Rotavirus nonstructural protein 1 suppresses virus-induced cellular apoptosis to facilitate viral growth by activating the cell survival pathways during early stages of infection J Virol 84 6834-6845

42 Roulston A Marcellus RC and Branton PE (1999) Viruses and apoptosisAnnu RevMicrobiol 53 577-628

43 Orrenius S and Nicotera P (1994) The calcium ion and cell death JNeuralTransmSupp l43 1-11

44Desagher S Osen-Sand A Nichols A Eskes R Montessuit S Lauper S Maundrell K Antonsson B and Martinou JC (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis J Cell Biol 144 891-901

45 Gross A Jockel J Wei MC and Korsmeyer SJ (1998) Enforced dimerization of BAX results in its translocation mitochondrial dysfunction and apoptosis EMBO J 17 3878-3885

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46 Korsmeyer S J Wei MC Saito M Weiler S Oh KJ and Schlesinger PH (2000) Pro-apoptotic cascade activates BID which oligomerizes BAK or BAX into pores that result in the release of cytochromec Cell Death Differ 7 1166-1173

47 Wei MC Zong WX Cheng EH Lindsten T PanoutsakopoulouVRoss AJ Roth KA MacGregor GR Thompson CB and Korsmeyer SJ (2001)Proapoptotic BAX and BAK a requisite gateway to mitochondrial dysfunction and death Science292 727-730

48 Smaili SS Hsu YT Sanders KM Russell JT and Youle RJ(2001) Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potentialCell Death Differ 8 909-920

49 Vaux DL and Korsmeyer SJ (1999) Cell death in development Cell 96 245-254

50 Wang X (2001) The expanding role of mitochondria in apoptosis Genes Dev15 2922-2933

51 Green DR and Reed JC (1998) Mitochondria and apoptosis Science 281 1309-1312

52 Bergmann CC Maass D Poruchynsky MS Atkinson PH and Bellamy AR (1989) Topology of the non-structural rotavirus receptor glycoprotein NS28 in the rough endoplasmic reticulum EMBO J 8 1695ndash1703

53 De Pinto V Guarino F Guarnera A Messina A Reina STomasello FM Palermo V and Mazzoni C (2010) Characterization of human VDAC isoforms a peculiar function for VDAC3BiochimBiophysActa17971268-1275

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55 Li P Nijhawan D Budihardjo I Srinivasula SM Ahmad M Alnemri ES and Wang X (1997) Cytochrome canddATP-dependent formation of Apaf-1caspase-9 complex initiates an apoptotic protease cascade Cell 91 479ndash489

56 Srinivasula SM Ahmad M Fernandes-Alnemri T and Alnemri ES (1998)Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization Mol Cell 1949ndash957

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63 Rao L Debbas M Sabbatini P Hockenbery D and Korsmeyer S (1992)The adenovirus E1A proteins induce apoptosis which is inhibitedby the E1B 19-kDa and Bcl-2 proteins Proc Natl Acad Sci USA 89 7742ndash7746

Footnotes

This study was supported by the Indian Council of Medical Research (ICMR) New Delhi and the Program for Funding Research Centers for Emerging and Reemerging Infectious Diseases (Okayama University-National Institute of Cholera and Enteric Diseases India) from the Ministry of Education Culture Sports Science and Technology Japan

1 These authors were supported by Junior Research Fellowships from the Council of Scientific and Industrial Research (CSIR) India 2 These authors were supported by Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR) India 3 supported by Senior Research Fellowship from ICMR 4 supported by Junior Research Fellowships from the University Grants Commission (UGC) India 5 supported by Dr DSKotharipost doctoral fellowships from the University Grants Commission (UGC) India Antibody of NSP 4 was kindly donated by Koki Taniguchi Antibody of Trap-alpha was kindly donated by Dr RS Hegde George Banik is acknowledged for his technical support The abbreviations used are NSP Nonstructural protein aa Amino acid MTS Mitochondria translocating signal PTPC permeability transition pore complex moi Multiplicity of infection hpi Hours post infectionVP viral protein ER Endoplasmic reticulum IVT invitro coupled transcription and translation VDAC Voltage dependent anion channel ANT Adenine nucleotide translocator Cyt c Cytochrome cCsA CyclosporineAsupe supernatantFRETFluorescence resonance energy transfer Figure legends

Figure1

NSP 4 induced apoptosis is not fully dependent on ca+2 ion efflux and Bax activation

(A) Ectopic expression of Nonstructural protein 4 (NSP 4) in 293T cells pcDNA6or pcDNSP 4 were transfected in 293T cells andat 16h and 24h cells were harvested Whole cell lysateswereresolved by SDS-PAGE and subjected to immunoblotting with NSP 4 antisera for confirming expression of NSP 4 Actin was used as an endogenous control for equal protein loading (B) Comparision between expression levels of NSP 4 during ectopic expression and infection (C) Analysis of apoptosis by NSP 4 in 293T cells Apoptosis was measured by TUNEL assay using flowcytometry Cells were harvested after 24h of transfection and incubated with Tdt and FITC-conjugated anti-BrdU monoclonal antibody as per kit protocol (APO-BRDUtrade Kit) 293T cells transfected with pcDNSP 4 showed 44 apoptotic cells compared to only vector control cells (III) 293T cells transfected with pcDNSP 4 were treated with BAPTA-AM (50microM) [6h after transfection] for 24h As shown in (IIIIV) NSP 4 expressing cells revealed reduced

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apoptosis (244) in presence of BAPTA-AM compared to only pcDNSP 4 expressing cell (II) In 293T cells pretreated with Bax-siRNA for 24 h followed by transfection with pCDNSP 4 or pcDNA vector control reduced apoptosis was observed compared to only pcDNSP 4 expressing cells (II) In cells treated with both Bax-siRNA and BAPTA-AM expression of pcDNSP 4 (VIII) resulted in significant reduction in of apoptotic cells (215) compared to only pcDNSP 4 expressing cells (II) In cells transfected with pcDNA6 only 37-74 apoptotic cells were observed (I III V VII) irrespective of Bax-siRNA or Ca+2chelator treatment (D) NSP 4 expression results in cleavage of caspase-9 and PARP 293T cells were transfected with either pcDNA6 or pcDNSP 4 and cell lysates were prepared after 16 h and 24 h Immunoblotting was done with caspase-8 caspase-9 caspase-3 caspase-7 and PARP antibodies for assessing there cleavage Actin was used as an endogenous control for equal protein loading (E) NSP 4 expression elevated BAPTA-AM titrable ca+2 ion levels in 293T cells Cells were transfected with pcDNSP 4 and intracellular Ca+2 ion conc was measured during 1-24 h using fura-2 in absence or presence of BAPTA-AM (50microM) [added 6 h after transfection] (F) Knockdown of Bax expression in 293T cells Cells were transfected with either specific Bax-siRNA or Scrambled Bax-siRNA Expression of Bax protein was then assayed by immunoblotting the cell lysates after 24 h Actin was used as an endogenous control for equal protein loading

Figure 2

NSP 4 depolarizes mitochondria and induces release of mitochondrial cyt c

(A) Effect of NSP 4 on mitochondrial membrane potential 293T cells were transfected with pcDNSP 4 or vector control and 18 h post transfection cells were incubated with 100 nM TMRE for 30 min at 37oC followed by analysis for TMRE accumulation within mitochondria using flowcytometry Compared to Vector control (I) pcDNSP 4 expressing cells showed significant dissipation of mitochondrial potential (II) In cells pretransfected with Bax-siRNA followed by transfection with pcDNSP 4 (IV) or pcDNSP 4 expressing cells treated with 50microM BAPTA-AM [6h posttransfection] (VI) depolarization of mitochondria was asymp 20 to 40 less compared to only pcDNSP 4 expressing cells (II) In presence of both Bax-siRNA and BAPTA-AM (VIII) significant reduction (50) in NSP 4 mediated dissipation of mitochondrial potential (II) was observed (B) IVT NSP 4 depolarized purified mouse mitochondria Mouseliver mitochondria (50μg total protein) were incubated for 10 min at RT with invitro coupled transcripted and translated (IVT) NSP 4 (0-02microM) followed by incubation with TMRE (50nM) for 10 min at RT followed by measurement of mitochondrial membrane potential dissipation fluorometrically Data is represented as percent fluorescence relative to mock treated mitochondria (C) Cyt c released by NSP 4 is partly independent of Ca+2 ion and Bax activation 293T cells were transfected with either Bax-siRNAor nonspecific control siRNA After 24 h both sets of cells were transfected with with pcDNSP 4 or pcDNAcontrol After 6 h cells were treated with BAPTA-AM (50 microM) After 24 h of transfection cells were harvested and subcellular fractions were separated followed by immunoblotting of cytosolic extract with cyt c antibody Pellet was immunoblotted with Cox 4 and β actin as control (D) IVT NSP 4 alsoreleasescyt c from mouse liver mitochondriaMouse liver mitochondria (50 microg) were incubated with IVT NSP 4 (0-02μM) for 1 h at 30oC Reaction mix was centrifuged

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(7000gX10min) to pellet down mitochondria and supe was immunoblotted to assess cyt c release in cytosol IVT reaction mixture control and IVT pcDNA control were used as negative control Mitochondrial pellet was immunoblotted with anti Cox 4 antibody as internal control (E) NSP 4 induced cyt c release is independent of caspase activation 293T cells transfected with pcDNSP 4 cells were either treated with broad spectrum caspaseinhibitor z-vad-fmk (10 μ ) or DMSO control followed by subcellular fractionation after 24 h Cyt c released into cytosol was detected by immunoblot analysis

(F) NSP 4 induced cell death is dependent on caspase activation 293T cells transfected with pcDNSP 4 cells were either treated with broad spectrum caspaseinhibitor z-vad-fmk (10μ ) or DMSO control followed by cell death analysis by TUNEL assay

Figure 3

NSP 4 translocates to mitochondria

(A)NSP 4 is localized in mitochondria enriched fraction during infection MA104 cells were either infected with SA11 (2 moi) or mock infected Mitochondrially enriched fractions were isolated resolved in SDS-PAGE and immunoblotted with NSP 4 antisera Cox 4 was used as internal control for equal protein loading

(B) Localization of NSP 4 to mitochondria is independent of other viral proteins 293T cells were transfected with either pcDNSP 4 or empty vector After 24 hmitochondrially enriched fractions were isolated resolved in SDS-PAGE and immunoblotted with NSP 4 antisera and anti Cox 4 antibody (loading control) anti Calnexin antibody (endoplasmic reticulum marker) anti Lamp2 antibody (lysosome marker)

(C) IVTNSP 4 translocates into mitochondria Purified IVT NSP 4 and IVT reaction mixture control IVT pcDNA control (as negative control) were incubated with rat liver mitochondria (50μg total protein) for 1h at 30oCReaction mix was centrifuged (7000gX10min) and pellets were immunoblotted with NSP 4 antisera and anti Cox 4antibody (loading control)

(D) Fluorescence resonance energy transfer (FRET) analysis to assess localization of NSP 4 within mitochondria Using confocal system images of donor acquired using a 488-nm excitation line and emission between 500 and 550 nm obtained pre and post bleaching showed dequenching of the donor Using a 568 nm excitation line and emission between 580 and 620 nm sequential scan images of the acceptor were also acquired pre and post bleaching showing effective bleaching of the acceptor Spot bleaching of the acceptor were performed as described in material and mehod ROI is indicated by arrow

(E) Intensity of NSP 4-GFP (donor) increased concomitantly with the bleaching-induced decrease in the TMRE (acceptor) signalThese panels depict the mean pixel intensity profiles over time of the bleached region bleaching point is marked by arrows

Figure 4

Identification of mitochondria translocation sequence of NSP 4

(A) Schematic representation of N-terminal c-Terminal NSP 4 mutants generated in pACGFP1-c2 vector as described in method section

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(B) 60-83 aa is the shortest region of NSP 4 that acts as MTS HeLa cells were transfected with either empty GFP expressing vector or wild type NSP 4 or mutant constructs of NSP 4 as GFP fusion protein (green) After 16 h of transfection cells were fixed and stained with anti α MnSOD antibody (mitochondrial marker) followed by rhodamine labeled (red) secondary antibody and DAPI (blue) The cells were visualized by fluorescence microscope to assess localization of NSP 4 (green) and mitochondria (red)

(C) Helical wheel representation (Helical wheel projection Don Armstrong) of a predicted helix formed by residues 74 to 83 of NSP 4Hydrophilic residues as circles hydrophobic residues as diamonds potentially negatively charged as triangles and potentially positively charged as pentagons The most hydrophobic residue is green and the amount of green is decreasing proportionally to the hydrophobicity with zero hydrophobicity coded as yellow Hydrophilic residues are coded red with pure red being the most hydrophilic (uncharged) residue and the amount of red decreasing proportionally to the hydrophilicity The potentially charged residues are light blue

(D) Putative helix prediction (H helix) within NSP 4 sequence usingPSIPRED (PSIPRED Server) and sspro (Phyre Server) programs

Figure 5

NSP 4 is present in both outer and inner membrane of mitochondria

(A) Trypsin sensitivity assays reveal presence of NSP 4 on integral membrane Either mitochondria enriched fraction isolated after 24 h from 293T cells transfected with pcDNSP 4 (Left) or pelleted rat liver mitochondria after being incubated with IVT NSP 4 (Right) were treated with trypsin (25-50μgμl) in presence or absence of Tritonx100 Immunoblot analysis for NSP 4 Hexokinase (marker for peripheral membrane) and VDAC (integral membrane marker) revealed no effect of trypsin on NSP 4 and VDAC in absence of Tritonx100 (Top panel) whereas in presence of Tritonx100 both NSP 4 and VDAC were degraded Peripheral membrane marker protein hexokinase is degraded both in presence or absence of Tritonx100

(B) Alkaline treatment confirmed presence of NSP 4 on both inner and outer mitochondrial integral membrane Mitochondria enriched fraction isolated after 24 h from 293T cells transfected with pcDNSP 4 (Left) or pelleted mouse liver mitochondria after being incubated with IVT NSP 4 (Right) were subjected to alkaline treatment (NaHCO3) Reaction mix was centrifuged to pellet down mitochondrial integral membrane Both supe and pellets were immunoblottedusing NSP 4 antisera anti VDAC and anti Hexokinase antibodies (C) KCl treatment confirms co purification of NSP 4 with both mitochondrial outer and inner membrane integrated proteins As described previously (B) mitochondria enriched fractions were subjected to KCl treatment Treated fractions were fractionated by differential centrifugation followed by immunoblotting with NSP 4 antisera anti VDAC and anti Cox 4 antibody

Figure 6

NSP 4 interacts with VDAC1 and ANT3

(A) Confirmaion of expression and localization of ANT3 and VDAC1 proteins in transfected cells 293T cells transfected with pcDVDAC1 pcDANT3for 24 h fractionated to cytosol and mitochondrial fraction followed by immunoblottingwitrh anti VDAC1 anti ANT3 antibody Cox 4 and actin served as protein loading control for mitochondrial and cytosolic fraction respectively

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(B) NSP 4 specifically interacts with VDAC1 and ANT3His tagged VDAC1 and ANT3 (pcDVDAC1pcDANT3) were either expressed separately or with FLAG tagged NSP 4 (pFLAG-CMV6-NSP 4) in 293T cells NSP 4 was immunoprecipitated (IP) using an anti FLAG antibody and probed for VDAC1 and ANT3 with an antiHis antibody (left) In reciprocal immunoprecipitation VDAC1 and ANT3 were immunoprecipitated (IP) using an anti-His antibody and probed for NSP 4 with an anti FLAG antibody Cox 4 was used as mitochondrial immunoprecipitation control

(C) NSP 4 expressed during infection interacts with VDAC1 and ANT3 293T cells transfected with pcDVDAC1 pcDANT3 were either infected with SA11 (2 moi) or mock infected After 8hpi lysates were immunoprecipitated with NSP 4 antisera and probed with anti His antibody to confirm co immunoprecipitation with His tagged VDAC1 and ANT3

Figure 7

Rotavirus (SA11) infection induces mitochondrial depolarization in MA104 cells

(A) SA11 induced dissipation of mitochondrial membrane depolarization MA104 cells were either infected with SA11 at 2 moi or mock infected After 2-14 hpi cells were incubated with TMRE (100nM) for 30 min at 37oC and TMRE accumulation was analyzed using flow cytometry Representative bar diagrams showed cells with depolarized mitochondria For positive controls cells were treated with 2microM STS for 6 h followed by TMRE staining

(B) Rotavirus (SA11) induced mitochondrial depolarization is not fully dependent on Ca+2 ion elevation and Bax activation MA104 cells were transfected with Bax-siRNA for 24 h followed by either SA11 infection at 2 moi or mock infected At 2 hpi cells were treated with BAPTA-AM (50 microM) 2-14 hpi cells were harvested and stained with TMRE as described previously (A)

(C) SA11 induced dissipation of mitochondrial membrane depolarization inhibited by NSP 4 siRNA MA104 cells were either infected with SA11 at 2 moi or mock infected after 24 h transfection with either NSP 4 siRNA or scrambled NSP 4 siRNA After 2-14 hpi cells were incubated with TMRE (100nM) for 30 min at 37oC and TMRE accumulation was analyzed using flow cytometry Representative bar diagrams showed cells with depolarized mitochondria

Figure 8

NSP 4 mediated apoptotic signaling is counteracted by virus involved pro-survival pathways

(A) Co-relation of cyt c release into cytosol with Bax translocation to mitochondria during rotavirus (SA11) infection At 0 hpi to 14 hpi MA104 cells infected with SA11 (2 moi) were harvested and subjected to subcellular fractionation Western blot analysis was done on cytosolic and mitochondrial fraction using anti cyt c and anti Bax antibodies Cox 4 and GAPDH were used as loading control for mitochondrial and cytosolic fractions respectively

(B) Downregulation of NSP 4 by siRNA delays cyt c release into cytosol MA104 cells were treated with NSP 4 siRNAor scrambled NSP 4 siRNAfor 24 h followed by infection with SA11 at 2 moi At increasing time point (o-14 hpi) cells were subjected to subcellular fractionation Cytosolic extracts were analyzed for cyt c by immunoblotting GAPDH was used as protein loading control

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(C) NSP 4 siRNAdownregulates NSP 4 during infection MA104 cells were transfected with NSP 4 siRNA or Con siRNA for 24 h followed by infection with SA11 at 2 moi and after 8 hpi whole cell lysates wereimmunoblotted with NSP 4 antiseraGAPDH was used as protein loading control

(D) Rotavirus (SA11) infection activates both apoptotic and antiapoptotic proteins MA104 cells were infected with SA11 at 2 moi and cells were harvested at increasing time points during 0-14 hpi Caspase-9 PARP pAKT AKT XIAP proteins were analyzed by western blot analysis GAPDH was used to confirm equal protein loading

(E) Inhibition of rotavirus induced prosurvival pathway induces early apoptosis MA104 cells were infected with SA11 at 2 moi and after virus absorption PI3K inhibitor LY294002 (10 microM) was added to media At increasing time points cells were harvested and pAKT AKT XIAP caspase-9 PARP GAPDH were analyzed by immunoblotting

(F) Downregulation of NSP 4 counteracts early apoptosis initiation by LY294002 during rotavirus infecrtion MA104 cells were transfected with NSP 4 siRNA for 24 h followed by infection with SA11 at 2 moi After viral absorption cell were treated with 10 microM of LY294002 and harvested at increasing time points (0-14 hpi) Caspase-9 clevage was assessed by immunoblotting

Figure 9

Rotavirus encoded protein NSP1 counteracts proapoptotic signaling of NSP 4

(A) Absence of NSP1 does not affect rotavirus induced cyt c release but facilitates early apoptosis MA104 cells were infected with NSP1 mutant strain A5-16 (2 moi) and at increasing time points (0-14 hpi) cells were harvested Immunoblot analysis of either cytosolic fraction or whole cell lysate for either cyt c released into cytosol or caspase-9 caspase-3 cleavage was done GAPDH was used as loading control for cytosolic fraction

(B) Co-overexpression of NSP 4 and NSP1 293T cells were transfected with either non expressing vector control or pcDNSP 4 or cotransfected with pcDNSP 4 and pcDNSP1 followed by western blot analyses using NSP1 and NSP 4 antisera β-Actin was used to confirm equal protein loading

(C) NSP1 minimizedcyt c releasing effect of NSP 4 293T cells were transfected with either non expressing vector control or pcDNSP1 or cotransfected with pcDNSP 4 and pcDNSP1 followed by subcellular fractionation and western blot analyses of the cytoplasmic fraction using anti cyt c antibody β-Actin was used to confirm equal protein loading

(D) NSP1 diminished proapoptotic stimuli elicited by NSP 4 293T cells were transfected with empty vector control or pcDNSP 4 or cotransfected with pcDNSP 4 and pcDNSP1 and incubated with terminal deoxynucleotidyltransferase and FITC-conjugated anti-BrdU monoclonal antibodiesand DNA fragmentation was measured after 24h by TUNNEL assay using flowcytometry

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Table 1

List of primers

Vector Gene RE site Primer sequence pcDNA6v5 (Invitrogen)

NSP 4 EcoR1 EcoRv

5rsquo-GAATTCATGGAAAAGCTTACCGACC-3rsquo 5rsquo-GATATCCACATTGCTGCAGTCACTTCT-3rsquo

pcDNA6v5 (Invitrogen)

VDAC1 EcoR1 Xho1

5rsquo-GATATCATGGCTGTGCCACCC-3rsquo 5rsquo-CTCGAGCGTGCTTGAAATTCC-3rsquo

ANT3 EcoRI EcoRv

5rsquo-GAATTCTATGACGGAACAGGC-3rsquo 5rsquo-GATATCGATCACCTTCTTGAG-3rsquo

pAcGFP1-C2 (Clontech)

NSP 4 EcoR1 Sal1

5rsquo-GAATTCATGGAAAAGCTTACCGACC-3rsquo 5rsquo-GTCGACCACATTGCTGCAGTCACTTCT-3rsquo

NSP 4Δ1-25 EcoRI Sal1

5rsquo-GAATTCTG GAGGATCCAGGAATGGC-3rsquo 5rsquo-GTCGACCACATTGCTGCAGTCACTTCT-3rsquo

pAcGFP1-C2 (Clontech

NSP 4Δ1-48 EcoR1 Sal1

5rsquo-GAATTCTGGCATCCATTCCAACAATG-3rsquo 5rsquo-GTCGACCACATTGCTGCAGTCACTTCT-3rsquo

pAcGFP1-C2(Clontech)

5rsquo-GAATTCTGTCAAAATGTTCATATAAAG-3rsquo 5rsquo-GTCGACCACATTGCTGCAGTCACTTCT-3rsquo

5rsquo-GAATTCTGGGTTATAAAGAGCAGATAAC-3rsquo 5rsquo-GTCGACCACATTGCTGCAGTCACTTCT-3rsquo

NSP 4Δ143-175

EcoR1 Sal1

5rsquo-GAATTCATGGAAAAGCTTACCGACC-3rsquo 5rsquo-GTCGACTATTTCGCCTGTCGTTTGCAC-3rsquo

NSP 4Δ92-175

EcoR1 Sal1

5rsquo-GAATTCATGGAAAAGCTTACCGACC-3rsquo 5rsquo-GTCGACAGTAGTTATCTGCTCTTTATAA-3rsquo

NSP 4Δ74-175

EcoR1 Sal1

5rsquo-GAATTCATGGAAAAGCTTACCGACC-3rsquo 5rsquo-GTCGACCTTTATATGAACATTTTGAC-3rsquo

NSP 4(61-91) EcoR1 Sal1

5rsquo-GAATTCTGTCAAAATGTTCATATAAAG-3rsquo 5rsquo-GTCGACAGTAGTTATCTGCTCTTTATA-3rsquo

5rsquo-GAATTCTGTCAAAATGTTCATATAAAG-3rsquo 5rsquo-GTCGACTGCCAATTTTAACAACGTATTA-3rsquo

5rsquo-GAATTCACAATTTTTATACGTTGTTAAA -3rsquo 5rsquo-GTCGACAGTAGTTATCTGCTCTTTATAA-3rsquo

pFLAG-CMV6 (Sigma-aldrich)

NSP 4 EcoR1 EcoRv

5rsquo-GAATTCAATGGAAAAGCTTACCGACC-3rsquo 5rsquo-GATATCCACATTGCTGCAGTCACTTCT-3rsquo

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

Mr(K) Time (h) 16 24

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

pcDNSP4pcDNSP4 +BAPTA-AM

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

by guest on June 4 2018 httpwwwjbcorg Downloaded from

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

Cox 4 (pellet) 17 Cox 4

FIGURE-2

17pcD pcDNSP 4 pcDNSP 4

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

N terminal truncation

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

C-terminal truncationDAPI GFP α MnSOD MERGED DAPI GFP α MnSOD MERGED

NSP4 ∆143-17549

6184

175175

175

111

14291

73

C terminal truncationNSP4 ∆1-83

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

Fine mapping61 9161 83

9174

NSP4(61-91)

N-terminal truncation

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

MEKLTDLNYT LSVITLMNNT LHTILEDPGM AYFPYIASVL TVLFALHKAS IPTMKIALKT SKCSYKVVKY CIVTIFNTLL KLAGYKEQITHHHH HHHHHHH HHHHHHHH

D

psipred

sspro

HHHHH HHHHHHH HHHHHHH HHH

HHHH HHHHHHHHHH HHHHH HHHHHHHHHH HHHHHHHH HHHHHHHH

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

TKGEIEKQMD RVVKEMRRQL EMIDKLTTRE IEQVELLKRI YDKLTVQTTG EIDMTKEINQ KNVRTLEEWE SGKNPYEPRE VTAAMHHHHHHHHH HHHHHHpsipred

sspro

FIGURE-4

HHHHHHHHHH HHHHHHHHHH HHHHHHHHHH HHHH HHHHHHHHHH

HHHH HHHHHHHHHH

HHHHHHHH HHHHH HHHHHHHH

91 101 111 121 131 141 151 161 171

HHHHHHHHHH HHHHHHHHHH HHHHHHHHHH HHHHHHHHHH

Hexokinase (Peripheral)

293 T IN VITRO ASSAYTrypsin (microgml) 0 25 50 0 25 50Trypsin (microgml)

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

VDAC (OMM Integral)35KD

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

0 25 50 0 25 50Trypsin (microgml)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

Pellet PelletSupe Supe

20KD

35KD 35KD

20KD

Mr(K) Mr(K) B Hexokinase (Peripheral)

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

CCox 4 (IMM Integral)

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

- + -pcDNA-VDAC1pcDNA-ANT3 - - +

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

IP Flag IP HisBC IP NSP 4

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

FLAG-NSP 4pcDVDAC1pcDANT3

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

35 KD (VDAC)32 KD (ANT)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

INP WBHis 35 KD (VDAC)

32 KD (ANT)32 KD (ANT)IN

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

C SA11 (8 hpi) - + +NSP4 si-RNA - - +CON si-RNA - + -

hpi

hpiMr(K)

M (K)

Mr(K)

28

Mitochondrial fraction

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

Scrambled + + + + + + fractionScrambled + + + + + +NSP4 si-RNA

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Cytoplasmicfraction Mr(K)

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

Kobayashi and Mamta Chawla-SarkarSatabdi Nandi Parikshit Bagchi Mukti Kant Nayak Oishee Chakrabarti Nobumichi

Rahul Bhowmick Umesh Chandra Halder Shiladitya Chattopadhyay Shampa Chandaapoptosis during infection

Rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of

published online August 10 2012J Biol Chem

101074jbcM112369595Access the most updated version of this article at doi

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R Bhowmick et al JBC 2012 revised 01_08
final FIGURE (7) - Copy 10812

2

Capsule

Background Rotaviral Nonstructural protein 4 (NSP-4) disrupts Ca+2 ion homeostasis by translocating to endoplasmic reticulum

Result In this study we show translocation of NSP 4 to mitochondria dissipation of mitochondrial potential and initiation of apoptosis which NSP1 counteracts during early infection

Conclusion NSP 4 and NSP1 regulate apoptosis during infection

Significance Study signifies modulation of cellular survival and apoptotic machinery by rotavirus for their own benefit

Abstract

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins Rotavirus is known to express six nonstructural and six structural proteins Among them NSP 4 is the enterotoxin known to disrupt cellular Ca+2 homeostasis by translocating to endoplasmic reticulum In this study we have observed translocation of NSP 4 to mitochondria resulting in dissipation of mitochondrial membrane potential both during viral infection as well as in NSP 4 overexpression Furthermore transfection of N-terminal and C-terminal truncated NSP 4 mutants followed by analyzing NSP 4 localization by immunofluorescence microscopy identified 61-83 amino acid (aa) region as the shortest mitochondria targeting signal (MTS) NSP 4 exert its proapoptotic effect by interacting with mitochondrial proteins ANT and VDAC resulting in dissipation of mitochondrial potential release of cytochrome c (cyt c) from mitochondria and caspase activation During early infection apoptosis activation by NSP 4 is inhibited by the activation of cellular survival pathways (PI3KAKT) since PI3Kinase inhibitor results in early induction of apoptosis However in presence of both PI3Kinase inhibitor and NSP 4 siRNA apoptosis was delayed suggesting that early apoptotic signal is initiated by NSP 4 expression This proapoptotic function of NSP 4

is balanced by another viral encoded protein NSP1 which is implicated in PI3KAKT activation since over expression of both NSP 4 and NSP1 in cells resulted in reduced apoptosis compared to only NSP 4 expressing cells Overall the study reports mechanism by which enterotoxin NSP 4 exerts cytotoxicity and mechanism by which virus counteracts it at the early stage for efficient infection

Introduction

Rotavirus a nonenveloped double stranded RNA virus belonging to the family reoviridae is the major cause of severe gastroenteritis in children under age of 5 years and causes two million hospitalizations and four lakh fifty thousand deaths per year worldwide (1) The virus with its 11 segmented dsRNA genome encodes six structural proteins (VP1 to VP4 VP6 and VP7) which form the virion and six nonstructural proteins (NSP1 to NSP6) which are translated only in host cells after virus infection and do not form part of the mature infectious virus (2) Nonstructural proteins play a crucial role in infection by establishing host virus interaction by carrying out diverse function Among them NSP 4 is identified as viral enterotoxin (3)

NSP 4 is a 175 aa long protein having fundamental role in both viral morphogenesis and pathogenesis (3 4) It was reported that exogenous addition of NSP 4 can induce an age and dose dependent diarrhea in mouse model (5) NSP 4 localizes in endoplasmic reticulum and acts as an intracellular receptor of the double layered viral particle during the entry of sub viral particle into endoplasmic reticulum (6 7 8 and 9) NSP 4 has a membrane destabilizing activity due to its viroporin domain by which it can create transmembrane aqueous pore in endoplasmic reticulum and increase the cytoplasmic Ca+2 concentration (10) Increase in cytoplasmic Ca+2 concentration and activation of Bax have been shown to play a role in rotavirus induced cell death (11 12 and 13) It is natural for virus an obligatory parasite to modulate the cell death machinery for winning the struggle between the virus and host (14) During the early stage of infection virus inhibits apoptosis

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Discussion

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REFERENCES

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Footnotes

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Figure 2

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Figure 3

Figure 4

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Figure 6

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

Alerts

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Discussion

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

Alerts

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Discussion

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 6

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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Discussion

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REFERENCES

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 6

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Figure 8

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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Discussion

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REFERENCES

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Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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Discussion

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 8

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

Alerts

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Discussion

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

Alerts

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Discussion

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

Alerts

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Discussion

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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Discussion

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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REFERENCES

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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25

Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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19

Footnotes

Figure1

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Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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20

Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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Figure 9

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Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

Alerts

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21

Figure 3

Figure 4

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Figure 5

Figure 6

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Figure 7

Figure 8

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24

Figure 9

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25

Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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22

Figure 5

Figure 6

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Figure 7

Figure 8

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24

Figure 9

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25

Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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23

Figure 7

Figure 8

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24

Figure 9

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25

Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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24

Figure 9

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25

Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

ww

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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25

Table 1

List of primers

NSP 4 EcoR1 EcoRv

VDAC1 EcoR1 Xho1

ANT3 EcoRI EcoRv

NSP 4 EcoR1 Sal1

NSP 4Δ143-175

EcoR1 Sal1

NSP 4Δ92-175

EcoR1 Sal1

NSP 4Δ74-175

EcoR1 Sal1

NSP 4 EcoR1 EcoRv

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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A

16 24

pcD

NSP

4

B

Mr(K) Time (h)

pcD

NA

28

(II)

(VI)

)

its

C

NSP4

β-Actin

20

424

2826

(I) (III)

(IV)(VIII)

Cel

l dea

th (

Arb

itrar

y u

niD

Caspase 8 52

pcD

NSP

pcD

NA

(I) (III) (V) (VII)

DN

SP4

h sa

11

h sa

11

h sa

11

Caspase-9

Caspase-8

4835

5224

h pc

D 5 11 14Caspase-3

Caspase-73531

32

E F

onc

[nM

]

Mr(K)

PARP

C p 31917

11885

Bax

szlig-actin

18 24

42

20

Ca+

2co

szlig-actin 42 1 3 6 9 12 15 18 24

FIGURE-1

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

Alerts

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ownloaded from

Page 27: Rotaviral Enterotoxin Nonstructural Protein 4 Targets … · · 2012-08-102 Capsule: Background: Rotaviral Nonstructural protein 4 (NSP-4) disrupts Ca+2 ion homeostasis by translocating

A CB(II)

rization()

ntial ()

cyt c

BAPTA-AM

Bax SiRNA

+ + -

+ + - Mr(K)

(IV)

(VI)(VIII)

mem

brne

depo

la

al m

embrne

poten

cyt c

szlig-actin 42

15

Cox 4 17000

02

000

2

0020

IVT NSP4 (microM)

02(I)

(III) (V) (VII)

Mito

chon

drial m

Mito

chon

dria

ol

D

n m

ixtu

re c

ontr

o

ontr

ol

VT N

SP4

D DN

SP4E F

cyt c (supe)

IVT

reac

tio

IVT

pcD

Co IV

000

02

0

002

02

15

cyt c

szlig-actin

z-vad-fmk

pcD

+ - +

15

42

Mr(K)

FIGURE-2

z-vad-fmk + - +

0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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0 2 64 8 10 12 14

NSP 4 24 24

pcD

NSP

4

xtur

e co

ntro

l

CA B

20 kD

pcD

NA

Mr(K)

M (K)

Time (h)

Cox 4

β-Actin

NSP4

Cox 4

β-Actin IVT

reac

tion

mix

IVT

pcD

Con

trol

IVT

pcD

NSP

4

17 kD

42kD

20 kD

17 kD

42kD M (K)

Mr(K)

COX- 4

NSP 4 20 kD

17 kDACCEPTOR DONOR

Mr(K) Calnexin

Lamp2110kD110kD

D

BLE

AC

H

E

BLEACHING

110kD

PRE

OST

BLE

AC

H BLEACHING

PO

FIGURE-3

ACCEPTORDONOR

1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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1 17526 175

49 175

A

NSP4∆1-25NSP4∆1-48

WILD TYPE

B

GFP

NSP4 ∆143-17549

6184

175175

175

111

14291

73

NSP4∆1 48NSP4 ∆1-60

NSP4 ∆74-175

NSP4 ∆143-175NSP4 ∆92-175

WILD TYPE

NSP4 ∆92-175

9174

NSP4(61-91)

Fine mapping

C

NSP4(74-91)NSP4(61-83) NSP4∆1-25

NSP4 ∆74-175

C

NSP4∆1-48

NSP4 ∆1-60

NSP4(61-91)

NSP4(61-83)

NSP4 ∆1-83 NSP4(74-91)

D

psipred

sspro

1 11 21 31 41 51 61 71 81

H HHHHHHH HHHp

sspro

FIGURE-4

HHHH HHHHHHHHHH

91 101 111 121 131 141 151 161 171

A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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A

120 KD 120 KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KD

Trypsin (microgml)

VDAC (OMM I t l)

Triton x-100

120 KD 120 KD

Mr(K) Mr(K)

+ + + Triton x-100 + + +

VDAC (OMM Integral)

NSP 4

20KD

35KD 35KD

20KD

120 KD 120 KD

35KD

r( )Mr(K)

NSP 4

PelletSupe

20KD

17KD

PelletSupe

20KD

17KD

Mr(K) Mr(K)

NSP 4

20KD

35KD

20KDFIGURE-5

Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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Cytosolic

+ - -

pcDNA

WBANT

A

32 KDMr(K)

Cytosolicfraction

Mitochondrial f ti

WBVDAC

β-Actin

WBANT

42 KD

35 KD

32 KD

C IP NSP 4

fractionWBVDAC

WBCox 4

35 KD

17 KD

pcDVDAC1

Sa 11Mock

pcDANT3

+ - + -- + - ++ + - -- - + +

WB NSP 4

ag

+ + + +

- - - +

- + - -

- + - +- - + +

+ + - -

20KD (NSP4)

Mr(K) Mr(K)

WBNSP 4

WBHis

WBCOX 4

WBFLAG

WBHis

WBCox 4

20KD (NSP4)

17KD (Cox4)

20KD (NSP4)

17KD (COX4)

r( )

WBCOX 4

WBNSP 4

WBHisUT

LYS

ATE WBCox 4

WBFLAG

17KD (Cox4)

20KD (NSP4)

35 KD (VDAC)

17KD (COX4)

20KD (NSP4)

35 KD (VDAC)PUT

LYS

ATE

s

FIGURE-6

A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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A

olarization (

)ial m

embrne

dep

Mito

chon

dri

RN

A

d N

SP4

siR

NA

zatio

n (

)

B C+ NSP4 siRNA

zatio

n (

)

A A RN

A

NSP

4 si

RN

A

ambl

ed N

SP4

siR

Scra

mbl

ed

+BAPTA-AM+Bax siRNA

mem

brne

depo

lari

mem

brne

depo

lari

siR

NA

bled

NSP

4 si

RN

A

siR

NA

bled

NSP

4 si

RN

A

siR

NA

ram

bled

NSP

4 si

R

siR

NA Sc

ram

bled

N

4 si

RN

ASc

r a

NSP

4 si

RN

A

Mito

chon

drial m

Mito

chon

drial m

NSP

4 s

Scra

mb

NSP

4 s

Scra

mb

NSP

4 s

Scr

NSP

4 s

NSP

4 N

FIGURE-7

Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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Cytoplasmicfraction

Cyt c

0 2 5 8 11 14

15

SA11A B

0 2 5 8 11 14

NSP4 si-RNA + + + + + +

SA11 + + + + + +

NSP4

hpi

hpiMr(K)

M (K)

Mr(K)

28

fraction

Cox 4

Bax

GAPDH

20

17

37

15

0 5 8

Cyt c

GAPDH

NSP4

GAPDH 37

20

Cytoplasmic

Mr(K) 2628

SA11 + + + + + +

15

0 2 5 8 11 14

Caspase-9 4835

SA11D

0 2 5 8 11 14

E

SA11 + + + + + + LY29004 + + + + + +

LY29004 + + + + + +

F

hpi

Mr(K)

p-AKT

PARP 118

85

60

p-AKT

AKT

XIAP

60

Caspase-9

0 2 5 8 11 14

48

SA11 + + + + + + NSP4 siRNA + + + + + +

hpiMr(K)

r( )

AKT

XIAP57

60

Caspase-9

XIAP 57

48

35

GAPDH 37

35

37GAPDH GAPDH 37

FIGURE-8

A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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A516

pcDNA-NSP4

pcDNA-NSP1pcDNA

NSP1 59

- + +

- - +

+ - -

BA

Cyt c

0 2 5 8 11 14

GAPDH

15

37

Mr(K)

NSP1

NSP4

β Actin

20

42

59

32

4835

Caspase-9

Whole cell

2628

D37

32

1917

GAPDH

Caspase-3

17

32

19

37

lysate

D

C

ell d

eath

()

β Actin 42

Cyt C 15

pcDNA-NSP4

pcDNA-NSP1

- + +

+ - +

Cytoplasmicfraction

Mr(K) C

e

FIGURE-9

42

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