RESEARCH ARTICLE

HES factors regulate specific aspects of chondrogenesis andchondrocyte hypertrophy during cartilage developmentTimothy P. Rutkowski1,2,*, Anat Kohn1,2,*, Deepika Sharma1,2, Yinshi Ren3, Anthony J. Mirando1,3 andMatthew J. Hilton1,3,4,‡

ABSTRACTRBPjκ-dependent Notch signaling regulates multiple processesduring cartilage development, including chondrogenesis,chondrocyte hypertrophy and cartilage matrix catabolism. Selectmembers of the HES- and HEY-families of transcription factors arerecognized Notch signaling targets that mediate specific aspects ofNotch function during development. However, whether particularHES and HEY factors play any role(s) in the processes duringcartilage development is unknown. Here, for the first time, we havedeveloped unique in vivo genetic models and in vitro approachesdemonstrating that the RBPjκ-dependent Notch targets HES1 andHES5 suppress chondrogenesis and promote the onset ofchondrocyte hypertrophy. HES1 and HES5 might have someoverlapping function in these processes, although only HES5directly regulates Sox9 transcription to coordinate cartilagedevelopment. HEY1 and HEYL play no discernable role inregulating chondrogenesis or chondrocyte hypertrophy, whereasnone of theHES or HEY factors appear tomediate Notch regulation ofcartilage matrix catabolism. This work identifies important candidatesthat might function as downstream mediators of Notch signaling bothduring normal skeletal development and in Notch-related skeletaldisorders.

KEY WORDS: HES1, HES5, Chondrogenesis, Chondrocytehypertrophy, SOX9

INTRODUCTIONThe limb skeleton largely comprises endochondral bones, whichinitially form as cartilage templates and are ultimately replaced bybone. Cartilage formation of the limb skeleton begins with themigration of mesenchymal progenitor cells (MPCs) from the lateralplate mesoderm into the developing limb field. MPCs undergo rapidproliferation to expand the limb bud, followed by the formation ofmesenchymal condensations that give rise to individual cartilageelements through chondrogenesis. This process, which generatesmature chondrocytes or cartilage cells from MPCs throughdifferentiation, is primarily driven by the expression and activityof the transcription factor Sry box 9 (SOX9). SOX9 induces and

maintains the expression of numerous cartilage-related genes,including collagen type II (Col2a1) and aggrecan (Acan), andalso drives growth of cartilage elements (Akiyama et al., 2002;Horton, 2003). As cartilage rudiments continue to develop,chondrocytes near the center of the elements undergo phenotypicand molecular changes known as pre-hypertrophy and hypertrophy,which are regulated and marked by the sequential activationof genes, including Indian hedgehog (Ihh), runt-relatedtranscription factor 2 (Runx2), collagen type X (Col10a1) andmatrix metalloproteinase 13 (Mmp13), and the concomitantdownregulation of Sox9. The cartilage matrix is ultimatelyremoved by the activity of terminally hypertrophic chondrocytes,which secrete MMP13 to catabolize or degrade the cartilage matrix,creating a scaffold for newly formed osteoblasts to lay down bonematrix (Zuscik et al., 2008).

The Notch signaling pathway is a known regulator ofchondrogenesis, chondrocyte hypertrophy, cartilage matrixcatabolism and osteoblastogenesis (Dong et al., 2010; Zanotti andCanalis, 2010; Kohn et al., 2012; Liu et al., 2015). Activation of theNotch pathway requires receptor–ligand interactions that initiate acascade of cleavage events, leading to the release of the Notchintracellular domain (NICD) and translocation to the nucleus, whereit forms a ternary transcriptional complex with recombination signalbinding protein for immunoglobin κJ Region (RBPjκ; also knownas RBPJ) and Mastermind-like (MAML) to activate downstreamtarget genes (Bray, 2006). Recently, several groups have utilizedvarious Notch pathway component loss-of-function (LOF) andgain-of-function (GOF) genetic approaches to study the roles ofNotch signaling during cartilage and bone development. Forexample, utilization of the Prx1Cre transgene to remove RBPjκfloxed alleles (Notch LOF) within MPCs demonstrates anacceleration in chondrogenic and osteoblastic differentiationwithin the limb skeleton, whereas overexpression of NICD (NotchGOF) within MPCs potently inhibits chondrogenesis andosteogenesis while maintaining and expanding MPCs (Donget al., 2010). Genetic removal of various Notch signalingcomponents (presenilin1, presenilin2, Notch1, Notch2 andRBPjκ) within MPCs using Prx1Cre or within cartilageprogenitor cells using a Col2Cre transgene delays the onset andprogression of chondrocyte hypertrophy and cartilage matrixcatabolism (Hilton et al., 2008; Kohn et al., 2012), whereasactivation of NICD in committed chondrocytes both in vivo andin vitro promotes chondrocyte hypertrophy and cartilage matrixcatabolism (Mead and Yutzey, 2009; Kohn et al., 2012). Recently,we have also demonstrated that several of the Notch-mediatedeffects on cartilage development occur in an RBPjκ-dependentmanner (Dong et al., 2010) and are likely to be the consequence ofan indirect transcriptional regulation of Sox9 (Kohn et al., 2015).Although the importance of Notch signaling in cartilagedevelopment has been well documented, the precise molecularReceived 2 October 2015; Accepted 5 April 2016

1Department of Orthopaedics and Rehabilitation, The Center for MusculoskeletalResearch, University of Rochester Medical Center, Rochester, NY 14642, USA.2Department of Biomedical Genetics, University of Rochester Medical Center,Rochester, NY 14642, USA. 3Department of Orthopaedic Surgery, DukeOrthopaedic Cellular, Developmental and Genome Laboratories, Duke UniversitySchool of Medicine, Durham, NC 27710, USA. 4Department of Cell Biology, DukeUniversity School of Medicine, Durham, NC 27710, USA.*These authors contributed equally to this work

‡Author for correspondence (matthew.hilton@dm.duke.edu)

M.J.H., 0000-0003-3165-267X

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mechanism(s) by which Notch regulates these distinct processesremain unclear or unknown.Hairy and Enhancer of Split (HES) and Hairy and Enhancer of

Split Related (HEY) proteins are bHLH transcription factors, ofwhich several are molecular targets of RBPjκ-dependent Notchsignaling and mediate aspects of Notch function within cells. Themost well-documented RBPjκ-dependent Notch targets are Hes1,Hes3, Hes5, Hes7, Hey1 and HeyL. HES and HEY transcriptionfactors are largely classified as transcriptional repressors that bindto unique N-box (CACNAG) and E-box (CANNAG) DNAsequences in the promoters of target genes (Kageyama et al.,2007). To determine whether specific HES and HEY factorsfunction during cartilage development and mediate some aspects ofNotch signaling in this process, we (1) analyzed HES and HEYgene expression using an in vitro model of chondrogenesis andchondrocyte hypertrophy, followed by further gene and proteinexpression analyses using in vivo models; (2) developed andanalyzed individual and combined HES or HEY gene LOF andGOF mouse models for defects in cartilage development; and (3)tested the ability of specific HES factors to transcriptionallyregulate Sox9 during chondrogenic differentiation in vitro andin vivo.

RESULTSHES and HEY genes are expressed during chondrogenesisand chondrocyte hypertrophyPreviously, we have analyzed the expression and function of Hes1,Hey1 and HeyL during in vitro chondrogenesis using limb-budmicromass cultures and small hairpin (sh)RNA knockdownexperiments. These data suggested that Hes1 is important insuppressing MPC differentiation and chondrogenesis, whereasHey1 and HeyL are dispensable during chondrogenic differentiation(Dong et al., 2010). Here, we have further analyzed the expressionof several RBPjκ-dependent Notch target genes of the HES andHEY families throughout both the processes of chondrogenesis andchondrocyte hypertrophy using a different in vitro system. ATDC5cells were cultured in the presence of insulin–transferrin–selenium(ITS) supplements in order to induce chondrogenic differentiationand maturation. RNAwas isolated for gene expression analysis at 7,14, 21 and 28 days following chondrogenic induction. To monitorthe progression of chondrogenesis and chondrocyte hypertrophy,we first examined the expression of the early chondrogenic markersSox9, collagen type II (Col2a1) and Acan by performingquantitative PCR (qPCR). All early chondrogenic genes peaked inexpression at day 21 and decreased by day 28, correlating with theramping up of chondrogenic differentiation and the transition tohypertrophy (Fig. 1A). The hypertrophic chondrocyte markersCol10a1 and Mmp13 were most highly expressed at day 28,indicating that the majority of cells had reached hypertrophy(Fig. 1A). We next analyzed the expression of Hes1, Hes3, Hes5,Hes7 and Hey1. Hes1 reached peak expression at day 21 and thendecreased at day 28 (Fig. 1A). Hes5 was highly expressed at day 7followed by a reduction in expression at days 14 and 21, and thenwas elevated again at day 28 (Fig. 1A). When comparing theexpression of Hes5 to that of Sox9, there appeared to be an inverserelationship between the two genes such that if one was highlyexpressed, the other decreased in expression. Hey1 expression washighest at day 28 and lower at earlier stages of chondrogenesis,suggesting a potential role in chondrocyte hypertrophy (Fig. 1A).Other HES genes were also analyzed but largely could not bedetected during chondrogenesis and chondrocyte hypertrophy ofATDC5 cells in culture.

To determine whether HES factors demonstrate similarexpression profiles during chondrogenesis in vivo, we isolatedRNA and protein from wild-type (WT) embryonic day 10.5 and11.5 (E10.5 and E11.5) limb buds. Using qPCR, we observed asimilar trend between Hes1 and Sox9, as well as between Hes5 andSox9; that is, increased Hes1 expression was observed as Sox9increased between E10.5 and E11.5, whereas Hes5 expressiondecreased as Sox9 expression increased between E10.5 and E11.5(Fig. 1B).We then analyzed HES1 and HES5 protein expression inE10.5 and E11.5 limb buds using western blot analysis. Similar tothe qPCR data, we again observed an increase of HES1 as SOX9increased between E10.5 and E11.5, whereas HES5 levelsdecreased as SOX9 increased between E10.5 and E11.5 (Fig. 1C).Collectively, these data demonstrate that both Hes1 and Hes5are expressed throughout chondrogenesis and chondrocytehypertrophy, and suggest that HES5 is a negative regulator ofSox9, consistent with the transcriptional repressive role for mostHES factors and the suppressive role of RBPjκ-dependent Notchsignaling during chondrogenesis (Kageyama et al., 2005; Donget al., 2010).

HES1 is dispensable for MPC differentiation, potentiallyowing to compensatory expression of HES5As stated previously, HES1 is an RBPjκ-dependent Notch targetgene that is capable of suppressing in vitro chondrogenesis in limb-bud micromass cultures (Dong et al., 2010). To determine whetherthe specific loss of Hes1 in MPCs can induce a similar accelerationof chondrogenesis in vivo, we generated and analyzed Prx1Cre;Hes1f/f (Hes1 LOF) embryos. This genetic targeting strategyallows for the specific removal of Hes1 floxed alleles withinMPCs of the developing limbs. Using whole-mount in situhybridization (WISH), we analyzed WT and Hes1 LOF mutantembryos at E12.5; however, we did not observe any change in Sox9(Fig. S1Aa,Ab) or Col2a1 (Fig. S1Ac,Ad) expression. RNA wasthen isolated from whole limb buds of both WT and Hes1 LOFE12.5 embryos. Using qPCR analyses, we did not observe anychange in expression of the chondrogenic markers Sox9,Col2a1 andAcan (Fig. S1B). This data was surprising because we had expectedto observe an acceleration in chondrogenesis as previously observedin Hes1 LOF in vitro models of chondrogenesis and other NotchLOF in vivomodels (Dong et al., 2010). To determine whether otherHES or HEY genes were compensating for the loss of Hes1, weisolated RNA from WT and Hes1 LOF whole limb buds at E11.5.HES and HEY genes are prominently expressed in undifferentiatedMPCs of the developing limb bud at E11.5 (Dong et al., 2010).Using qPCR analyses, we observed increasedHes5 gene expressionin Hes1 LOF limb buds compared to that in WT controls (Fig. S1B).Additionally, we analyzed the expression of Hes3, Hes7, Hey1 andHeyL, but did not observe any change in expression in Hes1 LOFlimb buds compared to WT controls, or the expression levels weretoo low to be reliably detected. These data suggest the potential forHES5 to compensate for the lack of HES1 during MPCdifferentiation in our Hes1 LOF embryos. This compensatoryeffect of HES factors has been well documented in other cellssystems, such as neural progenitor cells (Hatakeyama et al., 2004).

Removal of multiple HES factors in MPCs acceleratesdifferentiation and chondrogenesisOwing to the compensatory increase in Hes5 expression followingthe conditional removal of Hes1 (Fig. S1B), we analyzed embryosin which both HES factors had been deleted from MPCs. Wegenerated Prx1Cre;Hes1f/f;Hes5−/− (Hes1,5 LOF) andWTembryos

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to analyze cartilage development of the limb skeleton. We firstconfirmed genetic removal of Hes1 and Hes5 by analyzing theirexpression using qPCR on RNA isolated from E10.5 Hes1,5 LOFand WT limb buds (Fig. 2A). Using immunohistochemistry todetect SOX9 on E10.5 Hes1,5 LOF and WT control limb-budsections, we observed a subsequent increase in the expression ofSOX9 in Hes1,5 LOF limb buds as compared to that in WT mice(Fig. 2Ba,Bb). These data demonstrate an early acceleration in MPCdifferentiation and chondrogenesis. We next analyzed E11.5 Hes1,5LOF and WT embryos. Similar to E10.5 limb-bud sections, weperformed immunohistochemistry for SOX9 and observedincreased SOX9 in the Hes1,5 LOF limb buds as compared tothat in WT (Fig. 2Bc,Bd). We also performed qPCR on RNA thathad been isolated from E11.5 Hes1,5 LOF and control limb buds. AtE11.5, we observed a significant increase in both Sox9 and Col2a1expression in Hes1,5 LOF limb buds (Fig. 2A), further suggestingan acceleration in MPC differentiation and chondrogenesis in theabsence of Hes1 and Hes5. Lastly, we analyzed expression ofCOL2A1 and ACAN protein and RNA at E12.5 in WT and Hes1,5LOF limb buds. Alcian Blue–Hematoxylin–Orange-G (ABH–OG)staining, which stains cartilage matrix blue, showed that Hes1,5LOF mutant limbs displayed enhanced or accelerated cartilageformation in the radius and ulna, with more clearly defined jointformation in regions of the developing humerus and ulna whencompared to WT sections (Fig. 2Ca,Cb). Protein analysis usingimmunohistochemistry demonstrated altered COL2A1 expressionthat was associated with advanced secondary chondrogenesis andjoint formation (Fig. 2Cc–Cf) and an increase in ACAN expression(Fig. 2Cg–Cj) in E12.5 Hes1,5 LOF forelimbs as compared to WTcontrols. Further analysis at this time point revealed a change incellular morphology. As chondrocytes mature prior to hypertrophy,they begin to flatten (Zuscik et al., 2008). In E12.5 Hes1,5 LOF limbbuds, we observed more flattened cells compared to those in WTcontrols (Fig. 2Ci,Cj). This indicates that the cells in the Hes1,5LOF limb bud are beginning to form the columnar zone of

proliferating chondrocytes, which occurs just before hypertrophy.This was not observed as clearly in the WT control sections. Lastly,we analyzed RNA from E12.5 WT and Hes1,5 LOF mutant limbbuds using qPCR. Gene expression analysis showed that the loss ofHes1 andHes5 resulted in a significant increase inCol2a1 and Acanexpression (Fig. 2A). We did not observe any change in Sox9expression at this time point, which is probably owing to the fact thatas chondrocytes begin to mature, Sox9 levels decrease (Fig. 2A).Collectively, these data demonstrate that Hes1 and Hes5 arenecessary for appropriate MPC differentiation and chondrogenesis.

HES1 overexpression in MPCs delays chondrogenesis andinduces MPC proliferationTo determine whether Hes1 is sufficient to suppresschondrogenesis, we analyzed Prx1Cre;Rosa-Hes1f/f [Hes1 gain-of-function (Rosa-Hes1)] embryos across multiple time points.These experiments were conducted using a mouse line in whichHes1 was targeted to the Rosa26 locus containing a transcriptionalstop sequence flanked by loxP sites upstream of the Hes1 cassette.When crossed with the Prx1Cre mouse line, Hes1 is continuouslyoverexpressed in MPCs of the developing limb buds (Kobayashiand Kageyama, 2010). We first analyzed Hes1 and chondrogenicgene expression from E12.5 Rosa-Hes1 andWT limb buds by usingqPCR. We observed a significant overexpression of Hes1 and asignificant decrease in both Col2a1 and Acan expression in Rosa-Hes1 limb buds as compared to WT (Fig. 3A). Interestingly, Sox9,Sox5 and Sox6 gene expression was largely unchanged in Rosa-Hes1 limb buds as compared toWT (Fig. 3A). Histological analysesof E12.5 Rosa-Hes1 and WT limb bud sections using ABH–OGstaining demonstrated a reduction in proteoglycan content inRosa-Hes1 developing hindlimbs as compared to WT sections(Fig. 3Ba,Bb). Similar to the decrease in ABH–OG staining,immunohistochemistry analyses showed decreased expression ofCOL2A1 (Fig. 3Bc,Bd) and ACAN (Fig. 3Be,Bf ) in the Rosa-Hes1hindlimbs compared to WT sections.

Fig. 1. HES and HEY expression duringchondrogenesis and chondrocytehypertrophy. (A) qPCR gene expressionanalyses for Sox9, Col2a1, Acan, Col10a1,Mmp13, Hes1, Hes5 and Hey1 on RNA isolatedfrom ATDC5 cells cultured for 7–28 days in ITS-supplemented medium. (B) qPCR analysis forSox9, Hes1 and Hes5 on RNA isolated from WTlimb buds at E10.5 and E11.5. The y-axisrepresents relative gene expression normalized tothat of β-actin and then to expression in cultures atday 7. Bars represent means±s.d. *P

To determine whether overexpression of Hes1 also affects MPCproliferation, we used BrdU immunohistochemistry to analyzeRosa-Hes1 and WT forelimbs. Similar to Notch gain-of-function inMPCs (Dong et al., 2010), we observed an increase in BrdU-positive MPCs in the Rosa-Hes1 forelimbs compared to WT(Fig. 3Ca,Cb). The red box outlines the area in which cells (bothBrdU positive and BrdU negative) were counted. This region of thelimb bud is just beyond the highly proliferative apical zone, whereMPCs normally begin the differentiation process. Statisticalanalysis verified a significant increase in the percentage of BrdU-positive cells in the Rosa-Hes1 forelimbs compared to WT controls(Fig. 3Cc). We also isolated protein from E11.5 Rosa-Hes1 andWTlimb buds to perform western blot analysis of the proliferativemarker cyclinD1 (CYCD1). Rosa-Hes1 limb buds demonstratedincreased CYCD1 protein as compared to WT controls, further

validating the observed increase in proliferation (Fig. 3D).Collectively, these data demonstrate that Hes1 is sufficient todelay chondrogenesis by suppressing Col2a1 and Acan geneexpression, and is also capable of expanding the MPC populationduring early limb development.

HES, but not HEY, factors regulate early chondrocytehypertrophy, potentially through suppression of SOX9We have shown previously that RBPjκ-dependent Notch signalingis an important regulator of the onset and progression ofchondrocyte hypertrophy (Hilton et al., 2008; Kohn et al., 2012;Kohn et al., 2015). To determine whether HES1 regulates the onsetand progression of chondrocyte hypertrophy, we first analyzed Hes1LOF andWT embryos at E14.5. Hes1 LOF forelimbs were analyzedusing histological staining and in situ hybridization for markers ofchondrocyte maturation. ABH–OG staining of E14.5 Hes1 LOFmutant forelimbs revealed a mild and largely inconsistent decreasein the length of the hypertrophic zone (Fig. S1Ca,Cb), which wasalso revealed by reduced domains of Col10a1 (Fig. S1Cc,Cd) andMmp13 (Fig. S1Ce,Cf) expression as compared to WT controls.Collectively, these data suggest that Hes1 plays a limited role inregulating the onset of chondrocyte hypertrophy at E14.5 or thatcompensation by other HES factors, such as HES5, blunt the effectof Hes1 LOF alone.

To determine whether Hes5 compensatory expression also affectschondrocyte hypertrophy in Hes1 LOF mutants, we generated andanalyzed E13.5 Hes1,5 LOF mutant and WT embryos usinghistological approaches. Hematoxylin and eosin (H&E) staining ofhumerus sections demonstrated that Hes1,5 LOF mutants have asmaller hypertrophic zone compared toWT controls (Fig. 4Aa,Ab). Insitu hybridization for the hypertrophic chondrocyte marker Col10a1indicated a clear lack of expression in E13.5 Hes1,5 LOF mutanthumerus sections compared to WT (Fig. 4Ac,Ad). To understand thepotential mechanism underlying this delay in the onset of hypertrophy,we used immunohistochemistry to analyze the protein expression ofSOX9. Analysis of E13.5 Hes1,5 LOF mutant and WT humerussections demonstrated that Hes1,5 LOF mutants exhibited morecontinuous expression of SOX9 compared toWT controls within cellsof the central regions of elements poised for hypertrophy (Fig. 4Ae,Af). These data suggest that the delay in the onset of chondrocytehypertrophy is due to the maintenance of SOX9 expression.

To determine whether the loss of Hes1 and Hes5 results in acontinuous delay in chondrocyte hypertrophy, we analyzed E14.5Hes1,5 LOF mutant andWT limb skeletons. H&E staining revealeda decrease in the length of the hypertrophic zone in E14.5 Hes1,5LOF humerus sections as compared to WT (Fig. 4Ba,Bb).Furthermore, Hes1,5 LOF forelimbs exhibit a decrease in theCol10a1 (Fig. 4Bc,Bd) and Mmp13 (Fig. 4Be,Bf) expressiondomains at E14.5. To determine whether this continued delay inhypertrophy is due to altered SOX9 expression, we usedimmunohistochemistry analysis. Similar to the Hes1,5 LOF dataat E13.5, we observed maintenance of SOX9 expression deeper intothe hypertrophic zone of E14.5 mutant forelimbs (Fig. 4Bg–Bj). Todetermine whether this decrease in the size of the hypertrophic zonewas statistically significant, we measured the length of thehypertrophic zones and normalized those values to the totallengths of the cartilage elements for WT and Hes1,5 LOFmutants. Quantitative analysis showed a significant decrease inthe length of the hypertrophic zone in the Hes1,5 LOF mutantforelimbs compared to that in WT (Fig. 4C). To ensure this delay inhypertrophy was not due to changes in proliferation, we utilizedBrdU immunohistochemistry analysis and did not observe any

Fig. 2. Loss of Hes1 and Hes5 in MPCs accelerates chondrogenesis.(A) qPCR gene expression analyses for Hes1, Hes5, Sox9, Col2a1 and Acanfrom RNA isolated from Prx1Cre;Hes1f/f;Hes5−/− (Hes1,5 LOF) and WTforelimbs at E10.5, E11.5 and E12.5. The y-axis represents relative geneexpression normalized to that for β-actin then to the WT control. Bars representmeans±s.d. *P

change in the percentage of BrdU-positive cells in Hes1,5 LOFhumerus sections as compared to WT (Fig. S2A,B). Combined,these data demonstrate that HES1 and HES5 control the pace ofchondrocyte hypertrophy, potentially through regulation of SOX9.We next analyzed terminal chondrocyte hypertrophy and

cartilage matrix catabolism using E18.5 Hes1,5 LOF and WThumerus sections. Interestingly, no delay in terminal chondrocytehypertrophy or cartilage matrix turnover was observed betweenHes1,5 LOF and WT cartilage elements, as indicated by similarzones of hypertrophy in ABH–OG-stained sections (Fig. S2Ca,Cb)and a lack of any change in MMP13 expression (Fig. S2Cc,Cd).This was consistent with data demonstrating that genetic removal ofHes1 alone within MPCs caused no obvious defects in terminalchondrocyte hypertrophy and cartilage matrix turnover, as indicatedby similar zones of hypertrophy in E18.5 Hes1 LOF and WT H&E-stained sections (Fig. S2Da,Db), and similar expression domainsof Ihh (Fig. S2Dc,Dd), Col10a1 (Fig. S2De,Df) and Mmp13(Fig. S2Dg,Dh). Surprisingly, neither Hes1 LOF or Hes1,5 LOFmutants appeared to exhibit an expanded hypertrophic zone atE18.5, which has been observed previously in several Notch LOFmutant mice and is indicative of a continuous delay in terminalchondrocyte hypertrophy and cartilage matrix catabolism (Hilton

et al., 2008; Mead and Yutzey, 2009; Kohn et al., 2012; Kohn et al.,2015). Based on these data, combined with the observation thatHEY factor expression increases in maturing and hypertrophicchondrocytes (Fig. 1A), we obtained and analyzed E14.5 andE18.5 Hey1−/−; HeyL−/− double mutant (Hey1,HeyL LOF) andcontrol embryos for defects in the onset and progression ofchondrocyte hypertrophy and cartilage matrix catabolism. H&Estaining (Fig. S3Aa,Ab,Ba,Bb) and in situ hybridization for Ihh(Fig. S3Ac,Ad,Bc,Bd), Col10a1 (Fig. S3Ae,Af,Be,Bf) andMmp13(Fig. S3Ag,Ah,Bg,Bh) on tibia sections at E14.5 and E18.5demonstrate no obvious changes in the hypertrophic zonesbetween Hey1,HeyL LOF and control cartilage elements.Collectively, these data demonstrate that HES factors (particularlyHES5) primarily control the onset of chondrocyte hypertrophyduring cartilage maturation, potentially through regulation of SOX9,whereas neither the HES nor HEY factors appear to control terminalchondrocyte hypertrophy or cartilage matrix catabolism.

HES1 overexpression in MPCs delays chondrocytehypertrophy and inhibits skeletal growthTo determine whether Hes1 overexpression in MPCs affectschondrocyte proliferation and hypertrophy during cartilage

Fig. 3. Overexpression ofHes1 increases MPC proliferation and delays chondrogenesis. (A) qPCR gene expression analyses forHes1, Sox9, Sox5, Sox6,Col2a1 and Acan from RNA isolated from WT and Prx1Cre;Rosa-Hes1f/f [Hes1 gain-of-function (Rosa-Hes1)] limb buds at E12.5. The y-axis represents geneexpression normalized to that for β-actin then to WT control. Bars represent means±s.d. *P

development and maturation, we first examined Rosa-Hes1 andWTE14.5 skeletal preparations with Alcian-Blue staining. Rosa-Hes1cartilage rudiments were shorter, and the limbs as a whole weresmaller than those of WT controls (Fig. 5Aa–Ad). In the mostseverely affected Rosa-Hes1 forelimbs (Fig. 5Ab) and hindlimbs(Fig. 5Ad), we observed a hypoplastic or missing radius and/orfibula (black arrows). Distal cartilage rudiments appeared to bemore severely affected as compared to proximal elements, andhindlimbs were more affected than forelimbs (Fig. 5Aa–Ad).Analysis of Alcian-Blue-stained hindlimbs showed the formation ofa defined hypertrophic zone in WT cartilage rudiments (Fig. 5Ac)(red asterisks), although these were largely absent in severelyaffected Rosa-Hes1 mutants at this stage (Fig. 5Ad). ABH–OGstaining of E14.5 humerus sections (the least affected proximalelement) (Fig. 5Ba,Bb) demonstrated that Rosa-Hes1 mutantsexhibited only a mild delay in chondrocyte hypertrophy ascompared to WT controls, with only minor changes in Col10a1(Fig. 5Bc,Bd) and Mmp13 (Fig. 5Be,Bf ) expression within thehypertrophic zone.To assess whether changes in chondrocyte proliferation could

contribute to the Rosa-Hes1 skeletal phenotype, we performedBrdU staining on E14.5 Rosa-Hes1 and WT humerus sections(Fig. 5Ca,Cb). Consistent with the reduced size observed in mostelements of Rosa-Hes1 mutants, we observed a decrease in thepercentage of BrdU-positive chondrocytes (Fig. 5Cc). We nextanalyzed overall growth changes of skeletal elements at E18.5 usingRosa-Hes1 and WT forelimb and hindlimb skeletal preparations(Fig. 5Da,Db,Dd,De,Dg,Dh,Dj,Dk). Analyses indicated that thetotal lengths of these bones were significantly shorter in Rosa-Hes1mutants as compared to those of WT controls, with the mostprominent effects occurring on distal elements (Fig. 5Dc,Df,Di,Dl).Interestingly, Rosa-Hes1 mutant mice survive to adulthood andpresent with various skeletal anomalies, including alterations toskeletal patterning, bone ridge or tuberosity development, and digitnumber. These phenotypes are the likely result of the early andbroad effects of the Prx1Cre transgene controlling Hes1overexpression in skeletal progenitors. The precise cellular and

molecular mechanisms underlying each of these peripheralphenotypes will be explored and described elsewhere. The datapresented here suggest thatHes1 overexpression in MPCs can delaychondrocyte hypertrophy and reduce chondrocyte proliferation,although these effects might be secondary to the delay inchondrogenesis described above.

HES5 directly regulates Sox9 expressionBecause chondrogenic differentiation from MPCs and earlychondrocyte hypertrophy are coordinated by the expression andactivity of SOX9, we next examined whether HES factors cantranscriptionally regulate Sox9 expression. Above, we demonstratedthat Hes1 overexpression in MPCs in vivo is sufficient to repressCol2a1 and Acan expression without affecting Sox9 expression(Fig. 3A). To determinewhether HES5 is capable of regulating Sox9expression, we first transfected ATDC5 chondrogenic cells witheither Flag (control) or Hes5 overexpression constructs. After sevendays in chondrogenic differentiation medium, RNAwas isolated forqPCR analysis from each group. Hes5 overexpression resulted in anotable reduction of Sox9, Sox5 and Sox6, as well as chondrogenicgenes such as Col2a1 and Acan, although to a lesser degree at thistime point (Fig. 6A). These data suggest that HES5 is sufficient todownregulate or delay chondrogenesis in vitro and that it mightdirectly regulate Sox9 expression.

Previous studies have demonstrated RBPjκ-dependent Notchregulation of Sox9; however, the exact mechanism remainsunknown or controversial (Mead and Yutzey, 2009; Dong et al.,2010; Kohn et al., 2012; Chen et al., 2013; Kohn et al., 2015).Recent data have suggested that the Notch-mediated transcriptionalregulation of Sox9 occurs indirectly through secondary effectors(Kohn et al., 2015). Therefore, we first used a bioinformaticsapproach to search the Sox9 promoter for HES binding sites –N-boxor E-box sequences (Fig. S4A). We identified two N-box and/or E-box sequences within the first kilobase of the Sox9 promoter thatwere 100% conserved between the mouse and human genomes(Fig. S4C). To determinewhether HES5 directly binds to this regionof the Sox9 promoter in MPCs and in chondrogenic cells in vivo, we

Fig. 4. Loss of Hes1 and Hes5 delayschondrocyte hypertrophy owing toprolonged SOX9 expression. (A) H&Estaining (Aa,Ab), in situ hybridization forCol10a1 (Ac,Ad) andimmunohistochemistry for SOX9 (Ae,Af) onWT and Prx1Cre;Hes1f/f;Hes5−/− (Hes1,5LOF) humerus sections at E13.5. Blackcircles outline the hypertrophic zones.(B) H&E staining (Ba,Bb), in situhybridization for Col10a1 (Bc,Bd) andMmp13 (Be,Bf) and immunohistochemistryfor SOX9 (Bg–Bj) on WT and Hes1,5 LOFhumerus sections at E14.5. Red boxdenotes higher magnification images shownin Bi,Bj. (C) Statistical assessment of thehypertrophic zone lengths relative to thetotal lengths of the cartilage rudiments forWT and Hes1,5 LOF forelimbs. Barsrepresent means±s.d. *P

utilized chromatin immunoprecipitation (ChIP) assays on DNAisolated from WT E10.5 and E11.5 limb buds. As previouslyindicated, Hes5 and Sox9 expression demonstrated an inverserelationship in E10.5 and E11.5 limb buds (Fig. 1Ba,Bb). Primerswere designed to amplify the region of the Sox9 promotercontaining the N-box and/or E-box (Fig. S4B, red arrows), and anegative control region approximately 19 kb upstream of the N-boxand/or E-box sequences (Fig. S4B, green arrows). ChIP analysis atE11.5 using an antibody against HES5 revealed amplification ofDNA when using primers flanking the N-box and/or E-boxsequence (Primer 2; P2) (Lane 8), and no amplification whenusing primers targeting an upstream region of the Sox9 promoter(Primer 1; P1) (Lane 7) (Fig. 6Ba). Interestingly, no amplification ofeither primer set was observed when ChIP analyses were performedwith an antibody against HES1 (Lanes 5 and 6) (Fig. 6Ba),indicating specificity of binding to the N-box and/or E-box site forHES5. We also observed amplification of the positive control – thesheared genomic DNA (Lanes 1 and 2) – when using P1 and P2primers, whereas IgG pull downs showed no amplification (Lanes 3and 4) (Fig. 6Ba). By performing qPCR on DNA pulled down

during ChIP assays from E10.5 and E11.5 limb buds, we were ableto determine that the occupancy of HES5 on the Sox9 promoterregion was greater at E10.5 than at E11.5 (Fig. 6Bb). These datasuggest that RBPjκ-dependent Notch regulation of Sox9 works, inpart, through direct HES5 transcriptional activity.

Finally, we utilized a 1.0-kb Sox9-promoter-driven luciferaseconstruct that included the N-box and/or E-box sequence todemonstrate the direct transcriptional regulation of Sox9 by HES5.When this construct was co-transfected with Flag, Notch 1intracellular domain (NICD1) and HES5 expression vectors, weobserved a significant and similar level of suppression of luciferaseactivity between NICD1- and HES5-transfected groups whencompared to the Flag-transfected control (Fig. 6Ca). However,when we co-transfected a 1.0-kb Sox9-promoter-driven luciferaseconstruct containing a mutated N-box sequence with Flag or HES5expression vectors, we observed no change in luciferase activity(Fig. 6Cb). Collectively, these data demonstrate that the RBPjκ-dependent Notch target HES5 directly binds to the Sox9 promoterthrough the N-box sequence and is capable of downregulating Sox9expression in MPCs and chondrogenic cells.

Fig. 5. Overexpression of Hes1 delayschondrocyte hypertrophy, and reduceschondrocyte proliferation and skeletalgrowth. (A) Alcian-Blue-stained skeletalanalysis of Prx1Cre;Rosa-Hes1f/f (Rosa-Hes1) and WT forelimbs (Aa,Ab) andhindlimbs (Ac,Ad) at E14.5. Black arrowsdepict the missing radius in the forelimbs,and missing fibula in the hindlimbs of Rosa-Hes1 embryos. The red asterisks depictformation of the hypertrophic zone in the WThindlimbs. (B) ABH–OG (ABH/OG) staining(Ba,Bb) and in situ hybridization for Col10a1(Bc,Bd) and Mmp13 (Be,Bf) on WT andRosa-Hes1 humerus sections at E14.5.(C) BrdU immunohistochemistry on E14.5WT and Rosa-Hes1 humerus sections(Ca,Cb). Statistical analysis showing thepercentage of BrdU-positive cells in WT andRosa-Hes1 humerus sections (Cc) at E14.5.(D) Alcian-Blue and Alizarin-Red skeletalanalyses of WT and Rosa-Hes1 proximalforelimb (FL) elements (Da,Db), distalforelimb elements (Dd,De), proximalhindlimb (HL) elements (Dg,Dh) and distalhindlimb elements (Dj,Dk) at E18.5.Statistical analysis of the lengths of WT andRosa-Hes1 humeri (Dc), ulnae (Df), femurs(Di) and tibiae (Dl). Bars representmeans±s.d. *P

DISCUSSIONHES and HEY factors are well-known RBPjκ-dependent Notchtarget genes, which are capable of mediating several aspects of Notchfunction in various settings (Cau et al., 2000; Hirata et al., 2001; Zineet al., 2001; Kageyama et al., 2007). Previous in vitro studies haveimplicated HES1 as a potential suppressor of chondrogenesis, as wellas a potential transcriptional regulator of the Col2a1 and Acanpromoters (Grogan et al., 2008; Dong et al., 2010); however, the invivo evidence for HES regulation of cartilage development has beenlacking. Our results demonstrate that MPC-specific deletion of Hes1alone is not sufficient to affect cartilage development, and that theadditional removal of Hes5 is required to alter both chondrogenesisand the onset of chondrocyte hypertrophy, potentially owing to thecompensatory expression of Hes5 in vivo. Interestingly, mutant micein which Hes1had been deleted in more committed osteo-chondroprogenitors and combined with conventional deletion of Hes5

(Col2Cre;Hes1f/f;Hes5−/−) failed to show any defects in cartilagedevelopment (Karlsson et al., 2010). Importantly, this study onlyanalyzed embryos at E16.5 and later time points during endochondralbone development, thereby potentially missing the earlier defects wehave described here. Alternatively, Col2Cre;Hes1f/f;Hes5−/− mutantmice might not have developed defects such as those observed in ourstudy at E10.5–15.5 because the Col2Cre transgene targets a morecommitted osteo-chondro progenitor population. Prx1Cre;Hes1f/f ;Hes3−/−;Hes5−/− mutant mice have also previously been generatedand shown to have increased postnatal bone mass that is consistentwith other Notch LOF mutant mice (Hilton et al., 2008; Tu et al.,2012), although only limited late-stage embryonic skeletal analyseswere performed that showed no obvious phenotype (Zanotti et al.,2011). Similarly,Hey1+/−; HeyL−/−mutant mice have been shown tohave increased bone mass as compared to controls at late postnataland adult time points (Tu et al., 2012). Therefore, although these

Fig. 6. HES5 inhibits Sox9 expression through direct transcriptional regulation and is sufficient to delay chondrogenesis. (A) qPCR gene expressionanalyses for Hes5, Sox9, Sox5, Sox6, Col2a1 and Acan from RNA isolated from ATDC5 cultures at day 7. The y-axis represents relative gene expressionnormalized to that for β-actin then to Flag controls. Bars represent means±s.d. *P

studies have implicated HES and HEY factors in the regulation ofpostnatal bone development and homeostasis, they missed theimportant roles of specific HES factors during cartilage developmentof the limb skeleton.Here, we report the first in vivo genetic evidence demonstrating

that the RBPjκ-dependent Notch target genes Hes1 and Hes5 act asregulators of chondrogenesis and chondrocyte hypertrophy duringcartilage development. We have demonstrated that HES1 isdispensable for normal MPC differentiation and chondrogenesis,probably owing to compensatory expression of Hes5. However,HES1 is sufficient to delay chondrogenesis by acting downstreamof the SOX trio (SOX9, SOX5, SOX6), in addition to inducingMPC proliferation. Therefore, removal of both Hes1 and Hes5 inMPCs accelerates chondrogenesis and delays the onset ofchondrocyte hypertrophy. Consistent with recent Notch LOFstudies, the accelerated chondrogenesis and delay in the onset ofchondrocyte maturation is likely to be the result of increasedSOX9 expression (Kohn et al., 2015). We have identified HES5 asa direct transcriptional modifier of Sox9 gene expression, which inturn has direct influences on Sox5 and Sox6 gene regulation tocoordinate chondrogenesis. However, HES1 is likely to mediatecontrol of chondrogenesis and cartilage development through directdownstream regulation of other chondrogenic genes, such asCol2a1 and Acan (Grogan et al., 2008). Our work has alsodemonstrated that neither the removal of individual nor multipleHES or HEY genes alters terminal chondrocyte hypertrophy orcartilage matrix catabolism during normal development, therebysuggesting that the delayed terminal hypertrophy and cartilagematrix catabolism observed in other Notch LOF mutants (Hiltonet al., 2008; Mead and Yutzey, 2009; Kohn et al., 2012, 2015) arethe result of RBPjκ-dependent and HES- and HEY-independentsignaling mechanisms. Interestingly, genetic removal of Hes1within postnatal cartilages following joint injury in a murinemodel of osteoarthritis is capable of reducing Mmp13 expressionas well as that of other cartilage-catabolizing enzymes, whereasprolonged overexpression of Hes1 has also been shown to inducesome of these same catabolic genes in vitro (Sugita et al., 2015).Therefore, it is possible that HES, and potentially HEY, factorregulation of catabolic gene expression might only be evident inpathological or injury and/or inflammation contexts, and thatduring development, Notch signaling regulates terminalchondrocyte hypertrophy and cartilage matrix catabolism throughalternative mechanisms. Alternatively, numerous HES and/or HEYfactors might contribute to the regulation of this aspect of Notchfunction in cartilage, and therefore would require the elimination ofnearly all HES and HEY genes simultaneously to uncover theirrequisite role in regulating cartilage catabolism during normaldevelopment.Although the importance of Notch signaling in skeletal

development, injury and disease has recently come to light, we arejust beginning to learn about the underlying Notch-mediatedmolecular mechanisms that control these distinct events.Identifying the molecular players and their function would not onlyprovide us with additional important molecules to consider whenexamining skeletal disorders but would also lead to the generation ofadditional drug targets for the treatment of these conditions orailments. For example, Hajdu Cheney Syndrome (HCS) is a rareheritable multi-organ connective tissue disorder that presents withskeletal features such as skull deformities, short stature, joint laxityand a severe reduction in bone mass or osteoporosis, and is caused byheterozygous mutations in the NOTCH2 receptor (Majewski et al.,2011; Simpson et al., 2011). It has been recently discovered that these

mutations lead to Notch GOF within connective tissue cells(Majewski et al., 2011; Simpson et al., 2011), but the precisedownstream effectors that drive the pathology are unknown. Adams-Oliver Syndrome (AOS) is another rare heritable disordercharacterized by skin and limb defects including hypoplastic orshortened digits, absence of bones in hands or the feet, as well aspartial or complete absence of the lower legs (tibia, fibula and digits).AOS is an autosomal dominant disorder caused bymutations inRBPJand/or NOTCH1 genes resulting in Notch LOF, although noadditional molecular mechanisms underlying this disease areknown (Hassed et al., 2012; Stittrich et al., 2014). Notch signalingdefects, either GOF or LOF, have also been implicated inosteoarthritis (Mahjoub et al., 2012; Hosaka et al., 2013; Mirandoet al., 2013; Sassi et al., 2014; Liu et al., 2015), rheumatoid arthritis(Nakazawa et al., 2001; Park et al., 2015) and osteoporosis (Enginet al., 2008; Hilton et al., 2008; Majewski et al., 2011; Simpson et al.,2011), and have been associated with a predisposition to pathologicfractures (Kung et al., 2010). Studies like the one presented herefurther our understanding of the molecular players and events thatNotch signaling might control during normal skeletal development,as well as our understanding of how they contribute to the pathologyof certain skeletal diseases and injury processes.

MATERIALS AND METHODSMouse strainsThe Prx1Cremouse line has been previously described (Logan et al., 2002).The Hes1f/f, Hes1f/f; Hes5−/− and Rosa-Hes f/f strains were a generous giftfrom Dr Ryoichiro Kageyama (Institute for Virus Research, KyotoUniversity) and have been described previously (Cau et al., 2000; Hirataet al., 2001; Imayoshi et al., 2008; Tateya et al., 2011; Kobayashi et al.,2009). Hey1−/−; HeyL−/− mutant and control embryos were provided byDr Manfred Gessler (Biozentrum Universitat Wurzburg) and have beenpreviously described (Fischer et al., 2007). All animal work was approvedby both the Duke University and University of Rochester InstitutionalAnimal Care and Use Committees (IACUC).

Real-time RT-PCRIsolation of RNA from limb-bud tissues and ATDC5 cultures wereperformed as previously described (Kohn et al., 2015). Real-time reverse-transcriptase (RT)-PCR was used to analyze relative gene expression withthe Bio-Rad CFX Connect Real-Time system. Gene expression wasnormalized to that of β-actin (Actb) before being normalized to controlsamples. Mouse specific primers for Sox9, Sox5, Sox6,Col2a1, Acan,Hes1,Hes3, Hes5, Hes7 and Hey1 were designed as described previously (Donget al., 2010). Primer sequences are available upon request. Gene expressionanalyses from limb buds are from representative experiments of at least threebiological replicates from pooled genotypes with statistical analysesperformed on technical replicates of an individual experiment.

Tissue analysisEmbryos were harvested at E10.0–18.5 in cold 1× PBS, fixed in 10% neutralbuffered formalin (NBF) and then processed. Embryos at >E12.5 were treatedovernight with 14%EDTA.After processing, tissues were paraffin embedded.For embryos at E10.0–11.5, the whole embryo was embedded; limbs fromembryos at E12.5–14.5 were dissected from a whole embryo beforeembedding. Tissue was then sectioned at 4 μm and 5 μm for limbs atE10.0–12.5 and E13.5–18.5, respectively. To analyze cartilage compositionand general cellular morphology, standard histological staining usingABH–OG and H&E was performed. To analyze protein expression,immunohistochemistry was performed using the VectaStain ABC kits anddeveloped with ImmPACT DAB (Vector Labs). Primary antibodies againstthe following proteins were used for immunohistochemistry analyses: ACAN(1:200; catalog AB1031, Chemicon), COL2A1 (1:100; catalog MS235-P,Thermo Scientific), SOX9 (1:100; catalog sc20095, Santa CruzBiotechnology) and MMP13 (1:200; catalog MS-825P, Thermo Scientific).

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Standard heat-induced and sodium citrate antigen retrieval was performedfor the previously listed antibodies, whereas no antigen retrieval wasperformed for SOX9 immunohistochemistry. Relative quantification of theintensities of some immunohistochemistry staining was calculated usingthe immunohistochemistry image analysis toolbox developed in ImageJ.Briefly, the software was first trained to build a statistical positive-staindetector using DAB-positive pixels. By selecting a region of interestin the image, pixels of the original image were displayed withoriginal color values, while all other pixels were set to 255 as backgroundand thus filtered out during the color detection process. Finally, only DAB-stained pixels were quantified and compared between groups. BrdUimmunohistochemistry was performed as previously described (Dong et al.,2010). For in situ hybridization, embryos were prepared, fixed, processed andsectioned as described previously (Hilton et al., 2005, 2007, 2008; Donget al., 2010). Dig-labeled whole-mount in situ hybridizationwas performed asdescribed previously (Rutkowsky et al., 2014). Skeletal staining wasperformed using the protocol as previously described (Dong et al., 2010).

Chromatin immunoprecipitation assayThe ChIP assay was performed using the MAGnify ChromatinImmunoprecipitation system (Invitrogen) on limb buds at E10.5 andE11.5. Limb buds were homogenized in cold PBS using a 24 g syringe andimmediately frozen using liquid nitrogen. Sonication was performed using aCovaris S2 sonicator according to manufacturer’s instructions in order toshear chromatin to the lengths of 100–300 base pairs. The protocol wasoptimized for the use of six to ten limb buds. Antibodies against HES1 andHES5 (sc-25392 and sc-13859, respectively, Santa Cruz Biotechnology)were used at a concentration of 10 µg. Data analysis was performed usingqPCR with primers specifically designed to amplify the region of interestwithin the Sox9 gene promoter.

ATDC5 cell analysisATDC5 cells (RIKEN BRC, Japan) were grown in a 12-well plate withDulbecco’s modified Eagle’s medium (DMEM) with F12 1:1 (Invitrogen)supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Once cells were 70–80% confluent, they were treatedwith ITS media: standard DMEM/F12 medium supplemented with1× ITS Premix [insulin (5 µg/ml), transferrin (5 µg/ml) and selenous acid(5 ng/ml)] (BD Biosciences). Treatment with ITS supplement has beenpreviously reported to induce chondrocyte differentiation (Watanabe et al.,2001). Cells were incubated for 4 days with ITS medium before transfectionwith Flag, NICD1 and/or HES5 overexpression plasmids. Transfection wasachieved using FuGENE HD (Promega) with 500 ng of each construct.Cells were cultured for 7 days, changing medium every 48 h. Luciferaseassays were also performed using ATDC5 cells with a 1-kb Sox9 luciferaseconstruct and an N-box mutant 1-kb Sox9 luciferase construct using thesame protocol and reagents as described previously (Kohn et al., 2015).Western and luciferase analyses are representative experiments of at leastthree biological replicates with statistical analyses performed on technicalreplicates of an individual experiment. Real-time RT-PCR statisticalanalyses were performed on the means of three biological replicates.

Western blotTotal proteins were isolated from WT and mutant limb buds. Limb buds weredissociated in a standard lysis buffer and protease inhibitor solutions.Approximately, 10 µg of protein was separated using NuPAGE Novex4–12% Bis-Tris pre-cast gels (Invitrogen), and the fractionated protein lysateswere transferred onto a nitrocellulose membrane using the iBlot system(Invitrogen). Antibodies against HES1 (1:1000; catalog sc-25392, Santa CruzBiotechnology), HES5 (1:1000; catalog ab194111 Abcam), SOX9 (1:1000;catalog sc20095, Santa Cruz Biotechnology), CYCD1 (1:1000; catalog 2926,Cell Signaling) and β-actin (1:2000; catalog 4970, Sigma-Aldrich) were usedwith the appropriate secondary antibody following themanufacturer’s protocol.Quantification was performed on individual blots, and representative blots areshown. Western blot images were first converted to 8-bit images and thenanalyzed using ImageJ. Band intensity peak values were calculated andnormalized to those of the loading control (β-actin) for comparison.

Statistical analysisStatistical analyses were performed using two-tailed Student’s t-test and oneway ANOVA; a P-value

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