1
Supplementary information to Asymmetric partitioning of transfected DNA during mammalian cell division By:
Xuan Wanga,b, Nhung Lea,b, Annina Denoth-Lippunera.b, Yves Barrala and Ruth Kroschewskia,1
a Institute of Biochemistry, Department of Biology, Swiss Federal Institute of Technology Zurich
(ETH Zurich) CH-8093 Zürich, Switzerland b Molecular Life Science PhD Program, Life Science Zurich Graduate School, CH-8057 Zürich,
Switzerland 1 Corresponding author: [email protected]
1) Supplementary Materials and Methods Plasmid labelling Plasmid encoding H2B-eGFP was labelled with RX-Rhodamine (MIR7022,
MIRUS), according to the supplied protocol. The free dye molecules were removed with gel
filtration (MicroSpin G-50, GE Healthcare) and the labelling efficiency was checked by UV-VIS
spectrometry (Nanodrop, Thermo Scientific).
DNA transfection Lipofection was conducted using X-tremeGENE 9 DNA Transfection Reagent
(06365787001, Roche) with a 1:3 (w:v) plasmid: transfection reagent ratio. Cells transfected with
labelled plasmids (Fig. 1A, B, S1A-C) were washed with 20U Heparin before fixation to remove
extracellular DNA. The concentration for pLacO, Rho-plasmid (Fig. 1A-C, S1A-C, S9H), pControl
(Fig. 1C), and the linear fragment of pLacO (Fig. S3A, B) was 25 ng per culture surface area
(square centimeter) if not otherwise indicated. Electroporation was conducted using an
ElectroMicroporator (MP-100, Digital-Bio Technology) according to the manufacturer's
instructions. If not indicated, the plasmid clusters were analyzed 24-44 h after transfection.
Bead transfection Polystyrene beads (1.0 µm in diameter, Carboxylate-modified Microspheres,
F8887, ThermoFischer Scientific) with red fluorescence (580/605 nm) were transfected into
HeLa cells with modifications according to (1). Briefly, one volume of beads was centrifuged
(3000 rpm, 5 min) and re-suspended in 10 volumes of PBS. The re-suspended beads were
transfected into cells with lipofection like the plasmids with a 1:3 bead-suspension: transfection
reagent ratio (v: v). 0.1 µl of that bead-suspension was added to one 3.5-cm cell culture dish.
For bead and pLacO co-transfections (Fig. 2E, 3A-C, S3E, F, S4), the bead and pLacO were
2
mixed in individual tubes with transfection reagent and added to the cells at the same time. Cells
were washed with 20 U Heparin before fixation or imaging to remove extracellular beads.
Cell Culture and stable cell generation Cells were grown at 37°C with 5% CO2 in a humidified
incubator. HeLa and MDCK cells stably expressing LacI-eGFP/-mCherry were obtained from
lentivirus transductions (Table S1) (2). To obtain HeLa cells expressing Centrin1-eGFP and
LacI-mCherry, cells were first lipofected with a plasmid encoding Centrin1-eGFP (from S.
Doxsey, Univ. Massachusetts Med. Sch., USA) (3) and colonies were selected with G418 to
establish clones. Subsequently lentivirus transduction followed using one of the clones with a
vector encoding LacI-mCherry (Table S1).
Live cell imaging In Fig. 2A, S7 and S11, cells were transfected with pLacO for 30 h (Fig. 2A,
S11) or 24 h (Fig. S7) and imaged. In Fig. 3D-H, S8 and S9, cells were transfected with pLacO
for 24 h, then partially synchronized for 20 h with thymidine (2 mM) and imaged 5 h after release
from thymidine. In Fig. 3A-C and S4, cells were co-transfected with beads and pLacO for 24 h,
washed with 20 U Heparin and imaged. For all experiments in Fig. 2A, 3, S4, S7-9, S11 and
images in Fig. 4A (panel 4) and Fig. S10A the imaging of the live cells was as follows: Cells
were seeded on Lab-TekTM II chambers (155409, Thermo Scientific) with CO2-independent
media (Leibovitz L-15, ThermoFischer Scientific) and placed in a 37°C incubator mounted on a
Spinning Disk microscope (Axio Observer Z1 withSpinning Disk Confocal Fluorescence, Carl
Zeiss). Cells were recorded every 30 min (Fig. 2A, S11), 1 s (Fig. 3A-C, S4), 5 min (Fig. 3D-H,
S8-9), 2 min (Fig. S7) or as still images (Fig. 4A (panel 4), Fig. S10A) with z stacks (6-33x 0.7μm
steps) using a 63x, 1.2 NA objective and an Evolve 512 camera (Photometrics). For images of
live cells in Fig.4A (panel 1-3), 3D stacks were acquired with 0.3 μm steps using a 60x NA 1.42
objective on a DeltaVision microscope (Olympus) equipped with a CoolSNAP HQ camera
(Roper Scientific).
Immunofluorescence (IF) and immunofluorescence coupled with fluorescence in situ hybridization (IF-FISH) and imaging For IF (Fig1A, B, 2C-F, S1A-C, S2, S3, S5, S6B, C, S9H,
S10A, B), Methanol- or formaldehyde-fixed (Table S1) cells were permeabilized with 0.2%
TritonX-100 and blocked with 5% Bovine serum albumin (BSA). Cells were then incubated with
primary antibodies followed by secondary antibodies and 2 μM Hoechst (Table S1). For IF-FISH
(Fig. S1D, E), after immuno-staining of cells with anti-GFP antibody, a second fixation with 3.7%
paraformaldehyde and a second block with 5% BSA containing 20 mM glycine followed.
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Afterwards, the cells were incubated at 80 °C (3 min), hybridized with the LacO PNA probe (Cy5-
aattgttatccgctcacaattc) (PANAGENE) (2 h), washed and stained with 2 μM Hoechst.
For images of fixed cells, 3D stacks were acquired in 0.3 μm steps using a 60x NA 1.42
objective on a DeltaVision microscope (Olympus) equipped with a CoolSNAP HQ camera
(Roper Scientific).
APC western blotting (Fig. S6A) Cells were washed twice with chilled PBS followed by a 5 min
incubation with 200 µl of lysis buffer (150 mM NaCl, 10 mM Tris (pH 7.2), 0.1% SDS, 1.0%
Triton X-100, 1% Deoxycholate, 5 mM EDTA containing 100 µM PMSF and protease inhibitor
cocktail (11697498001, Roche)) per 10 cm dish. Cells were scraped off and centrifuged at
14’000 rpm for 20 min at 4°C. The western blotting was according to a published method (4).
Briefly, the cell lysates were loaded on 4-12% gradient SDS-gels (NP0322, NuPAGE,
ThermoFischer Scientific), the gels run in MOPS running buffer (NP0001, ThermoFischer
Scientific) and proteins transferred to nitrocellulose membrane (0.1 μm pore size; 10600000,
Amersham). The membrane was blocked and incubated with primary followed by secondary
antibodies in blocking buffer (5% nonfat milk, 1% BSA and 0.02% Triton X-100 in Tris-buffered
saline) (Table S1).
RNAi Pooled siRNAs targeting human Ninein, APC or ODF2 (Table S2) were transfected to
cells 24 h prior to pLacO transfection using RNAiMAX Transfection Reagent (13778150,
ThermoFischer Scientific) (experimental schemes Fig. S5B, S6C). Pooled siRNAs targeting
canine Ninein were transfected twice (24 h and 12 h) to MDCK cells prior to pLacO transfection
using JetPrimeTM reagent (89129, Polyplus Transfection).
Correlative light and focused ion beam scanning electron microscopy (FIB-SEM) Stable
HeLa-LacI-mCherry cells were cultured on a dish with grid (P35G-2-14-C-GRID, MatTek) and
transfected with pLacO for 24 h. Cells were fixed and stained with Dioc6 (1 μg/ml). 3D image
stacks were obtained using a 63x 1.4 NA objective on a LSM 710 confocal microscope (Carl
Zeiss Microscopy) equipped with an MRm Rev.3 camera (AxioCam). FIB-SEM imaging of the
cells is described in (5). FIB-SEM microscopes were Helios NanolabTM 600i (FEI) (Fig. 4B) and
NVision 40 (Carl Zeiss Microscopy) (Fig. S10C). The imprinted grid of the dishes and the
fluorescence of the confocal images were used to find the relevant cell and match the region of
the pLacO cluster between the FIB and fluorescence images. The regions including the plasmid
clusters were milled by FIB with fine steps and imaged by SEM. The voxel sizes were 6.97 nm
and 9.45 nm in Fig. 4B and Fig. S10C, respectively.
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Image analysis
3D distance measurement: To obtain 3D distances between 2 objects or 1 object at 2 time
points, the x, y, and z (= (slice numer-1) x step thickness) coordinates of the objects were
detected using Fiji and the distance were calculated in 3D (Fig. 3E, G, H, S4, S5B-E). The
distances at metaphase were averaged if more than one frame were recorded in Fig. 3E and
S9D.
Intensity and diameter measurements: For foci intensity (Fig. 1B, S8D) and diameter (Fig. S8D)
measurements (Fiji), the raw image stacks were projected with sum. intensity (for intensity
measurement) or max. intensity (for diameter measurement). To measure the intensity, the
cluster area was segmented along the cluster’s fluorescence edge. Adjacent background areas
were segmented with the same size. The cluster intensity was calculated as (Intensity (cluster) –
Intensity (background)). The measurement of the cluster diameter was guided by the imaging of
the 1 µm beads, which are used as reference objects in light microscopy, and was approximated
as the cluster shape can be irregular.
Deconvolution: Displayed images (Fig. 1A, 2C, 4A (panel 1-3), S1B, S2A, B, S3C, E, S5A, D,
S6B, S10B) acquired with a DeltaVision microscope were deconvolved using Softworx (Applied
Precision). Images acquired at the LSM 710 confocal microscope (Fig. 4B, S10C) were
deconvolved using Huygens (Scientific Volume Imaging).
Confocal and EM correlation analysis: The correlation analyses between confocal and EM
images were performed using Amira (FEI).
Single particle tracking: For Fig. 3A-C and S4, the beads and clusters were tracked by single
particle tracker (6) within Fiji on max. intensity projected images. The trajectories were plotted
with Prism (GraphPad) with their (x, y) coordinates at t(0) normalized to the origin. The mean
square displacements (MSD) were calculated (41) and plotted with Prism (GraphPad).
Statistics The random expectations for the 2:0 and 3:0 possibilities (Fig. 2B) are based on
Pascal's triangle. The statistical methods are indicated in the figure legends. Data were with
Gaussian distribution normality test (α=0.05) if t-tests were used; otherwise as indicated. The
signs are as follows: (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; n.s.: not significant)
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Supplementary References 1. Kobayashi S, et al. (2010) Artificial induction of autophagy around polystyrene beads in
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decrease in cell migration and overall changes in microtubule stability. Mol Biol Cell
18(3):910-918.
5. Lucas MS, Guenthert M, Gasser P, Lucas F, & Wepf R (2014) Correlative 3D imaging:
CLSM and FIB-SEM tomography using high-pressure frozen, freeze-substituted
biological samples. Methods Mol Biol 1117:593-616.
6. Sbalzarini IF & Koumoutsakos P (2005) Feature point tracking and trajectory analysis for
video imaging in cell biology. J Struct Biol 151(2):182-195.
7. Lin CC, et al. (2006) Characterization and functional aspects of human Ninein isoforms
that regulated by centrosomal targeting signals and evidence for docking sites to direct
gamma-tubulin. Cell Cycle 5(21):2517-2527.
8. Kumaran RI & Spector DL (2008) A genetic locus targeted to the nuclear periphery in
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2) Supplementary Figure legends Fig. S1. The majority of transfected plasmid DNA localize as clusters in the cytoplasm, independently of the plasmid detection and transfection methods (related to Fig. 1). (A-C)
Related to Fig.1 A and B. (A) Number of nuclear and cytoplasmic clusters per cell after the
transfection of Rho-plasmid (n: pooled cell number of 3 experiments, 17-27 cells per
experiment). (B) Representative image of eGFP-H2B expressing and non-expressing cells
transfected with Rho-plasmid (value above: transfection efficiency). (C) The correlation of eGFP-
H2B expression with plasmid foci localization (n: pooled cell number of 3 experiments, 17-41
cells per experiment). (D) Representative IF-FISH image of HeLa cells expressing LacI-eGFP
after pLacO transfection. (E) Quantification of images like in D. 95.3% of the clusters recognized
by the LacO PNA probe were also recognized by the GFP antibody (n: pooled cell number of 3
experiments, 23-38 cells per experiment). (F, G) MDCK cells stably expressing LacI-eGFP were
electroporated with pLacO for 24 h (related to Fig. 1C and D). (F) Images of cells after no
plasmid and pLacO electroporation. (G) Number of cytoplasmic plasmid clusters per cell after
pLacO electroporation (mean with SEM of 3 experiments, n>50 per experiment). (max. intensity-
projected image stacks; scale bars: 10 µm)
Fig. S2. Centrosome classification. (A) Distinguishing old and young centrosomes by fluorescence
intensities of indicated reporters and corresponding differential interference contrast image
(DIC). 3D fluorescence images were projected with max. intensity. Then, in each ana- or
telophase cell, the areas of the two centrosomes (2 white squares; c1, c2) and their adjacent
backgrounds (2 yellow squares; b1, b2) of the same size were segmented (0.4-0.7 square
micrometer). Subsequently, the integrated intensity of each segmented area was measured (c1
and c2, b1 and b2). The intensity ratio of the young (less bright one) to old centrosome (bright
one), termed Y/O, was calculated: Y/O = (c2-b2) / (c1-b1). The ratio value of 0.8 was used as
the threshold for reliable classification, and only cells with (Y/O � 0.8) were analyzed. (B, C)
Evaluation of the centrosome classification method using stable Centrin1-eGFP expression as
centrosome-reporter in HeLa cells. Classification outcomes for Centrin1-eGFP and anti-ODF2
were compared in the same cell and categorized either as “consistent” or “not consistent”. (B)
Images illustrating “consistent” and “not consistent” examples. (C) The quantification of the
“consistent” and “not consistent” groups in cells with and without pLacO transfection. The old
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and young centrosome classification based on Centrin1-eGFP was in minimally 96% of the
cases consistent with the classification based on ODF2 immuno-staining (n: pooled cell number
of 3 experiments (cell number range per experiment: 9-25 (- pLacO), 25-34 (+ pLacO))
Fig. S3.
Centrosome correlation with linear DNA and beads (related to Fig.2B-E). HeLa cells
expressing Centrin1-eGFP and LacI-mCherry were transfected with pLacO (13.5 kb, A-F), a
linear fragment of pLacO (10.0 kb, A, B), fluorescent beads (C, D) or co-transfected with pLacO
and beads (E, F) for 20 h followed by thymidine (2 mM) addition for 20 h. To enrich for dividing
cells, cells were then released from thymidine for 8 h and fixed. (A) Number of cytoplasmic
clusters per cell in a population after pLacO (circle with fat part representing LacO repeats) and
linear LacO repeats (fat circle part) transfection (mean with SEM of 3 experiments, n>150 cells
per experiment). (B) Analysis of the correlation between the pLacO clusters and the centrosome
type in dividing cells with an x: 0 partition pattern (x=1, 2 and 3 clusters). (C) Representative
image of a telophase cell with an intracellular bead, used for analysis. The intracellular
localization is shown in yz-scan images along white dashed lines labelled with x* and y*.
LaminB1 was immuno-stained to visualize the nuclear border and is only shown in the yz-scan
images. The bead is inside the cell, close to the nucleus and can also be seen in the DIC image.
(D) Analysis of the correlation between the pLacO cluster/ bead and the centrosome type in
dividing cells with a 1: 0 partition pattern. (E) Representative image of a telophase cell co-
transfected with beads and pLacO. The red fluorescence of the beads (low and normal exposure
images) is of much higher intensity than that of LacI-mCherry bound to pLacO (normal exposure
image), thus beads and pLacO clusters can be distinguished. Only the bead can be seen in the
DIC image (arrowheads: bead, pLacO cluster). (F) Frequency of the four localization patterns
(schemes) of beads, pLacO cluster and centrosomes in individual cells. “Copartition” in case the
bead(s) and cluster(s) are all in the same cell half, otherwise “split” (related to Fig. 2E, mean with
SEM of 3 experiments; at least 16 cells per experiment; cells with x: 0 partition patterns for
clusters and beads (x=1, 2 and 3)). (n (B, D): pooled cell number of 3 experiments (cell number
range per experiment: 23-32 (B, Circular), 15-26 (B, Linear), 18-32 (D, pLacO), 15-30 (D,
Bead)); statistics: Binomial test to compare experimental to random frequency (B, D), χ2 test to
compare circular and linear pLacO (B) or pLacO and bead (D) and non-paired t-test (F))
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Fig. S4. The pLacO clusters are relatively immobile compared to beads in interphase cells (related
to Fig. 3A-C). HeLa cells expressing LacI-eGFP were co-transfected with pLacO and beads for
24 h and cells with beads and pLacO clusters were imaged every second. Trajectories (0 - 400
s) of beads (upper graph) and pLacO clusters (lower graph) of 7 cells are shown, whereby only 1
bead and 1 pLacO cluster of the same cell (same color) is depicted.
Fig. S5.
Effects and control of altered Ninein levels in HeLa and MDCK cells. (A, B) Evaluation of
the efficiency of RNAi-mediated Ninein downregulation (related to Fig. 2F). (A) Representative
images of HeLa cells expressing Centrin1-eGFP and LacI-mCherry after siNinein or siControl
treatments. Cells were immuno-stained with an antibody recognizing Ninein. (B-top) A scheme
indicates the siRNA procedure for siNinein in Fig. 2F, S5C. (B-bottom) Quantification of the
Ninein-negative cells in Centrin1-eGFP and LacI-mCherry expressing HeLa cells treated with
siControl and siNinein oligos (mean with SEM of 3 experiments; n>30 per experiment). (C)
Analysis of the correlation between the pLacO clusters and the centrosome type in dividing
MDCK cells with an x: 0 partition pattern (x=1, 2 and 3 clusters). Cells were pre-treated with
siNinein or siControl prior to pLacO transfection. (D, E) HeLa cells expressing LacI-mCherry
were co-transfected with pLacO and Ninein-eGFP, or pLacO and H2B-eGFP (control) for 20 h
followed by thymidine addition (2 mM) for 20 h. To enrich for dividing cells, cells were then
released from thymidine for 8 h and immuno-stained with anti-ODF2 and γ-Tubulin. Late
telophase cells with Ninein-eGFP localizing at the centrosomes, or expressing H2B-eGFP were
analyzed. (D) Representative image of a telophase cell transfected with pLacO and expressing
Ninein-eGFP. Upon high image exposure (bottom right) γ-Tubulin becomes visible on
centrosomes but then the LacI-mCherry fluorescence is extreme, as the two objects are imaged
in the same channel. Upon normal exposure only the pLacO cluster is visible (bottom left). (E)
Analysis of the correlation between the pLacO clusters and the centrosome type in dividing cells
over-expressing Ninein-eGFP and H2B-eGFP. Cells with an x: 0 partition pattern were analyzed
(x=1, 2 and 3 clusters). (max. intensity-projected image stacks; arrowheads: centrosomes, scale
bars: 10 µm, n (C, E): pooled cell number of 3 experiments (cell number range per experiment:
21-71 (C, siControl), 10-70 (C, siNinein), 14-21 (E, Ninein-eGFP), 13-14 (E, H2B-eGFP));
statistics: Binomial test to compare experimental to random frequency, and χ2 test to compare
siNinein with siControl and Ninein-eGFP with H2B-eGFP)
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Fig. S6. Evaluation of the efficiencies of RNAi-mediated APC and ODF2 downregulations (related
to Fig. 2F). (A) The efficiency of APC downregulation was validated by western blotting at the
time point of correlation analysis (72 h). The numbers under the blot are the intensity ratios of
the depicted bands for APC and αTubulin (loading control). (B) Representative images of HeLa
cells expressing Centrin1-eGFP and LacI-mCherry after siODF2 or siControl transfection. Cells
were immuno-stained with an antibody recognizing ODF2. (C-top) A scheme indicates the
siRNA procedure in Fig. 2F for siODF2 and siAPC. (C-bottom) Frequency of ODF2-negative
cells in Centrin1-eGFP and LacI-mCherry expressing HeLa cells treated with siControl and
siODF2 oligos (mean with SEM of 3 experiments; n>30 cells per experiment).
Fig. S7.
Asymmetric anaphase elongation does not correlate with centrosome age. HeLa cells
expressing Centrin1-eGFP and LacI-mCherry were transfected with pLacO for 24 h and
metaphase cells were imaged completing the mitosis. (A) Time-lapse images (max. intensity-
projected image stacks) showing that during anaphase the cell elongated towards the old
centrosome side (white arrowhead) whereas the pLacO cluster was relatively immobile (vertical
gray dashed line as reference for original pLacO cluster position) and partitioned with the young
centrosome (gray arrowhead) and the corresponding DIC images (0 min: the last frame before
elongation; white dashed lines: reference for left and right cell borders at metaphase; scale bar:
10 µm). (B) The method to calculate an elongation index is described schematically. (C) The
elongation indexes were calculated at the young (negative values) and old centrosome (positive
values) sides separately, in cells with and without plasmid clusters. Medians with interquartile
ranges are shown in addition to individual values. Distances in max. intensity-projected images
were analyzed. The red dot indicates the elongation index of the dividing cell in (A). (n: pooled
cell number of >3 experiments (cell number range per experiment: 7-25 (- clusters), 8-17 (+
clusters)); statistics: non-paired t-test)
Fig. S8. During centrosome splitting the old centrosome migrates further than the young centrosome and the cluster (related to Fig. 3D-H). (A) Averaged OC- and YC-distances from 5
min before centrosomes split until the second frame of anaphase. (B) Averaged speeds of
centrosomes and plasmid clusters from 5 min before split until max split. Note, the speed of the
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clusters was higher between 10 min to 20 min likely due to the rounding up of the entire cells
around this time displacing the clusters passively. (C) The distances of cluster to the centrosome
pair 5 min before the centrosomes split in cells with only one cluster (dashed red line: the
averaged short diameter of nuclei, measured 5 min before centrosome split (17.5 μm)). (D)
Intensity and approximate diameter (2D) measurements of individual clusters (close, far) in cells
analyzed in Fig. 3F. (E) The old versus young centrosome displacement at max split is shown in
cells with (right) and without (left) plasmid clusters. Displacements were measured relative to the
locations of the centrosomes 5 min before they split. (A-C, E) Distances were measured in 3D.
(C-E) Medians with interquartile ranges are shown in addition to individual values. (A, B) error
bars: SD; n: pooled cluster (A) or cell (B-E) numbers of >3 experiments (number range per
experiment: 16-31 (A), 6-19 (B-D), 11-24 (E, - clusters), 6-19 (E, + clusters)); statistics: (B)
Dunn's multiple comparisons test was performed for each time point separately, only significant
differences are indicated; (D, E) non-paired t-test (E with log2 (value)).
Fig. S9.
Ninein downregulation affects mainly the maintenance of the orientation of the centrosome pair later in mitosis. (A) Time-lapse images of one HeLa cell expressing Centrin1-
eGFP and LacI - mCherry, after downregulation of Ninein and transfection of pLacO. Note, that
during 35 min-90 min the mitotic spindle rotated heavily. (white and gray arrowheads: old and
young centrosomes; dashed circles: the original location of centrosome pair at 0 min; max.
intensity-projected image stacks; scale bar: 10 µm) (B, C, D and E) correspond to S8C, E, Fig.
3E and G, respectively, yet in cells with control siRNA and Ninein siRNA treatments. Distances
were all measured in 3D ((E) mean and SD). (B, C) Medians with interquartile range are shown
in addition to individual values. (F) Schematic summary of the behavior of cluster and
centrosome dynamics in cells with one plasmid cluster and the cluster being close to the
centrosomes before centrosome splitting. Number of cells after control siRNA and Ninein siRNA
treatments are depicted at indicated mitotic stages. Percentage indicates cell number relative to
all analyzed cells of the given condition. (G) At max split and anaphase, in individual cells after
control and Ninein siRNA treatments, the spatial association of the plasmid cluster with
centrosome-type (old, young) were analyzed and categorized either as “swapped” ((young to
old) or (old to young)) between the two stages or “maintained” ((young–young) or (old-old)). (H)
Analysis of the plasmid localization in cells with siControl and siNinein treatments and after the
transfection of Rho-plasmid (mean with SEM of 3 experiments, at least 25 (siControl) or 24
(siNinein) cells per experiment). (n: pooled cluster (B, D) or cell (C, E-H) number of 3
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experiments (number range per experiment: (B, D): 4-12 (siControl), 3-12 (siNinein); (C, E-G): 3-
12 (siControl), 2-11 (siNinein)); statistics: (B, C) non-paired t-test with log2 (value), (E) Dunn's
multiple comparisons test was performed for each time point separately; only significant
differences were indicated, (G) χ2 test, (H) non-paired t-test)
Fig. S10. Characterization of pLacO clusters in mitotic cells. Plasmid clusters do not obviously co-
localize with cytoskeletal proteins and are embraced by the ER in mitotic cells (related to Fig. 4).
(A) PLacO clusters in mitotic HeLa cells expressing LacI-mCherry and YFP-αTubulin, or
expressing LacI-eGFP and stained with Rhodamine-Phalloidin or anti-Vimentin (B): PLacO
clusters in mitotic HeLa cells expressing eGFP-KDEL or immuno-stained with antibodies
recognizing Emerin and Lap2β (inlets: enlarged areas of the pLacO clusters). ((A, B)
arrowheads: pLacO cluster). (C) Correlative light with focused ion beam scanning electron
microscopy (EM) of an anaphase HeLa cell expressing LacI-mCherry with one pLacO cluster.
The EM image corresponds to the confocal image at a perpendicular view (xz). The boxed area
of the regions of plasmid cluster (blue) and chromosome (gray) are shown as enlarged images
(yellow arrowheads: tubular membrane surrounding the cluster; red arrowhead: the connecting
bridge between the pLacO cluster with tubular membrane). (single z-focus images; scale bars if
not labeled: 10 μm)
Fig. S11. HeLa cells with plasmid clusters have in average a longer cell cycle duration compared to their sibling cells without plasmid. Live HeLa-LacI-eGFP cells were imaged 30 h after pLacO
transfection over two mitoses. In cells with asymmetric (x:0, x=1, 2 and 3) partition patterns, the
cell cycle duration (CCD) was compared between the two daughter cells, D1 (with plasmid) and
D2 (without plasmid). (A) Schematic lineage tree of the divisions depicting plasmid (red dot)
partition and CCDs (length of solid vertical line) (vertical dashed line: incomplete cell cycle;
horizontal line: mitotic event; grey square: schematized cell with nucleus). A CCD is here defined
as the time from the first frame after abscission until the first frame after abscission in the next
cell cycle. (B) The CCD difference of the two daughter cells (D1-D2). Mean with SD is shown in
addition to individual values. (n: number of sibling pairs from multi-positions of 1 time-lapse
experiment)
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3) Supplementary Table legends Table S1: Plasmids for transient expression and stable cell generation, dyes to stain indicated subcellular
organelles, antibodies and cell lines used in this research are listed if not mentioned in the
materials and method section.
Table S2: SiRNAs used in this research are listed.
LacI (1-358 aa, without the tetramerization sequence) plus a C-terminal linker (SSL) and a SV40 NLS (PKKKRKV) (D.L Spector, Cold Spring Harbor Lab, USA) (8) was amplified and inserted into lenvtiviral vector pLenti6/V5-D-TOPO
PlasmidEncoded Gene Source (affliation) Encoded Gene/
sequenceCloning/preparation process
YFP-αTubulin D. Gerlich (IMBA Vienna, Austria)mCherryCentrin1-eGFP S. Doxsey (Univ. Massachusetts
Med. Sch., USA) (3)
mCherry-Sec61β T. Kirchhausen (Harvard Medical School, USA)
eGFP-KDEL A. Helenius (ETHZurich, Switzerland)
pLenti6/V5-D-TOPOlenti-envelope pMD.G
ThermoFischer Scientific
lenti-packaging psPAX2
CMV-mCherry was amplified from pCMV-mCherry (Clontech) and cloned into the backbone of pLacO without LacO repeats
ThermoFischer ScientificThermoFischer Scientific
LacI-eGFP/mCherry
MEM-GlutaMAX (Minimum Essential Media, ThermoFischer Scientific) with 10% FCS 100U/ml penicillin and 100mg/ml streptomycin.
# used as mother cells to develop stable cell lines;
DMEM(Dulbecco’s modified medium, ThermoFischer Scientific) with 10% FCS, 100 U/ml penicillin and 100mg/ml streptomycin;$
Cell lineName Source (affliation)/generation method Culture medium HeLa Kyoto# S. Narumiya, (Kyoto Univ., Japan)Hek 293T W. Krek, (ETH Zurich, Switzerland) MDCK II# I. Mellmann, (Yale Univ., USA) HeLa_LacI-eGFP lentiviral transductionHeLa_LacI-mCherry lentiviral transductionHeLa_Centrin1-eGFP_LacI-mCherry lipofection followed with lentiviral transduction M1 with 2 μg/ml blasticidin and 500 μg/ml G418MDCK_LacI-eGFP lentiviral transduction M2 with 2 μg/ml blasticidin
M1 with 2 μg/ml blasticidinM1 with 2 μg/ml blasticidin
M1^
M1M2$
M1^
M2
DyeName Supplier (Cat. no.) Name Supplier (Cat. no.)Rhodamine phalloidin ThermoFischer Scientific (R415)Hoechst33342 ThermoFischer Scientific (62249)
3,3'-dihexyloxacarbo-cyanine Iodide (DIOC6(3)) ThermoFischer Scientific (D273)
Table S1 Plasmids for transient expression and stable cell generation, dyes to stain indicated subcellular organelles, antibodies and cell lines used in this research are listed if not mentioned in the materials section.
Antibody Recognized antigen Supplier (Cat. no.)/Source (affliation) Host Sample fixation DilutionLaminB1 Abcam (ab16048) rabbit formaldehyde 1:1000 Odf2 Abcam (ab43840) (for HeLa) rabbit MeOH 1:500Odf2 Sigma (HPA001874) (for MDCK) rabbit MeOH 1:500Ninein M. Bornens, (Institute Curie, France) rabbit MeOH 1:500Vimentin Abcam (EPR3776) rabbit formaldehyde 1:500Lap2β BD transduction laboratories (611000) mouse formaldehyde 1:500Emerin Abcam (ab40688) rabbit formaldehyde 1:500GFP Roche (11814460001) mouse formaldehyde 1:1000
mouse IgG, Alexa-FluorTM 647 ThermoFischer Scientific ( A21236) goat -- 1:500mouse IgG, Alexa-FluorTM 594 ThermoFischer Scientific (A11032) goat -- 1:500rabbit IgG, Alexa-FluorTM 647 ThermoFischer Scientific (A21245) goat -- 1:500rabbit IgG, Alexa-FluorTM 594 ThermoFischer Scientific (A11037) goat -- 1:500rabbit IgG, Alexa-FluorTM 488 ThermoFischer Scientific ( A11034) goat -- 1:500
αTubulin Sigma (T 9026) mouse -- (western blot) 1:5000
γ-Tubulin Abcam (ab40688) rabbit formaldehyde 1:500
APC Abcam (ALi, ab58) mouse 1:1000
Biorad (1706516 ) goat 1:5000
-- (western blot)
mouse IgG, HRPrabbit IgG, HRP Biorad (1706515 )
-- (western blot)-- (western blot)goat 1:5000
Sec61α-eGFP A. Helenius (ETHZurich, Switzerland)Ninein-eGFP Y. Hong (Kaosiung Med. U,Taiwan)
(7)
LacO repeat frag-ment
pLacO (13.5 kb) was linearized with HindIII and BamHI, and the part with the LacO repeats (10.0 kb) was purified from agarose gel after DNA electrophoresis
H2B-eGFP D. Gerlich (IMBA Vienna, Austria)
4) Supplementary Tables
siRNA oliogoes(5'-3')
Supplier (Cat. no.)
UCGUUAACUUAUGUUGUCUAA Qiagen (Hs_NIN_14)
GAAUAUUGAUGGAGAGAUA
UGCCUUUGAGAUAAUACGUUA Qiagen (Hs_NIN_15)
CAGAGAAGCUGGCCGAAUA
CAGCCUCGAUGGAAACAUCAA Qiagen (Hs_NIN_10)
CAAGAGAACAUGAAGCAAA
siControlON-TARGET plus Non-TargetingPool
Dharmacon (D-001810-10-20)
GAGCAGCAGUGUAGGGUAU
Human Ninein
Dog NineinSigma
(5'-3')
SigmaSigmaSigma
Table S2SiRNAs used in this research are listed.
Human Odf2 Dharmacon (L-017319-01-0005)
(5'-3')GGCACAACAUCGAGCGCAUCAAAUGACCUGCACGGACAUGGCUGAGACUGAGCACGACGAGACAAAGAGAGCUUGA
GAUGAUAUGUCGCGAACUUAUGAUAAGCUCCCAAAUAAGAGAAUACGUCCACACCUUGAACUAGAUACACCAAUAA
Human APC (5'-3')
Dharmacon (L-017319-01-0005)Dharmacon (L-017319-01-0005)Dharmacon (L-017319-01-0005)
Dharmacon (L-003869-00-0005)Dharmacon (L-003869-00-0005)Dharmacon (L-003869-00-0005)Dharmacon (L-003869-00-0005)
Fig. S1.
F
B+: 69%H2B-eGFP
LaminB1Rho-plasmid
D
CA
G
antiGFP Merge+HoechstLacO probe
0
50
100
Plas
mid
clu
ster
s (%
)probe anti GFP probe anti GFP -+
++
(n=97)
E
no plasmid + pLacO electroporation
nr. of clusters/cell:
12>2
0
50
100
Cel
ls (%
)
(n=43)
cytop
lasmic
nucle
ar
(n=26)
only c
yto-
plasm
ic foc
i
cytoplasmic + nuclear foci
Cel
ls (%
)
eGFPeGFP -
+
0
50
100
no fo
ci
(n=20)
only c
yto-
plasm
ic foc
i
cytop
lasmic +
nucle
ar foc
i
(n=43) (n=26)
Cel
ls (%
)
67
plasmid dose(ng/100,000 cells)
330
1300
0
50
100
nr. clusters/cell:
3456>6
12
5) Supplementary Figures
Fig. S2.
A
B
Y OY O
Centrin1-eGFP v.s. ODF2:consistant not consistant
Y OO Y
Hoechst
consistantnot consistant
0
50
100
9896
Cel
ls (%
)
pLacO
C
Merge+HoechstODF2Centrin1-eGFP
0.70 0.18Y/O:
+-(n=91)(n=51)
c1
b1
c2b2
c1
b1
c2b2
DIC
Y/O:
B
E
C D
F
A
cluster
old youngCentrosome
Bead
0
5
10
15
20
Cel
ls (%
)
12>2
Nr. cluster/cell Centrosome
youngold
(n=79) (n=63)
0
50
100
65.8
**
n.s.
Cel
ls (%
) 63.9
** *
LaminB1
top
top
Bead Centrin1-eGFP Hoechst
DIC
*0
50
100
68.0
**
*
Cel
ls (%
) 48.0
**
n.s.
Centrosome
youngold
pLacO (n=72)
Bead(n=75)
Centrin1-eGFP Hoechst
LacI-mCherry/Bead:
DIC
Bead BeadpLacO
Bead
normal exposure low exposure
x*
y*
xz at y*
yz a
t x*
0
10
20
30
40
50copartition split
n.s.
Cel
ls (%
)
Fig. S3.
-5 5
-5
5
cell_1cell_2cell_3cell_4cell_5cell_6cell_7
-5 5
-5
5
Bead
pLacO cluster
x(µm)
y(µm)
x(µm)
y(µm)
Fig. S4.
A
B
SiC
ontro
lSi
Nin
ein
Centrin1-eGFP Ninein Merge+Hoechst
C
n.s.
SiContro
l
SiNinein
0
50
100
MDCK
(n=12
8)
(n=99
)
64.0
**
43.4
**
Cel
ls (%
)
Centrosome
youngold
siRNA I
siRNA II
pLac
O
(MDCK)
0h 12h 48h24h
corre
lation
analy
sis
MDCKHeLa
Ninein-
97 5% 98 3%92 5% 89 9%
48h24hSiNinein
48h
3 2%5 3%
SiControl
D ECentrosome
youngold
ODF2 DIC
Ninein-eGFP Merge+Hoechst
LacI-mCherry/γTubulin
normal exposure
LacI-mCherry/γTubulin
high exposure
*
Ninein-
eGFP
H2B-eG
FP0
50
100
(n=55
)
(n=40
)
65.4
*
67.5
n.s.
Cel
ls (%
)
*
Fig. S5.
B
A
Centrin1-eGFP ODF2 Merge+Hoechst
SiC
ontro
lSi
OD
F2siControl
APC
αTubulin
250 kDa
50 kDa
siAPC72h48h24h
APC/αTubulin: 0.19 0.06 <0.0001
72h
C
siRNA
pLac
OThy
midine
0h 24h 64h44h
Thymidi
ne
relea
seco
rrelat
ion
analy
sis
72h
ODF2-
73 2% 99 1%
72h24hSiODF2
72h
11 1%
SiControl
13 µl 30 µl
<0.0001 <0.0001
loaded lysate
Fig. S6.
elongation index: (A-B)/B (%)
cell centriod at anaphase I
centrosome
B A
0 min
4 min
6 min
anap
hase
Ian
apha
se II
met
apha
se
8 min
-8 min
anap
hase
Ian
apha
se II A B
Centrin1-eGFP
C
LacI-mCherry
10 min
n.s.
+
-100
-50
0
50
100
Elon
gatio
n in
dex
(%)
old
cent
roso
me
youn
g ce
ntro
som
e
elon
gatio
n si
de
-(n=45)(n=60)
clusters:
elongation index: 58.4 (%)
Fig. S7.
splitmetaphase
anaphase
(min)
87 85 67 41 17 9 3n= (in 75 cells)
0 20 40 60 800
5
10
15
20
25
YC
OC C
lust
er-c
entro
som
e (μ
m)
A B
Spee
d ( μ
m/m
in)
Time (min)
0.0
0.5
1.0
1.5
2.0
0-5 5-10 10-15 15-20
****
(n) 60 5160 59
centrosome
youngold cluster
C D
40
(n=60)0
10
20
30
Clu
ster
-cen
troso
me
pair
(µm
)
17.5 µm
16
Cen
troso
me
disp
lace
men
t (o
ld/y
oung
)
0.25
1
4
0.0625
n.s.
1.66 1.70(mean)(n=60)(n=67)
****
****
*****
*
old
young cluster
YC
OC
cluster centrosome pair
max splitbefore split
young
old
+-clusters:
E
0
1
2
3
4
Clu
ster
inte
nsity
(x10
6 AU
)
0
1
2
3
4
Dia
met
er (µ
m)
n.s.
n.s.
close (n=44)
far(n=16)
close (n=44)
far(n=16)
Fig. S8.
D
C
siControl siNinein0.0625
0.25
1
4
16
(n=19) (n=15)siControl siNinein
0
10
2017.5µm
(n=21) (n=20)
Clu
ster
-cen
troso
me
pair
(µm
)
B
Dis
plac
emen
t (µm
)
10 200 5 15
5
10
15
(min)
E
(n) 1519 1819 19
siControl
5
10
15
Dis
plac
emen
t (µm
)
(n) 1515 1515 15
siNinein
(n=21) (n=20)
OC>YC:65% 55%65%
siNinein
max
spl
it
met
apha
se
anap
hase
befo
re s
plit
OC>YC:67% 67%67%
siControl
-20
-10
0
10
20
Cen
trosm
e - c
lust
er (
OC
-YC
) (µm
)
max
spl
it
met
apha
se
anap
hase
befo
re s
plit
Fspatial association:(max split - anaphase)
G
AsiNinein LacI-mCherry Centrin1-eGFP
45 min
0 min
55 min
10 min
65 min
25 min
90 min
35 min
before split split max split
anaphase
maintainedswapped
cluster centrosome pair
max splitbefore split
young
old
Cen
troso
me
disp
lace
men
t (o
ld/y
oung
)
anaphase
before split split
14
9
19 (100%)
14 (93%)
10
5
4
4
siControl
siNinein(n=15)
(n=19)
cluster-centrosom: <17.5µm
close displacement:old >young
old youngcentrosome
clusternucleus 0
50
100
Cel
ls (%
)
siControl siNinein(n=19) (n=15)
*
n.s.n.s.
0
10 200 5 15 (min)
cluster
centrosome
youngold
****
**
*******
****
**
old
young cluster
YC
OC
0
Honly cytoplasm
cytoplasm and nucleus
only nucleus
0
50
100
Cel
ls (%
)
siControl siNinein
n.s.n.s.
Fig. S9.
BLacI-mCherry
A
C
LacI-mCherry/eGFP merge+Hoechstmerge+Hoechst
YFP-αtubulin
Phalloidin
Vimentin
Dioc6 LacI-mCherry
1µm1µm
z-1 z-2
xz at y*
y*
1µm
1µm
eGFP-KDEL
Emerin
Lap2β 1µm
Fig. S10.
A B
-10
-5
0
5
10
Diff
eren
ce (D
1-D
2)(h
)
(n=24)
pLacO cluster
D2 D1
Fig. S11.