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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: ruth.kroschewski@bc.biol.ethz.ch

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

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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

nonphagocytic cells. Autophagy 6(1):36-45.

2. Dull T, et al. (1998) A third-generation lentivirus vector with a conditional packaging

system. J Virol 72(11):8463-8471.

3. Kuo TC, et al. (2011) Midbody accumulation through evasion of autophagy contributes to

cellular reprogramming and tumorigenicity. Nat Cell Biol 13(12):1214–1223.

4. Kroboth K, et al. (2007) Lack of adenomatous polyposis coli protein correlates with a

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

living cells maintains its transcriptional competence. J Cell Biol 180(1):51-65.

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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.