Cells were maintained at 37 °C in a 5% CO2 atmosphere. hTERT RPE-1 cells (ATCC cat. no. CRL-4000, RRID:CVCL 4388) and HEK 293 cells (ATCC cat. no. CRL-1573, RRID:CVCL 0045) were grown in Dulbecco’s modified medium (DMEM) F12 (11320-033 from Gibco) containing 10% fetal bovine serum (GE Healthcare), 100 U ml−1 penicillin, 100 U ml−1 streptomycin (15140-122 from Gibco). BJ cells (ATCC cat. no. CRL-4001, RRID:CVCL 6573) and HCT116 cells (ATCC cat. no. CCL-247, RRID:CVCL 0291) were grown in Dulbecco’s modified medium + GlutaMAX (61965-026 from Gibco) containing 10% fetal bovine serum (GE Healthcare), 100 U ml−1 penicillin, 100 U ml−1 streptomycin (15140-122 from Gibco).
All cells were routinely checked for mycoplasma infection and are negative for mycoplasma infection. Identity and purity of the human cell lines used in this study were tested and confirmed using STR authentication.
Generation of an RPE-1 PCNAchromo stable cell line
RPE-1 cells were transfected with 10 µg Cell Cycle-Chromobody plasmid (TagRFP) (from Chromotek) using JET PRIME kit (Polyplus Transfection, 114-07) according to the manufacturer’s protocol. After 24 h, 500 µg ml−1 G418 (4727878001 from Sigma Aldrich) was added to the cell culture medium and then a mixed population of clones expressing PCNA chromobodies were selected.
Generation of an RPE-1 FUCCI or RPE-1 CCNB1AID FUCCI stable cell line
To produce lentiviral particles, HEK 293 cells were transfected with 4 µg pBOB-EF1-FastFUCCI-Puro (Addgene 86849) + 4 µg pMD2.G (Addgene 12259) + 4 µg psPAX2 (Addgene 12260) using a FuGENE HD Transfection Reagent (Promega E2311) in OptiMEM medium (ThermoFisher 51985034). Cells were incubated at 37 °C in a 5% CO2 atmosphere for 16 h and then growth media were removed and replaced by 6 ml fresh OptiMEM. The following day, viral particles were isolated by filtering the medium containing them through a 0.45-μm filter (Sartorius Stedim Biotech 16537). Then, RPE-1 or RPE-1 CCNB1AID cells36 were incubated with viral particles in the presence of 8 μg ml−1 polybrene (Santa Cruz sc-134220) at 37 °C in a 5% CO2 atmosphere for 24 h. RPE-1 GFP and RFP-positive cells were then collected using Sony SH800 FACS (BD FACSDiva Software Version 8.0.1). RPE-1 or RPE-1 CCNB1AID clones expressing FUCCI were selected and the cell lines were established from one single clone.
pBOB-EF1-FastFUCCI-Puro37 was a gift from K. Brindle and D. Jodrell (Addgene 86849).
Generation of an RPE-1 GFP-53BP1 RFP-H2B stable cell line
This cell line was obtained as described below. In brief, to produce lentiviral particles, HEK 293 cells were transfected with 4 µg pSMPUW-IRIS-Neo-H2BmRFP (Fachinetti laboratory) + 4 µg pMD2.G (Addgene 12259) + 4 µg psPAX2 (Addgene 12260). Then, RPE-1 cells were incubated with viral particles and RPE-1 RFP-positive cells were collected using Sony SH800 FACS (BD FACSDiva Software Version 8.0.1). RPE-1 clones expressing RFP-H2B were selected, and the cell line was established from one single clone.
Then, new lentiviral particles were produced by transfecting HEK 293 cells with 4µg Apple-53BP1trunc (Addgene 69531) + 4 µg pMD2.G (Addgene 12259) + 4 µg psPAX2 (Addgene 12260). RPE-1 RFP-H2B cells were incubated with viral particles, and RPE-1 clones expressing both RFP-H2B and GFP-53BP1 were selected using flow cytometry (Sony SH800 FACS). The cell line was established from one single clone.
Apple-53BP1trunc was a gift from R. Weissleder38 (Addgene).
Generation of an RPE-1 shp53 stable cell lines
This cell line was obtained as described below. In brief, to produce lentiviral particles, HEK 293 cells were transfected with 4 µg short hairpin RNA (shRNA) p53-puromycin (Fachinetti laboratory) + 4 µg pMD2.G (Addgene 12259) + 4 µg psPAX2 (Addgene 12260). Then, RPE-1 cells were incubated with viral particles. After 24 h, 5 µg ml−1 puromycin (A1113803 from Gibco) was added to the cell culture medium and then a mixed population of clones expressing p53 shRNA was selected.
Induction of tetraploidy in human cell lines
To induce mitotic slippage, cells were incubated with DMSO (D8418 from Sigma Aldrich) or with 50 µM monastrol (S8439 from Selleckchem) + 1 µM MPI-0479605 (S7488 from Selleckchem) for at least 2 h. Alternatively, CCNB1 depletion in RPE CCNB1AID cells was induced as described36. In brief, cells were treated with 2 µg ml−1 doxycycline (D3447 from Sigma Aldrich) + 3 µM asunaprevir (S4935 from Selleckchem) for 2 h. Then, 500 µM auxin (I5148 from Sigma Aldrich) was added to the cell culture medium for at least 4 h. In the figures, mitotic slippage was induced by the combination of monastrol + MPI-0479605 treatment except for the following figures: Figs. 2i, 3a–h, j–o, Extended Data Figs. 2a, b, 7a, d, 8d–h, 9k, in which mitotic slippage was induced by CCNB1 depletion.
To induce cytokinesis failure, cells were incubated with 10 µM genistein (G6649 from Sigma Aldrich) for at least 2 h. Alternatively, cell were incubated with 0.75 µM dihydrocytochalasin D (DCD; D1641 from Sigma-Aldrich) or with 5 µM latrunculin (L5288 from Sigma-Aldrich) for 1 h. In the figures, cytokinesis failure was induced by genistein treatment except for the following figures: Extended Data Fig. 6j, k, in which cytokinesis failure was induced by DCD treatment and Extended Data Fig. 2c, d, in which cytokinesis failure was induced by latrunculin treatment.
To induce endoreplication, cells were incubated with 10 µM SP600125 (S1460 from Selleckchem) for at least 2 h. Alternatively, CCNA2 depletion in RPE CCNA2AID cells was induced as described36. In brief, cells were treated with 2 µg ml−1 doxycycline (Sigma Aldrich D3447) for 2 h. Then, 500 µM auxin (Sigma Aldrich I5148) + 3 µM asunaprevir (Selleckchem S4935) was added to the cell culture medium for at least 4 h. In the figures, endoreplication was induced by SP600125 treatment except for Figs. 3q, 4c, Extended Data Figs. 2e, f, 3f, 5d, j, 8c, in which endoreplication was induced through CCNA2 depletion.
Cell cycle synchronization and DNA replication inhibition
Cells were treated with 1 µM palbociclib (Cdk4/6 inhibitor, Selleckchem S1579), or with 0.5 µM abemaciclib (Cdk4/6 inhibitor, Selleckchem S5716) or with 1 µM K03861(Cdk2 inhibitor, Selleckchem S8100) for 16 h to synchronize cells at G1/S transition, and were collected (indicated by ‘G1 arrest’ in the figures). Alternatively, cells were then washed five times with PBS and released in S phase for 10 h before being collected. To extend G1 duration cells were treated with 160 nM palbociclib or with 50 nM abemaciclib or with 400 nM K03861 for 16 h and were collected (indicated by ‘G1 lengthening’ in the figures). Alternatively, cells were then washed 5 times in PBS and released in S phase for 10 h before being collected.
To inhibit DNA replication, cells were released in S phase in the presence of low doses of Aphidicolin (APH, A0781 from Sigma-Aldrich), a DNA replication polymerase inhibitor, or of PHA767491 (PZ0178 from Sigma-Aldrich), a Cdc7 inhibitor (indicated by ‘release in S phase + APH’ or ‘release in S phase + PHA’, respectively, in the figures). Doses were chosen to significantly decrease EdU incorporation without affecting the levels of DNA damage.
Cells were synchronized in G1 using 1 µM palbociclib and then released in S phase (see ‘Cell cycle synchronization and DNA replication inhibition’) in the presence of nucleosides at the following concentrations: dC 7.3 mg l−1 (Sigma Aldrich D0776); dG 8.5 mg l−1 (Sigma Aldrich D0901); dU 7.3 mg l−1 (Sigma Aldrich D5412); dA 8 mg l−1 (Sigma Aldrich D8668) and dT 2.4 mg l−1 (Sigma Aldrich T1895) (+ in the figures) or dC 14.6 mg l−1; dG 17 mg l−1; dU 14,6 mg l−1; dA 16 mg l−1 and dT 4,8 mg l−1 (++ in the figures).
The drugs were used at the following concentrations: Auxin (Sigma I5148), 500 µM; doxycycline (Sigma D3447), 2 µg ml−1; asunaprevir (Selleckchem S4935), 3 µM; monastrol (Selleckchem S8439), 50 µM; MPI-0479605 (Selleckchem S7488), 1 µM; genistein (Sigma G6649), 10 µM; SP600125 (Selleckchem S1460), 10 µM; abemaciclib (Selleckchem S5716), 50 nM or 0.5 µM; K03861 (Selleckchem S8100), 400 nM or 1 µM; palbociclib (Selleckchem S1579), 120 nM or 1 µM; aphidicolin (Sigma A0781), 0,4 µM or 1 µM; hydroxyurea (Selleckchem S1896), 2 mM; PHA767491 (Sigma PZ0178), 1 µM; RO3306 (Calbiochem 217699), 10 µM; dihydrocytochalasin D (Sigma D1641), 0,75 µM; latrunculin B (Sigma L5288), 5 µM; 5′-chloro-2′-deoxyuridine (CIdU) (Sigma C6891), 100 µM; 5′-iodo-2′-deoxyuridine (IdU) (Sigma I7125), 100 µM.
Fly husbandry and fly stocks
Flies were raised on cornmeal medium (0.75% agar, 3.5% organic wheat flour, 5.0% yeast, 5.5% sugar, 2.5% nipagin, 1.0% penicillin-streptomycin and 0.4% propionic acid). Fly stocks were maintained at 18 °C. Crosses were carried out in plastic vials and maintained at 25 °C. Stocks were maintained using balancer inverted chromosomes to prevent recombination. Stocks used in this study: sqh1,39, pavarotti RNAi (Pav RNAi) (Bloomington Drosophila Stock Center BL#42573)32, UAS-E2F1 (FlyORF F001065) and UAS-Rb (Bloomington Drosophila Stock Center BL#50746).
In all experiments, larvae were staged to obtain comparable stages of development. Egg collection was performed at 25 °C for 24 h. After development at 25 °C, third instar larvae were used for dissection.
Preparation and imaging of human cells
Cells were plated on cover slips in 12-well plates and treated with the indicated drugs. To label cells, they were fixed using 4% of paraformaldehyde (Electron Microscopy Sciences 15710) + Triton X-100 (2000-C from Euromedex) 0.1% in PBS (20 min at 4 °C). Then, cells were washed three times using PBS-T (PBS + 0.1% Triton X-100 + 0.02% Sodium Azide) and incubated with PBS-T + BSA (Euromedex 04-100-812-C) 1% for 30 min at room temperature. After 3 washes with PBS-T + BSA, primary and secondary antibodies were incubated in PBS-T + BSA 1% for 1 h and 30 min at room temperature, respectively. After 2 washes with PBS, cells were incubated with 3 μg ml−1 DAPI (Sigma Aldrich D8417) for 15 min at room temperature. After two washes with PBS, slides were mounted using 1.25% n-propyl gallate (Sigma P3130), 75% glycerol (bidistilled, 99.5%, VWR 24388-295), 23.75% H2O.
Images were acquired on an upright widefield microscope (DM6B, Leica Systems, Germany) equipped with a motorized xy stage and a 40× objective (HCX PL APO 40×/1.40–0.70 Oil from Leica). Acquisitions were performed using Metamorph 7.10.1 software (Molecular Devices) and a sCMOS camera (Flash 4V2, Hamamatsu). Stacks of conventional fluorescence images were collected automatically at a z-distance of 0.5 µm (Metamorph 7.10.1 software; Molecular Devices, SCR 002368). Images are presented as maximum intensity projections generated with ImageJ software (SCR 002285).
Whole-mount tissue preparation and imaging of Drosophila larval brains
Brains or salivary glands from third instar larvae were dissected in PBS and fixed for 30 min in 4% paraformaldehyde in PBS. They were washed 3 times in PBST 0.3% (PBS, 0.3% Triton X-100 (Sigma T9284), 10 min for each wash) and incubated for several hours in agitation at room temperature and overnight at 4 °C with primary antibodies at the appropriate dilution in PBST 0.3%. Tissues were washed three times in PBST 0.3% (10 min for each wash) and incubated overnight at 4 °C with secondary antibodies diluted in PBST 0.3%. Brains and salivary glands were then washed 2 times in PBST 0.3% (30 min for each wash), rinsed in PBS and incubated with 3 μg ml−1 DAPI (4′,6-diamidino-2-phenylindole; Sigma Aldrich D8417) at room temperature for 30 min. Brains and salivary glands were then washed in PBST 0.3% at room temperature for 30 min and mounted on mounting media. A standard mounting medium was prepared with 1.25% n-propyl gallate (Sigma P3130), 75% glycerol (bidistilled, 99.5%, VWR 24388-295), 23.75% H2O.
Images were acquired on a spinning disk microscope (Gataca Systems). Based on a CSU-W1 (Yokogawa), the spinning head was mounted on an inverted Eclipse Ti2 microscope equipped with a motorized xy stage (Nikon). Images were acquired through a 40× NA 1.3 oil objective with a sCMOS camera (Prime95B, Photometrics). Optical sectioning was achieved using a piezo stage (Nano-z series, Mad City Lab). The Gataca Systems’ laser bench was equipped with 405, 491 and 561 nm laser diodes, delivering 150 mW each, coupled to the spinning disk head through a single mode fibre. Multi-dimensional acquisitions were performed using Metamorph 7.10.1 software (Molecular Devices). Stacks of conventional fluorescence images were collected automatically at a z-distance of 1.5 µm (Metamorph 7.10.1 software; Molecular Devices SCR 002368). Images are presented as maximum intensity projections generated with ImageJ software (SCR 002285).
Primary and secondary antibodies were used at the following concentrations: guinea pig anti-CEP192 antibody40 (1:500; R.B. laboratory), rabbit anti-β catenin (1:250; Sigma-Aldrich C2206, RRID AB 476831), mouse anti-γH2A.X phospho S139 (1:1,000; Abcam ab22551, RRID AB 447150), mouse anti-XRCC1 (1:500; Abcam ab1838, RRID AB 302636), rabbit anti-Rad51 (1:500; Abcam ab133534, RRID AB 2722613), mouse anti-KU80 (1:200; ThermoFisher MA5-12933, RRID AB 10983840), rabbit anti-FANCD2 (1:150; Novusbio NB100-182SS, RRID AB 1108397), mouse anti-53BP1 (1:250; Millipore MAB3802, RRID AB 2206767), rabbit anti-γH2Av (1:500; Rockland600-401-914, RRID AB 11183655), Alexa Fluor 647 Phalloidin (1:250; ThermoFisher Scientific A22287, RRID AB 2620155), goat anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (1:250; ThermoFisher A21245, RRID AB 2535813), goat anti-guinea pig IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:250; ThermoFisher A11073, RRID AB 253411), goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (1:250; ThermoFisher A11003, RRID AB 2534071), goat anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (1:250; Thermo Fisher Scientific A-11035, RRID AB 2534093).
Quantitative analysis of DNA damage
Drosophila neuroblasts and 3D spheroids
Quantitative analysis of DNA damage was carried out as previously described32. In brief, DNA damage was assessed in Drosophila using a γH2Av primary antibody and in 3D spheroids with a γH2AX antibody, and detected with an Alexa Fluor secondary antibody. Confocal volumes were obtained with optical sections at 1.5-µm intervals. Image analysis was performed using Fiji and a custom plugin developed by QUANTACELL. After manual segmentation of the nuclei, a thresholding operation was used to determine the percentage of γH2Av- or γH2AX-positive pixels (coverage) and their average intensity in a single projection. Coverage and intensity were multiplied to obtain the γH2Av or γH2AX index. The threshold used to detect and quantify the γH2Av index in polyploid neuroblasts does not detect any damage in salivary glands. However, it is important to mention that in a fraction of these cells, γH2Av dots (small and of low fluorescence intensity) can be occasionally seen.
2D human cell lines
For DNA damage quantification, the signals obtained in cultured cells were different from the signals found in Drosophila neuroblasts. To asses DNA damage in human cells, we used an ImageJ software-based plugin developed by QUANTACELL, where γH2AX signals were measured using z-projection stacks after thresholding. Nuclear size, DAPI intensity, the number of γH2AX foci, γH2AX fluorescence intensity and the percentage of nuclear coverage by γH2AX signal were obtained for each nucleus.
Cells were plated on a dish (627870 from Dutscher) and treated with the indicated drugs. Images were acquired on a spinning disc microscope (Gataca Systems). Based on a CSU-W1 (Yokogawa), the spinning head was mounted on an inverted Eclipse Ti2 microscope equipped with a motorized xy stage (Nikon). Images were acquired through a 40× NA 1.3 oil objective with a sCMOS camera (Prime95B, Photometrics). Optical sectioning was achieved using a piezo stage (Nano-z series, Mad City Lab). Gataca Systems’ laser bench was equipped with 405-, 491- and 561-nm laser diodes, delivering 150 mW each, coupled to the spinning disk head through a single mode fibre. Laser power was chosen to obtain the best ratio of signal/background while avoiding phototoxicity. Multi-dimensional acquisitions were performed using Metamorph 7.10.1 software (Molecular Devices). Stacks of conventional fluorescence images were collected automatically at a z-distance of 0.5 µm (Metamorph 7.10.1 software; Molecular Devices, RRID SCR 002368). Images are presented as maximum intensity projections generated with ImageJ software (RRID SCR 002285), from stacks deconvolved with an extension of Metamorph 7.10.1 software.
Mitotic slippage on 3D cultures
To generate spheroids, 500 cells per well were seeded into 96 ultra-low-attachment well plates (Corning7007) in presence of DMSO (Sigma Aldrich D8418) or with 50 µM monastrol (Selleckchem S8439) and 1 µM MPI-0479605 (Selleckchem S7488). Plates were spin down at 200g for 3 min, to allow spheroid formation, and incubated for 24 h at 37 °C.
Spheroids were collected and washed quickly with PBS before fixation using 4% paraformaldehyde (Electron Microscopy Sciences 15710) in PBS for 40 min. Then, spheroids were permeabilized for 5 min using Triton X-100 (Euromedex 2000-C) 0.3% in PBS and blocked for 30 min using blocking buffer (PBS + 0.3% Triton X-100 + 0.02% sodium azide + 3% BSA). Aggregates were incubated with primary antibodies diluted into blocking buffer overnight. After 3 washes using blocking buffer, spheroids were incubated with secondary antibodies in blocking buffer for 3 h. Cells were then washed several times for 2 h in blocking buffer and mounted on glass with EverBrite (Biotium). For primary and secondary antibodies see ‘Immunofluorescence microscopy and antibodies’.
Imaging and DNA damage analysis
Spheroids were imaged using an inverted scanning laser confocal (Nikon A1RHD25) equipped with a 100× CFI Plan Apo Lambda S Sil objective (NA 1.35). z-stacks were acquired every 0.3 μm. Diploid and tetraploid cells were distinguished using cell and nuclear size and centrosome number. Then, quantitative analysis of DNA damage was carried out (see ‘Quantitative analysis of DNA damage’).
EdU incorporation into DNA was visualized with the Click-it EdU imaging kit (Life Technologies C10338), according to the manufacturer’s instructions. For human cell lines, EdU was used at a concentration of 1 µM (Extended Data Figs. 6e, 9h) or 10 µM (Extended Data Fig. 5g, h) for the indicated time. Cells were incubated with the Click-it reaction cocktail for 15 min. EdU incorporation in polyploid neuroblasts was done as previously described32 with a pulse of 2 h before fixation.
Comet assays were performed using Single Cell Gel Electrophoresis Assay kit (4250-050-ES from Trevigen) according to the manufacturer’s instructions. Comets were then imaged using an inverted Eclipse Ti-E Nikon videomicroscope equipped with a 40× CFI Plan Fluor objective. Images were analysed with OpenComet plugin on Fiji. Based on the comet DNA content of DMSO treated cells, a manual threshold was applied to identify diploid from tetraploid cells. The same threshold was applied on the cells treated for mitotic slippage.
FACS of diploid and tetraploid cells
A mix of diploid and tetraploid cells (see ‘Induction of tetraploidy in human cell lines’) were incubated with 2 µg ml−1 Hoescht 33342 (Sigma Aldrich 94403) for 1 h at 37 °C, 5% CO2. Then, a single cell suspension was generated. Cells were washed using PBS, the supernatant was removed and cells were resuspended in a cold cell culture medium at 1 × 107 cell per ml and kept at 4 °C during all the experiments. Fluorescence-activated cell sorting (FACS) was performed using Sony SH800 FACS (BD FACSDiva Software Version 8.0.1). Compensation was performed using the appropriate negative control samples. Experimental samples were then recorded and sorted using gating tools to select the populations of interest. RFP+GFP− cells (G1 cells) were first selected. Then, in this population, DNA content was used to segregate diploid (2n) and tetraploid (4n) G1 cells (Extended Data Fig. 8d). Once gates have been determined, the same number of diploid and tetraploid G1 cells were sorted into external collection tubes. The number of cells was then checked using a cell counter and the same number of diploid an tetraploid cells were collected for western blot analysis. In parallel, post-sort analysis was performed to determine the purity of the sorted populations (Extended Data Fig. 8e).
Cell cycle analysis and measure of RNA levels by flow cytometry
Cells were detached by treatment with Accutase (Sigma), immediately washed in PBS, fixed in 2 ml 70% ethanol and stored at −20 °C overnight. They were then washed in PBS and staining buffer (BD Pharmingen 554656).
For cell cycle analysis, DNA content was visualized by incubating the cells with 2 µg ml−1 Hoescht 33342 (Sigma Aldrich 94403) in staining buffer for 15 min at room temperature. Alternatively, to measure RNA levels, cells were incubated with 2 µg ml−1 Hoescht 33342 + pyronin 4 µg ml−1 (Santa Cruz sc-203755A) in a staining buffer for 20 min at room temperature. Flow cytometry analysis was done using LSRII (BD Biosciences), by analysing 10,000 cells per condition. Data were then analysed with FlowJo 10.6.0 software (Tree Star).
RPE-1 cells were transfected using 0.25 µg pCMVHA E2F1 (Addgene 24225) with a JET PRIME kit (Polyplus Transfection 114-07) according to the manufacturer’s protocol. Five hours later, cells were incubated with DMSO (D8418 from Sigma Aldrich) or with 50 µM monastrol (Selleckchem S8439) + 1 µM MPI-0479605 (Selleckchem S7488) to generate tetraploid cells. After 2 h, DMSO or 1 µM palbociclib (Selleckhem S1579) were added to the cell culture medium for 16 h. Cells were then fixed in G1 (T0) or washed five times using PBS and released in S phase and fixed after 10 h (T10). The immunofluorescence protocol is described in the corresponding section.
pCMVHA E2F1 was a gift from K. Helin41 (Addgene plasmid 24225).
For a whole-cell extract, cells were lysed in 8 M urea, 50 mM Tris HCl, pH 7.5 and 150 mM β-mercaptoethanol (Bio-Rad 161-0710), sonicated and heated at 95 °C for 10 min. For chromatin-bound fractions, cells were prepared using the Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher Scientific 78840), according to the manufacturer’s instructions. Then, samples (equivalent of 2 × 105 cells) were subjected to electrophoresis in NuPAGE Novex 4–12% Bis-Tris pre-cast gels (Life Technologies NP0321). The same number of cells (see ‘FACS sorting of diploid and tetraploid cells’) were loaded for diploid and tetraploid conditions, allowing us to compare one diploid cell with one tetraploid cell. Protein fractions from the gel were electrophoretically transferred to PVDF membranes (PVDF transfer membrane; GE Healthcare RPN303F). After 1 h saturation in PBS containing 5% dry non-fat milk and 0.5% Tween 20, the membranes were incubated for 1 h with a primary antibody diluted in PBS containing 5% dry non-fat milk and 0.5% Tween 20. After three 10-min washes with PBS containing 0.5% Tween 20, the membranes were incubated for 45 min with a 1:2,500 dilution of peroxidase-conjugated antibody. Membranes were then washed three times with PBS containing 0.5% Tween 20, and the reaction was developed according to the manufacturer’s specifications using ECL reagent (SuperSignal West Pico Chemiluminescent Substrate; Thermo Scientific 34080).
The background-adjusted volume intensity was calculated and normalized using a H2B signal (H2B was used as a readout of DNA content) for each protein, using Image Lab software version 6.0.1, Bio-Rad Laboratories. All the original uncropped blots (gel source data) are presented in Supplementary Fig. 1.
Primary and secondary antibodies were used at the following concentrations. Mouse anti-α-tubulin (1:5,000; Sigma T9026, RRID AB 477593), mouse anti-CDC45 (1:100; Santa Cruz Biotechnology sc-55569, RRID AB 831146), rabbit anti-PCNA (1:500; Santa Cruz sc56, RRID AB 628110), rabbit anti-actin (1:2,000; Sigma-Aldrich A5060, RRID AB 476738), mouse anti-H2B (1:1,000; Santa Cruz Biotechnology sc-515808), mouse anti-ORC1 (1:100; Santa Cruz Biotechnology sc-398734), mouse anti-MCM2 (1:500; BD Biosciences 610701, RRID AB 398024), mouse anti-E2F1 (1:2,000; Santa Cruz sc251, RRID AB 627476), mouse anti-CDC6 (1:500; Santa Cruz sc-9964, RRID AB 627236), rabbit anti-CDT1 (1:500; Cell Signaling 8064S, RRID AB 10896851), rabbit anti-treslin (1:500; Betyl A303-472A, RRID AB 10953949), goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (1:2,500; ThermoFisher G21234, RRID AB 2536530), Peroxidase AffiniPure goat anti-mouse IgG (H+L) (1:2500; Jackson ImmunoResearch 115-035-003, RRID AB 10015289).
3D reconstruction and analysis
3D videos (see ‘Time-lapse microscopy’) were imported into Imaris software v.9.6.0 (Bitplane, RRID SCR 007370). For chosen cells, the module ‘Spot tracking’ of Imaris v.9.6.0 was used to detect the foci, as spots of diameter 0.5 µm in the xy-direction and 1 µm in z-direction (modelling PSF elongation). Because the volume of the foci changes in time, the option ‘Enable growing regions’ was used. In each video, the threshold was chosen on the brightest frame (to detect a maximum of the correct spots) and then applied to the whole video. For each cell, at each time point, the number of spots and volumes were recorded. To determine DNA replication timing, we quantified the signal of PCNA fluorescence intensity in the nucleus. This replication timing was characterized independently of any particular behaviour of PCNA. As soon as PCNA fluorescence intensity was detected in the nucleus, t = 0 (beginning of S phase) was defined, and when PCNA fluorescence intensity was not detected anymore the last time point was defined (end of S phase). For each condition, at least ten cells (Supplementary Data 1) were studied and the statistics from Imaris v.9.6.0 were averaged at each time point using a MATLAB script.
Tetraploid HCT116 were generated by cytokinesis inhibition using 0.75 µM dihydrocytochalasin D (DCD, inhibitor of actin polymerization, Sigma-Aldrich D1641) for 18 h overnight. Afterwards, the cells were washed 3 times with PBS and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin for additional 20 h. Tetraploid RPE-1 and BJ cells were generated by mitotic slippage or endoreplication (see ‘Induction of tetraploidy in human cell lines’). Then, the cells were washed three times with PBS and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin for an additional 20 h. For each method, we determined that the proportion of tetraploid cells in the treated population is about 40–60%. Due to the presence of diploid cells in the treated population, the consequences of tetraploidization on replication fork speed, fork asymmetry and IOD are most probably underestimated.
Diploid controls and the tetraploid-enriched population were then pulse-labelled with 0.1 mM CIdU and 0.1 mM IdU for 30 min and 100,000–300,000 cells per condition were collected for further analysis. The DNA was extracted from cells and prepped following the manufacturer’s instructions using the FiberPrep DNA Extraction Kit (Genomic Vision). Subsequently, the prepped DNA was stretched onto coated glass coverslips (CombiCoverslips, Genomic Vision) by using the FiberComb Molecular Combing System (Genomic Vision). The labelling was performed with antibodies against ssDNA, IdU and CldU using the Replication Combing Assay (RCA) (Genomic Vision). The imaging of the prepared cover slips was carried out by Genomic Vision and analysed using the FiberStudio 2.0.1 Analysis Software by Genomic Vision. Replication speed was determined by measuring the combined length of the CldU and IdU tracks. Fork asymmetry was determined by measuring symmetry of the CldU and IdU incorporation by the forks (the length of the first track (CldU) is compared to the length of the second track (IdU)). IOD was determined by measuring distance between two origins on the same fibres.
Antibodies were used at the following concentrations. Rabbit anti-ssDNA (1:5; IBL International 18731, RRID AB 494649), rat anti-CldU (1:10; Abcam Ab6326, RRID AB 2313786), mouse anti-IdU (1:10; BD Biosciences 555627, RRID AB 10015222), mouse Alexa Fluor 647 donkey (1:25; Biozol JIM-715-605-151), rat Alexa Fluor 594 donkey (1:25; Biozol JIM-712-585-153), rabbit Brilliant Violet 480 donkey (1:25; Jackson Immuno Research 711-685-152, RRID AB 2651109).
Quantitative phase imaging and measurements
Cells were plated on glass-bottom dishes coated with 50 µg ml−1 fibronectin for 1 h and rinsed, and trypsinized cells were plated at a concentration of 1.5 × 106 cells per ml. The cells used for the experiments were seeded in T-25 dishes at a concentration of 0.7 × 106 cells per ml 2 days before the actual experiment. On the day of the experiment, the cells were detached with EDTA (versene), and plated at a concentration of 1.5 × 106 cells per ml. For inducing tetraploidy, cells were treated with 2 µg ml−1 doxycycline (Sigma Aldrich D3447) for 2 h. Then, 500 µM auxin (Sigma Aldrich I5148) + 3 µM asunaprevir (Selleckchem S4935) was added to the cell culture medium for at least 4 h. The cells were then imaged for 35 h every 20 min to track them throughout their cell cycle.
The cell cycle state was indicated by the FUCCI system; G1 cells express Cdt1–RFP while S/G2 cells express geminin–GFP and mitosis was indicated by the nuclear envelope break down with geminin being present through the cells42. To quantify the fluorescence of geminin in the nucleus, first a background subtraction was performed on the images. A region of interest (ROI) was used to define an area containing the background fluorescence in the image. An average value of the ROI was then subtracted from all the frames. Subsequently, a ROI was drawn as close as possible to the cell, and then the mean gray value was measured across all the frames. This helped identify the frames of birth and G1/S transition during the cell cycle.
A detailed protocol for the mass measurement with phasics camera is available in refs.43,44. Images were acquired by a Phasics camera every 20 min for 35 h for the duration of the experiment. To obtain the reference image, 32 empty fields were acquired on the dish and a median image was calculated. This reference image was subtracted from the interferograms (images acquired by phasics) by custom written MATLAB scripts to measure the optical path difference. They were then processed to calculate the phase, intensity and phase cleaned images (the background set to 1,000 and the field cropped to remove edges). Background normalization was performed using a gridfit method, and a watershed algorithm was used to separate cells which came in contact with each other. Mass was calculated by integrating the intensity of the whole cell.
Sequencing and AneuFinder analysis
A mixed population of diploid and tetraploid RPE-1 CCNB1AID FUCCI cells were synchronized in G1 using 1 µM palbociclib (Selleckchem S1579) for 16 h or released in S phase for 20 h in the presence of 10 µM RO3306 (Calbiochem 217699) in order to block cells in the subsequent G2/M. G1 and G2/M diploid and tetraploid cells were then isolated using cell sorting (see ‘FACS sorting of diploid and tetraploid cells’) and collected in a 96-well plate.
Sequencing was performed using a NextSeq 500 (Illumina; up to 77 cycles; single end). The generated data were subsequently demultiplexed using sample-specific barcodes and changed into fastq files using bcl2fastq (Illumina; version 1.8.4). Reads were afterwards aligned to the human reference genome (GRCh38/hg38) using Bowtie2 (version 2.2.4; ref. 45. Duplicate reads were marked with BamUtil (version 1.0.3; ref. 46. The aligned read data (bam files) were analysed with the copy number calling algorithm AneuFinder47 (https://github.com/ataudt/aneufinder). Following GC correction and blacklisting of artefact-prone regions (extreme low or high coverage in control samples), libraries were analysed using the dnacopy and edivisive copy number calling algorithms with variable width bins (average bin size = 1 Mb; step size = 500 kb). The G1 samples were analysed with an euploid reference48. The G1 samples were used as a reference for the analysis of the G2/M samples (G1 diploid for G2/M diploid and G1 polyploid for G2/M polyploid). Aneuploid libraries were not used as a reference and blacklists were constructed using the example from Bioconductor as a guideline. The RPE-1 diploid G1 sample (2n) was analysed with the standard version of AneuFinder (from Bioconductor) while the other samples were analysed with the developer version of AneuFinder (from GitHub; 4n and 8n samples). The ground ploidy for these samples was constrained between 3.5 and 4.5 (4n samples) or between 7.5 and 8.5 (8n samples; parameters: min.ground.ploidy and max.ground.ploidy). Results were afterwards curated by requiring a minimum concordance of 95 % (2n sample) or 90% (4n and 8n samples) between the results of the two algorithms. Libraries with on average less than 10 reads per chromosome copy of each bin (2-somy: 20 reads, 3-somy: 30 reads, etc.) were discarded. This minimum number of reads comes down to roughly 60,000 for a diploid genome in G1 phase (2n) up to 240,000 for a polyploid genome in G2/M phase (8n). Analysis of the BJ samples showed aberrations (wavy patterns) that resulted in wrongly called segments with a copy number which is either one higher or one lower than the expected state (when euploid). The means of the read counts (read counts of the bins) of these states were too close to the mean of the expected state (for example, mean 5-somy too close to mean 4-somy; 4n sample; Supplementary Methods 1). When more than 1 % of the genome was classified as such (for example, more than 1 % 5-somy), a non-rounded version of the copy number of the state was calculated using the mean of the expected state (ploidy of euploid sample) as a reference:
Non-rounded copy number.state = Mean state/(mean.expected state/copy number.expected state)
Example 5-somy (4n sample):
Non-rounded copy number.5-somy = Mean.5-somy/(Mean.4-somy/4)
This was done to quantify the distance between the two states. The values are typically between −0.5 and +0.5 of the state under consideration (for example, 5-somy; between 4.5 and 5.5), which will result in a rounded value equal to the state. The libraries with aberrations have typically a deviation of 0.25 and more from the expected value (Supplementary Methods 1). Libraries that showed a deviation of more than 0.25 were therefore discarded (For 5-somy; a value lower than 4.75 or higher than 5.25). By applying this cut-off, we eliminated libraries that clearly showed this aberration (Supplementary Methods 1) while preserving true aneuploid libraries (Supplementary Methods 1). This specific method was only used for the BJ samples.
GSEA with TCGA PanCancer data
GSEA was performed using GSEA software v.4.2.149,50. The normalized mRNA expression (Illumina HiSeq_RNASeqV2, RSEM) from pan cancer studies were downloaded from https://www.cbioportal.org/: detailed information about RNA sequencing experiment and tools used can be found at the NCI’s Genomic Data Commons (GDC) portal https://gdc.cancer.gov. The ploidy status for bladder urothelial carcinoma (156 near-diploid and 200 near-tetraploid samples), Lung adenocarcinoma (205 near-diploid and 240 near-tetraploid samples), and ovarian serous cystadenocarcinoma (116 near-diploid and 130 near-tetraploid samples) were extracted from35. In addition to ranked list of genes and ploidy status, we use gene sets derived from the GO Biological Process ontology to assess significant pathway enrichment between near -diploid and near tetraploid tumors in GSEA tool. GSEA is a computational method that determines whether a defined set of genes shows statistically significant concordant differences between two biological states (for example, two distinct phenotypes), using the algorithm based on the calculation of an enrichment score (ES), the estimation of significance level of ES (nominal P value) and adjustment for multiple hypothesis testing (ES normalization and FDR calculation)49.
Image analysis and quantifications were performed using Image J software V2.1.0/1.53c, https://imagej.net/software/fiji/downloads. To quantify the colocalizations between two signals (Extended Data Figs. 3i, m, 4g, j) we calculated the Manders coefficient using the JACOP plugin with Image J V2.1.0/1.53c software. We determined that the colocalizations between γH2AX signal and EdU, FANCD2 or RAD51 signals are not random using an home-made based Costes randomization on nuclear area with Image J software. 1000 randomizations of the pixel positions were performed for each condition (Supplementary data 2). 3D videos (Extended Data Figs. 3c, 6c, 9c, d) were corrected using the 3D correct drift plugin with Image J V2.1.0/1.53c software to keep the cell of interest at the centre of the region of interest. The nuclear area and DAPI intensity were measured using the wand tool with Image J V2.1.0/1.53c software. For the figures, images were processed on Image J V2.1.0/1.53c software, and mounted using Affinity Designer (https://affinity.serif.com/fr/designer/).
Statistics and reproducibility
At least two (n) independent experiments were carried out to generate each dataset, and the statistical significance of differences was calculated using GraphPad Prism (RRID SCR 002798) version 7.00 for Mac (GraphPad Software). The statistical test used for each experiment is indicated in the figure legends. Each representative image (Figs. 2a, c, 3a, e, g, k, n, 4a, Extended Data Figs. 2a, c, e, l, 3a, g, 4c, 5g, 6c, 9c, d, 10a) originates from a dataset composed of at least two (n) independent experiments.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.
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