All cell lines were maintained at 37°C with 5% CO2 atmosphere in DMEM (U20S, HT1080), DMEM:F12 (hTERT RPE-l), or DMEM:F12 medium without phenol red (H2B-GFP and H2B-mRFP U2OS, H2B-GFP and H2B-mRFP RPE-1, H2B-GFP Caco2, H2B-GFP SCC-114, H2B-GFP HeLa, H2B-mRFP MDA-231). All media was supplemented with 10% FBS, 100 IUml-1 penicillin and 100 μg ml-1 streptomycin. HT1080 cells bearing the alphoidtetO human artificial chromosome and expressing the tetracycline repressor TetR fused to the transcriptional silencer tTS (a gift from Vladimir Larionov) were also supplemented with 1 μg ml-1 doxycycline (Sigma), 0.5 mg ml-1 G418 (geneticin, Invitrogen) and 4 μg ml-1 blasticidin S (Invitrogen). U2OS cells stably expressing human NFATc1 fused to the C-terminus of EGFP (Thermo Scientific; R04-017-01) were supplemented with 0.5 mg/ml G418.
Generation of Whole-Chromosome Micronuclei
To generate cells with whole-chromosome MN, hTERT RPE-1 and U2OS cells were treated with 100 ng ml-1 nocodazole (Sigma) for 6 h. Mitotic cells were collected, washed twice with fresh medium containing 10% FBS, and then plated into medium containing 20% FBS where they completed cell division. To overcome the aneuploidy-induced p53-dependent G1 arrest associated with nocodazole washout experiments, RPE-1 cells were transfected with 50 nmol p53 siRNA (Smartpool, Dharmacon) using Lipofectamine RNAi Max (Invitrogen) 12 h prior to the nocodazole treatment, enabling cell cycle progression of the subsequent aneuploid daughter cells with MN. For Nuf2 and MCAK knockdown, U2OS cells were serum starved for two days prior to transfection with 50 nmol Nuf2 or MCAK siRNAs (Smartpool, Dharmacon). Cells were then washed in fresh medium containing 10% FBS and 24 h later treated with 4 μM dihydrocytochalasin B (DCB; Sigma) for 16 h to inhibit cytokinesis. The subsequent arrested tetraploid cells depleted of Nuf2 or MCAK (many of which harbored MN) were then treated for 6 h with 10 μM SB203580 (Sigma), a p38 inhibitor that promotes cell cycle progression of tetraploids. Sequence information of the small interference RNA (siRNA) pools used from Dharmacon are as follows: Human TP53 ON-TARGETplus SMARTpool siRNA L-003329-00-0005, (J-003329-14) GAAAUUUGCGUGUGGAGUA, (J-003329-15) GUGCAGCUGUGGGUUGAUU, (J-003329-16) GCAGUCAGAUCCUAGCGUC, (J-003329-17) GGAGAAUAUUUCACCCUUC; Human KIF2C/MCAK ON-TARGETplus SMARTpool siRNA L-004955-00-0005, (J-004955-06) GGCAUAAGCUCCUGUGAAU, (J-004955-07) CCAACGCAGUAAUGGUUUA, (J-004955-08) GCAAGCAACAGGUGCAAGU, (J-004955-09) UGACUGAUCCUAUCGAAGA; Human CDCA1/NUF2 ON-TARGETplus SMARTpool siRNA L-005289-00-0005, J-005289-06 GAACGAGUAACCACAAUUA, J-005289-07 UAGCUGAGAUUGUGAUUCA, J-005289-08 GGAUUGCAAUAAAGUUCAA, J-005289-09 AAACGAUAGUGCUGCAAGA.
Generation of Micronuclei Containing the Human Artificial Chromosome (HAC)
HT1080 cells carrying the HAC were cultured in doxycycline-free medium and treated with 100 ng ml -1 nocodazole for 6 h to synchronize cells in mitosis. The absence of doxycycline enables TetR binding to TetO, which induces inactivation of the HAC centromere by tTS and the subsequent formation of a MN following release from mitotic arrest. Cells released from mitotic arrest were synchronized at G0/G1 by serum starvation for 24 h, then released into the cell cycle with fresh medium containing 10% serum for further analysis.
Cells were fixed with 70% ethanol at 4° C followed by incubation with 250 μg ml -1 RNase A and 10 μg ml -1 propidium iodide at 37 °C for 30 min. FACS analysis was performed with a FACScalibur flow cytometer (Becton Dickinson) and data analysed with CellQuest software.
Indirect Immunofluorescence Microscopy
For most experiments, cells were seeded on glass coverslips, washed in CSK buffer (10 mM PIPES, 100 mM NaCl, 300 mM sucrose, 3mM MgCl2
), pre-extracted with CSK buffer containing 0.5% Triton X-100 for 5 min at 4° C, and fixed in PBS containing 4% paraformaldehyde for 15 min. After fixation, cells were permeabilised in PBS-0.2% Triton X-100 for 5 min, blocked in blocking buffer (PBS containing 5% FBS, 2% BSA and 0.1% Triton X-100) for 30 min, and then incubated with primary antibodies at room temperature for 1 h or overnight at 4° C. Cells were washed with PBS-0.1% Triton X-100 and incubated with fluorescence-conjugated secondary antibodies (1:1000, Molecular Probes) at room temperature for 1 h. Cells were also stained for DNA with Hoechst 33342 (1:5000, Invitrogen) in PBS. More specific immunostaining protocols were as follows: for γ-H2AX and other DNA damage response proteins56
; for ELYS and mAb41457
; for Cyclin B1/γ- H2AX/BrdU58
and for Mcm2, Cdt1 and Orc259
. Images for most experiments were collected with a Yokogawa CSU-22 spinning disk confocal mounted on a Zeiss Axiovert microscope using 488, 561 and 640 nm laser light. A series of 0.3 μm optical sections were acquired using a 100X 1.4 NA Plan Apo objective with an Orca ER CCD camera (Hamamatsu Photonics). For nuclear pore quantitation, images were collected on a Leica SP5 laser scanning confocal microscope with a 405 nm laser and a white light laser tuned to 488 nm, 568 nm, and 647 nm using a 63X 1.4 NA Plan Apo objective. Z-stacks were collected with a 0.3 μm step size with pinhole at 1 Airy unit. Photomultipliers were calibrated to ensure linear range. For quantitation of Mcm2 and BrdU signals, mean fluorescence intensity within the primary nuclei or micronuclei was measured using ImageJ software. For quantitation of γ-H2AX, primary nuclei were classified as positive when five or more foci were detected, with the threshold of foci intensity set against the irradiated sample. The same threshold was applied to score γ-H2AX foci in MN. Acquisition parameters, shutters, filter positions and focus were controlled by Slidebook software (Intelligent Imaging Innovations, Denver, CO). Images presented in figures are maximum intensity projections of entire z
-stacks, unless otherwise stated.
Antibodies for Immunofluorescence
Samples were incubated with primary antibodies for human anti-centromere ACA (1:1000; Antibodies, Inc.), rabbit γ-H2AX-Ser139 (1:500, Cell Signaling), mouse γ-H2AX (clone JBW301, 1:500, Upstate), rabbit 53BP1 (1:500, Cell Signaling), rabbit phospho-53BP1-Ser1778 (1:500, Cell Signaling), rabbit phospho-Chk1-Ser317 (1:500, Cell Signaling), rabbit phospho-ATM-Ser1981 (1:500, Cell Signaling), rabbit ATR (1:500, Cell Signaling), rabbit Mre11 (1:300, Abcam), rabbit Rad51 (1:100, Santa Cruz), mouse Brca1 (1:300, Santa Cruz), mouse RPA2 (1:500; Abcam), rabbit phospho-RPA2-S33 (1:500, Bethyl Laboratories), Mcm2 and Orc2 (1:300, gifts from Bruce Stillman), rabbit Cdt1 (1:500, Bethyl Laboratories), rabbit geminin (1:500; Santa Cruz), mouse mab414 (1:5000, Abcam), ELYS/MEL28 (1:1000, gift from Iain Mattaj), mouse Lamin A/C (1:300, Abcam), rabbit cyclin A (1:300; Santa Cruz), rabbit cyclin B1 (1:500; Santa Cruz), rabbit cyclin D1 (1:300; Santa Cruz) and mouse BrdU (Sigma). Secondary antibodies used were Alexa Fluor 488 (green), 594 (red) and 647 (far red) from Molecular Probes.
Terminal deoxynucleotidyl-transferase-mediated dUTP-nick end-labelling (TUNEL) was performed according to the manufacturer’s instructions (In Situ cell death detection kit, Roche).
Cells were pre-labelled with 10 μM BrdU for 30 min and subsequently fixed and permeabilised according to the manufacturers’ conditions (5-Bromo-2-deoxyuridine Labelling and Detection Kit 1, Roche). Cells were then stained with anti-BrdU (1h, diluted 1:300) and processed for IF. In the box and whiskers plot, the box represents upper and lower quartiles, line within box represents median and the whiskers extend to the highest and lowest value data sets.
Long-term live-cell imaging
Labelled cells were grown on glass-bottom 12-well tissue culture dishes (Mattek) and imaged on either a Nikon TE2000-E2 or Nikon Ti-E inverted microscope. Both microscopes were equipped with a cooled CCD camera (TE2000, Orca ER, Hamamatsu; Ti-E, Coolsnap HQ2, Photometrics), a precision motorized stage (Bioprecision, Ludl), and Nikon Perfect Focus, and both were enclosed within temperature and CO2-controlled environments that maintained an atmosphere of 37° C and 3-5% humidified CO2. GFP or RFP images were captured at multiple points every 5-10 minutes for 3-5 days with either 10X (0.3 NA) or 20X (0.5, 0.75 NA) objectives, and exposure to fluorescent light was minimized to the greatest extent possible (all image acquisition utilized neutral density filters and/or 2×2 binning). Cell viability was confirmed by the continuous observation of mitotic cells throughout the duration of experiments. Captured images from each experiment were analyzed using NIS-Elements software.
Imaging of H2B-Kaede
Tandem repeats of Kaede (a kind gift of Atsushi Miyawaki, RIKEN Institute) were fused to H2B and cloned into the pLenti6/V5 lentiviral vector (Invitrogen). A U2OS cell line stably expressing H2B-Kaede was generated by lentiviral infection followed by repeated FACS sorting of GFP-positive cells. For tracking experiments, pre-converted micronuclei (GFP-positive) were identified and photoconverted with a brief (1-3s) pulse of 350-400 nm UV light from a DAPI filter cube. Micronuclei that efficiently converted (RFP-positive) were subsequently imaged through the completion of the following mitosis using the same microscope setup as described above. Images were acquired every 10 minutes with a 20X objective and 2×2 binning.
Chromosome Spreads/Spectral Karyotyping
RPE-1 cells were treated with p53 siRNA (as previously described) for 16 h and then grown in medium supplemented with nocodazole (100 ng ml-1) or vehicle control (DMSO) for 6 h. Mitotic cells were collected by shake-off, washed thoroughly in fresh medium without nocodazole, and re-plated in culture medium with 20% FBS to complete mitosis. FACS analysis demonstrated that the subsequent daughter cells progressed to S/G2-phase 18-20 h post-release. At this point, interphase samples were collected and fixed in order to observe the fraction of cells containing MN. In parallel, 100 ng ml-1 colcemid (Invitrogen) was added to the DMSO or nocodazole-treated cells for an additional 6 h and then fixed. All samples were similarly processed for fixation: cells were pelleted and resuspended in a hypotonic solution of 0.075 M KCL for 18 min, fixed in Carnoy’s fixative (3:1 methanol:glacial acetic acid) and washed four times with Carnoy’s fixative. All fixed samples were spread on slides for staining or hybridisation. Mitotic samples were stained with Giemsa or Hoechst to visualize chromosomal abnormalities. Spectral karyotyping (SKY) was performed on interphase and mitotic samples (from both DMSO and nocodazole treated samples) according to the SkyPaint™ DNA kit H-5 for human chromosomes procedure (Applied Spectral Imaging, SKY000029) and imaged on a Nikon Eclipse E6000 microscope equipped with the SD300 Spectracube and Spectral Imaging acquisition software. To determine if pulverized chromosomes were derived from late-replicating chromosomes in MN, the above procedure was also performed with addition of BrdU labeling reagent (Roche Applied Science) only in the last two hours of colcemid treatment before fixation. Mitotic samples from the BrdU samples were treated according to the 5-Bromo-2’-deoxy-uridine Labeling and Detection Kit I protocol (Roche Applied Science, 11296736001), except that the anti-BrdU antibody was diluted 1:50.
Single Cell Gel Electrophoresis (Comet) Assay
Single cell comet assays were performed according to the manufacturer’s instructions (Trevigen). Briefly, U2OS cells were harvested at G1
, S and G2
phases of the cell cycle after nocodazole washout was used to induce MN. As controls, G1
cells were irradiated at 10 Gy. Cells were resuspended in cold PBS at 2×105
, mixed with low-melt agarose (1:10 ratio) and spread on frosted glass slides. After the agarose solidified, the slides were sequentially placed in lysis and alkaline solutions (Trevigen). Slides were then subjected to electrophoresis at 12 V for 10 min in 1x TBE buffer, fixed with 70% ethanol, and stained with DAPI (4’,6-diamidino-2-phenylindole). Nuclei were visualized using epifluorescent illumination on a Zeiss microscope and images were analyzed with the NIH Image J program. DNA damage was quantified for 50 cells with MN for each experimental condition by determining the tail DNA percentage using Comet Score (TriTek) software.
Quantitative Analysis of Nuclear Pore Complexes (NPCs)
To determine nuclear pore complex density in primary nuclei and MN, cells were pre-extracted, fixed, permeabilised and immunostained with mAb414 and ELYS antibodies. Confocal images were collected on a Leica SP5 laser scanning confocal with both a 405 nm and white light laser (at 488nm and 568nm) using a 63X Plan Apo 1.4 NA oil objective. Z-stacks were collected with a 0.3 μm step size with pinhole at 1 Airy unit. Photomultipliers were calibrated to ensure linear range. Single layers of the Z-stack showing optimal nuclear pore staining for the primary nucleus and the MN were chosen for quantitative analysis using MetaMorph. The areas of primary nuclei and MN were judged from Hoechst staining and used to determine the perinuclear rim area. Total fluorescence intensity of mAb414 and ELYS was measured in the perinuclear rim area. To compare nuclear pore densities between primary nuclei and MN, the total nuclear pore fluorescence was divided by the perinuclear rim area of primary nuclei and MN, resulting in an average intensity ratio.
To measure nuclear import, we used U2OS cells stably expressing the fusion protein NFATc1-EGFP (Thermo-Fisher Scientific). The inactive transcription factor NFATc1 resides in the cytosol. Elevated calcium levels in the cytosol lead to the dephosphorylation of NFATc1 by calcineurin thereby inducing its rapid translocation into the nucleus. To increase the calcium level in the cytoplasm, U2OS cells released from nocodazole washout were treated with 0.2 μM thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase. Nuclear import of NFATc1 was then measured in the primary nuclei and MN of U2OS cells at both 6 h and 20 h after nocodazole release. Cells were subsequently fixed with 2% paraformaldehyde and import was quantified based on nuclear fluorescence. To measure nuclear import of IBB-DiHcRed60
, U2OS cells were transiently transfected with the IBB-DiHcRed-plasmid (gift from Jan Ellenberg) using Lipofectamine 2000 (Invitrogen). Cells were then synchronized for 10 h with nocodazole, washed as previously described, and released into the cell cycle. Cells were fixed with 2% paraformaldehyde both 6 h and 20 h later, and import was quantified based on nuclear fluorescence.