Chromosome segregation errors: a double-edged sword - TI Pharma

Live cell imaging
Cells were plated in 2, 4 or 8-well chambered glass bottom slides (LabTek) and imaged in a heated
chamber (37ºC and 5% CO2) using a 40X/1.3NA oil objective on a Zeiss Axiovert 200M microscope
equipped with a 0.55NA condenser and controlled by a lambda-DG4 (Roper Scientific) and MetaMorph
software. Green fluorescent (80 msec exposure) and red fluorescent images (200msec exposure) were
acquired every 12 min using a Photometrics CoolSnap HQ CCD camera (Roper Scientific) for 48-64
hours. Images were processed using MetaMorph software. For live analysis of Monastrol release
(Fig.3F, S2B-E), RPE-1 cells stably expressing H2B-RFP and 53BP1-GFP were imaged using a 40X/0,75NA
UPLFLN objective on an Olympus 1X-81 microscope, controlled by Cell-M software (Olympus). Green
fluorescent and red fluorescent images were acquired every 12 minutes for 12 hours following release
from Monastrol. Images were processed using Cell-M software. Segregation errors in daughter
cells were categorized in three classes: no, mild (metaphase plate formed properly, some lagging
chromosomes in anaphase) and severe (no proper metaphase plate has been formed when cells enter
anaphase) (Fig.1A, G). To avoid analysis of 53BP1 foci formation that might be associated with the
onset of DNA replication, we analyzed only 53BP1 foci that formed within 5 hours after mitotic exit
(anaphase) in all live cell imaging experiments shown.
Immunofluorescence Microscopy
Cells were plated on 10 or 12mm coverslips. For all Monastrol release samples, mitotic cells were
collected by shake-off, washed 3 times with PBS using centrifugation for 5 minutes at 1000 rpm
and replated in a new well. Cells were harvested 6 hours after Monastrol release or 16 hours after
Thymidine release. Fixation was done using 4% PFA in PBS. Antibodies were incubated O/N in PBS 3%
BSA. Secondary antibodies and DAPI were incubated in PBS 0, 1% Tween. DAPI was used to visualize
DNA. Stained coverslips were mounted with Vectashield Mounting Medium (Vector). Images were
acquired on a Zeiss 510 Meta confocal laser scanning microscope with a 63X/1.4NA Plan-ApoChromat
objective using the Zeiss LSM software or on a DeltaVision RT system (Applied Precision) with
100x/1.40NA UplanSapo objective (Olympus) using SoftWorx software.
Chromosome spreads and COBRA FISH
RPE-1 cells were transfected with either luciferase or p53 siRNA on day 0, treated for 2 days with
or without Mps1-IN-1 and harvested on day 4 (Fig.4). CIN tumor cell lines MCF7, SW480 and U2OS
were grown asynchronously (Fig.S10). Calyculin A (80nM) was added for 25 minutes to the medium
to enrich for G2 and Mitotic cells with condensed chromosomes. Cells were treated with 0.075 M KCl
at 37ºC for 10 minutes and centrifuged at 750 rpm for 8 minutes. Cells were fixed with Methanol:
Acetic Acid (4:1) and centrifuged at 750 rpm for 8 minutes. The fixation procedure was repeated 3
times. Samples were collected in Methanol: Acetic Acid (4:1). Chromosome spreads were created by
allowing the drops to fall from 30 cm height onto glass slides. Spreads were left at room temperature
for 2 days before hybridization. COBRA-FISH staining and analysis was performed as described before
433 . Structural chromosomal aberrations include all aberrations; breaks, deletions and translocations.
RPE-1 cells harbor an endogenous translocation derived from chromosome X and 10. Therefore, this
specific translocation has been left out of the quantifications shown in Fig.4C.
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Chromosome Segregation Errors cause DNA Damage
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