Fig. 1

Replication stress induced by low-dose aphidicolin or hydroxyurea results in chromosome mis-segregation and micronuclei comprised of acentric chromatin. A Image of RPE1 prometaphase cell; DNA damage foci detected using γH2AX antibody. Scale bar in this and all subsequent microscopy images represents 5 μm. B Quantification of DNA damage in RPE1 prometaphase cells after indicated treatments; “noc w/o” indicates a nocodazole washout and release (see “Methods”). Statistical test was an unpaired t-test, comparing DMSO control to each individual condition. Data from at least three experiments, each in a separate color with mean for each experiment represented by large circle; n=94–147 total cells per treatment. C Immunofluorescence image of RPE1 anaphase cell with acentric lagging chromosome; centromeric proteins stained with CREST, DNA damage foci detected by γH2Ax staining. D Segregation error rates in RPE1 anaphase cells after indicated treatments (summary of three to seven experiments; n=105–313 cells, respectively; combined data from immunofluorescent and FISH analysis (see Figure S2f)); statistical test was an unpaired t-test. Each dot indicates mean of individual experiment. Error bars here and in all other figures indicate standard deviation. E Centromeric status of lagging chromosomes in RPE1 cells (as determined by CREST staining) after indicated treatments (n=2, 46, 7, or 37 lagging chromosomes, respectively, taken from at least three experiments). F Percentage of lagging chromosomes in RPE1 anaphase cells with DNA damage detectable on chromosome ends or within chromosome mass (n=0, 35, 12, and 37 lagging chromosomes scored in DMSO, aphidicolin, hydroxyurea, or nocodazole washout treatment, summary of three experiments). G Representative image of RPE1 cell with an acentric micronucleus. CREST antibody was used to stain for presence of centromeric proteins. H Quantification of micronuclei rates in RPE1 cells after indicated treatments (n=658–2150 cells respectively, taken from three to seven experiments) (see also Figure S2f for FISH staining). I Centromere status of micronuclei in RPE1 cells after indicated treatments (n=86–122 micronuclei per condition from three experiments), as determined by CREST staining (see also figure S2f). J Representative images of RPE1 cells treated with specific chromosome FISH probes to identify chromosomal identity of micronuclei. K Quantification of frequency with which indicated chromosomes were detected in micronuclei in RPE1 cells (summary of at least three experiments per chromosome tested, 50–100 micronuclei scored per chromosome per experiment). Statistical test was a one-way ANOVA