p53 promotes genomic stability in E1A;HrasG12V;H11Cas9 MEFs. (A) Representative metaphase spreads from sgNTC and sgp53(2) E1A;HrasG12V MEFs. Scale bar, 25 μm. (B) Quantitation of chromosomes per cell from metaphase spreads. n = 3 cell lines/sgRNA. >30 cells analyzed per cell line. Data are presented as mean ± SD; *, P < 0.05, nested t test. (C) Percentage of E1A;HrasG12V MEFs with >40 chromosomes. Data are mean ± SD, n = 3 cell lines/sgRNA. >30 cells analyzed per cell line. *, P < 0.05, Mann–Whitney unpaired t test. (D) Generation of FUCCI cell cycle marker–expressing variants of E1A;HrasG12V MEFs. (E) Quantitation of G1 tetraploid and polyploid populations in sgRNA-targeted E1A;HrasG12V MEFs. Data represent the mean ± SD of n = 3 cell lines/sgRNA. *, P < 0.05, two-way ANOVA, Dunnett’s multiple comparison test. (F) Montage of normal and abnormal mitotic events in sgp53(2)-targeted E1A;HrasG12V MEFs from time-lapse imaging. Cells of interest are indicated with black arrows. Scale bar, 50 μm. (G) Quantitation of abnormal mitotic events in E1A;HrasG12V MEFs. n = 3 cell lines/sgRNA, with >50 mitotic events analyzed per cell line. Data are mean ± SD; *, P < 0.05, one-way ANOVA, Bonferroni’s multiple comparison posttest. (H) Nucleus number in E1A;HrasG12V MEFs. Data are mean ± SD of n = 3 cell lines/sgRNA. *, P < 0.05; **, P < 0.005, two-way ANOVA, Tukey’s multiple comparisons test. (I) Representative image of a multinucleated sgp53(2)-targeted E1A;HrasG12V MEF. WGA marks cell membrane, and DAPI marks nuclei. Scale bar, 25 μm. (J) Representative BrdU proliferation analysis of multinucleated sgNTC and sgp53(2) E1A;HrasG12V MEFs. White lines indicate outlines of multinucleated cells. White arrows indicate BrdU+ multinucleated cells. Scale bar, 25 μm. (K) Quantitation of BrdU-positive multinucleated sgRNA-targeted E1A;HrasG12V MEFs. Data are mean ± SD, n = 3 cell lines/sgRNA; *, P < 0.05, two-way ANOVA, Dunnett’s multiple comparison test. For A–K, all experiments were performed in physiological (5%) oxygen.
