Extended Data Fig. 1: Chromosome mis-segregation triggers a p53-p21 response.
a. Number of mitotic CENP-AAID RPE-1 cells from live imaging post-G1 release. b. RPE-1 cells with micronuclei. Each dot: mean of one experiment. n = 3 or 4 (CENP-AAID 48 h) independent replicates with 100 cells/experiment/condition. Ordinary one-way ANOVA: *p = 0.0801; **p = 0.0026; ****p < 0.0001. c. Distribution of the number of micronuclei/cell from (b). n = 2 independent replicates with 40 cells/experiment/condition. d. p53 immunofluorescence in RPE-1 cells. N = 64, 82, 65 and 91 cells for NT, 24hIAA, 2nd G1 arrest and IAA+2nd G1 arrest, respectively, from one experiment. Unpaired two-sided t-test: ∗∗∗p = 0.0003; ∗∗∗∗p < 0.0001. e. p21 immunofluorescence in RPE-1 cells. N = 211, 222, 218, 116, 162 and 120 cells for the treatment groups shown from left to right, from one experiment. Kruskal-Wallis test: ***p = 0.0008; ****p < 0.0001. f. p21 immunofluorescence in RPE-1 cells. N = 40, 46, 58 and 52 cells for the treatment groups shown from left to right, from one experiment. Unpaired two-sided t-test: ns, not significant. g. FACS plots of p21-positive and -negative sorted (Alexa Fluor-647) CENP-AAID RPE-1 cells (IAA: 36 h). h. Chromosome-specific FISH in p21-negative and p21-positive cells. N = 460, 942, 753, 559 cells for chromosomes X, 3, 9 and 6, respectively, from one experiment. i. p21 intensity (from Fig. 1g) in eGFPp21 RPE-1 live-cell imaging (normalized to T = 0 for untreated cells). Coloured lines: eGFPp21 intensity in single CENP-Ei+Mps1i-treated cells; black lines: in untreated cells; grey lines: background. n = 3 independent replicates with 3, 8 and 20 cells for Bkg, NT and CENP-Ei+Mps1i, respectively. j. p21 immunofluorescence in G1 RPE-1 cells. N = 57, 50, 62, 67, 57, and 72 cells for the treatment groups shown from left to right, from one experiment. Kruskal-Wallis test: ***p = 0.0002; ****p < 0.0001. k. Percentages of cells in G1, S and G2/M phases. N = 201, 216, 355 and 235 cells for 0 h, 24 h, 48 h and 72 h IAA respectively, from two independent replicas. Two-sided Fisher’s exact test: ****p < 0.0001. l. Percentages of S-phase cells (EdU positive) of p53 and scramble shRNA in RPE-1 cells. N = 50 cells/condition from one experiment. m. Percentages of BrdU-negative and -positive CENP-AAID RPE-1 cells. n = 3 independent replicates with 10,000 cells. In d–f and j, the center line is the median, the lower and upper bounds are the 25th and 75th percentiles, the whiskers are the furthest observations within 1.5× the interquartile range and the dots inside and outside the whiskers are the mean of one experiment and outlier data points, respectively. In b and m, the error bars represent s.e.m.
