Extended Data Fig. 2: Chromosome mis-segregation triggers p53/p21 response independent of DNA damage, replication stress and extended mitotic duration.
a. γH2A.X immunofluorescence intensity in RPE-1 cells. N = 75, 65, 94 and 105 cells for the treatment groups shown from left to right, from one experiment. Unpaired two-sided t-test. b. Number of 53BP1 foci/nucleus in RPE-1 cells. N = 211, 222, 136 and 162 cells for the treatment groups shown from left to right, from one experiment. Unpaired two-sided t-test. c. p21 immunofluorescence intensity in RPE-1 cells. N = 56, 52 and 61 cells for NT, IAA and IAA+ATMi, respectively, from one experiment. Kruskal-Wallis test: *p = 0.0207. d. Immunoblot analysis of RPE-1 cells. IAA: 72 h. Loading control: Vinculin. e. Immunoblot analysis in RPE-1 cells for ATMi efficacy. Positive control: Etoposide. Loading control: Vinculin. f. p21 immunofluorescence intensity in micronuclei-negative and -positive RPE-1 cells treated with IAA (24 h). N = 73, 47, 73 and 47 cells forthe treatment groups shown from left to right, from one experiment. Unpaired two-sided t-test. g. p21 immunofluorescence intensity in RPE-1 cells, normalized to the untreated group. n = 3, 3, 3, 4, 7 and 4 independent replicates with 30 cells/experiment/condition for the treatment groups shown from left to right. Wilcoxon t-test: **p = 0.0060, 0.0021, 0.0011 and 0.0062 from left to right; ****p < 0.0001. h. Immunoblot analysis of RPE-1 cells. Loading control: Vinculin. i. p21 immunofluorescence intensity in CENP-AAID (circle) or CENP-CAID (square) RPE-1 cells (MPSi: 24 h MPI-0479605 or Reversine). n = 3, 7, 7, 7 and 12 independent replicates with 79, 91, 74, 68 and 63 cells/experiment for NT, IAA, IAA + 0.2 μM Mps1i, IAA + 0.5 μM Mps1i and 0.5 μM Mps1i, respectively. Kruskal-Wallis test: **p = 0.0051. j. p21 immunofluorescence intensity. N = 70 and 47 cells for −/+ IAA respectively, from one experiment. Unpaired two-sided t-test: ****p < 0.0001. k. Immunoblot analysis. Positive control: Etoposide. Loading control: Vinculin. l. 53BP1 immunofluorescence intensity. N = 70 and 46 cells for −/+ IAA respectively, from one experiment. Unpaired two-sided t-test. In a–c, f, j and l, the center lines are median values, the lower and upper bounds are 25th and 75th percentiles, the whiskers are the furthest observations within 1.5× the interquartile range and the dots outside the whiskers are the outlier data points. In g and i, the error bars represent s.e.m. Each dot is the mean of one experiment.
