Extended Data Fig. 4: Restoration of heterochromatin and nuclear shape, but not perturbation of the cytoskeleton, prevent p53 activation.
a. p21 immunofluorescence signal of RPE-1 CENP-AAID cells (methylstat 3 μM). N = 87, 82, 51 and 67 cells for the indicated conditions (left to right in the graph), from one experiment. Kruskal-Wallis test: **p = 0.0016, ****p < 0.0001. b. p53 immunoblot analysis in RPE-1 CENP-AAID cells. Loading control: Vinculin. c. EFC ratio of CENP-A/CAID RPE-1 cells. N = 85, 114, 101, 34, 66 and 59 cells for the treatment groups shown from left to right, from one experiment. Kruskal-Wallis test: *p = 0.0398 and 0.0398. d. H3K9me3 immunofluorescence intensity in RPE-1 CENP-AAID cells (Remodelin: 50 μM). N = 103, 98 and 99 cells for the treatment groups shown from left to right, from one experiment. Kruskal-Wallis test: ****p < 0.0001. e. Nuclear stiffness of RPE-1 CENP-AAID cells (Remodelin: 50 μM, 24 h). N = 19, 25, 24, 24, 23, 22, 22, 27, 26, 23, 22 and 25 cells for the indicated conditions (left to right in the graph), from 3 independent replicates. Kruskal-Wallis test: *p = 0.0264; ***p = 0.0006. f. p21 immunofluorescence intensity in RPE-1 CENP-AAID cells (IAA 72 h). N = 87, 82, 69 and 59 cells for the indicated conditions (left to right in the graph), from one experiment. Kruskal-Wallis test: ****p < 0.0001. g. p21 immunofluorescence intensity of CENP-AAID RPE-1 cells (Blebbistatin: 100 μM; Dihydrocytochalasin B: 4 μM; Nocodazole: 100 ng/ml). N = 56, 52, 51, 60 and 71 cells for the indicated conditions (left to right in the graph), from one experiment. Kruskal-Wallis test. In a and c–g, 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 outside the whiskers are the outlier data points.
