Fig. 4: Alteration of nuclear membrane mechanics triggers p53/p21 activation in response to chromosome mis-segregation.
a, Schematic of the rationale behind the experiments presented in this figure. b,c, Distribution of fluorescence lifetimes with a Gaussian fit, measured using the ER Flipper-TR membrane tension probe, in CENP-CAID (b) and CENP-AAID RPE-1 cells (c) treated for 24 h with IAA. Sucrose treatment (1 M) served as a control. n = 3 independent replicates with 157, 152, 109, 169, 158 and 56 cells for NT, IAA and sucrose in CENP-AAID cells and NT, IAA and sucrose in CENP-CAID cells, respectively. Statistical significance was determined by Kruskal–Wallis test (***P = 0.0038; ****P < 0.0001). d, NE fluctuations measured in RPE-1 cells expressing LAP2β-GFP. n = 93, 91, 149 and 169 cells for NT and IAA in CENP-AAID cells and NT and IAA in CENP-CAID cells, respectively, from three independent replicates depicted with different shapes. Statistical significance was determined by Kruskal–Wallis test (***P = 0.0002; ****P < 0.0001). e, Representative images (left) and quantification (right) of p21 activation (eGFP–p21) in compressed RPE-1 cells (3 h). Each dot represents the mean of one experiment. n = 1,221 and 2,290 cells for 10 and 3 μm, respectively, from three independent replicates depicted with different shapes. Statistical significance was determined by unpaired two-sided t-test (*P = 0.0354). Scale bars, 30 μm. f, p21 intensity (eGFP–p21) in RPE-1 cells in suspension following compression. n = 82, 90, 98, 160, 90, 147, 63, 133, 79 and 90 cells for the conditions depicted from left to right, from two independent replicates. Etoposide (50 μM) was used as a positive control. g, Live p21 intensity (eGFP–p21) in RPE-1 cells in hypotonic versus isotonic medium. Continuous lines denote the average values of 28 (NT) and 35 (hypotonic buffer-treated) cells from n = 3 independent replicates. The horizontal dashed line marks the basal signal intensity to which the data have been normalized (T0). The vertical dashed line marks the time of hypotonic buffer addition. Statistical significance was determined by unpaired two-sided t-test (****P < 0.0001), calculated at the last time point. h, Left, cPLA2 immunofluorescence in CENP-CAID RPE-1 cells treated or not with IAA. Scale bars, 5 μm. Right, cPLA2 nuclear signal in CENP-AAID (circles) and CENP-CAID cells (squares). Hypotonic medium was used as the positive control. n = 627, 628, 650, 1,065 and 653 cells for 0, 18, 22 and 48 h IAA and hypotonic buffer, respectively, from four (48 h release) or six independent replicates. Statistical significance was determined by Kruskal–Wallis test (****P < 0.0001). In d, f and h, the central lines represent median values, the lower and upper bounds represent the 25th and 75th percentiles, the whiskers represent the furthest observations within 1.5× the interquartile range and the dots inside and outside the whiskers represent the means of one experiment and outlier data points, respectively. In e and g, the error bars represent s.e.m. NS, not significant.
