Fig. 3: p53 activation is halted by restoration of heterochromatin and nuclear shape.
a, Schematic of the rationale behind the experiments presented in this figure. b, Immunoblot of p53/p21 in CENP-AAID RPE-1 cells. The loading control was GAPDH. c, Quantification of p53 from immunoblots of CENP-AAID RPE-1 cells treated as indicated for 72 h at 3 μM and normalized to GAPDH and NT (dashed line). n = 9 independent replicates for IAA and n = 6 for methylstat, JIB-04, IAA + methylstat and IAA + JIB-04. Statistical significance was determined by Kruskal–Wallis test (**P = 0.0024; ****P < 0.0001). d, p21 immunofluorescence signals of CENP-AAID (circles) and CENP-CAID RPE-1 cells (squares) treated for 72 h with IAA and/or methylstat/JIB-04 (3 μM). n = 335, 274, 229 and 376 cells for the indicated conditions (left to right in the graph) from four independent replicates. Statistical significance was determined by Kruskal–Wallis test (****P < 0.0001). e, Elliptic Fourier coefficient (EFC) ratios (DAPI) of CENP-AAID (circles) and CENP-CAID RPE-1 cells (squares). Both IAA and remodelin were administered for 24 h at 50 μM. n = 284, 337 and 316 cells for NT, IAA and remodelin + IAA, respectively, from four independent replicates. Statistical significance was determined by Kruskal–Wallis test (**P = 0.0015; ****P < 0.0001). f, H3K9me3 immunofluorescence signal of CENP-AAID RPE-1 cells treated with IAA, remodelin or both for 24 h at 50 μM. n = 260, 233 and 242 cells for NT, IAA and remodelin + IAA, respectively, from three independent replicates depicted with different shapes. Statistical significance was determined by Kruskal–Wallis test (****P < 0.0001). g, Nuclear stiffness of CENP-AAID RPE-1 cells treated with IAA, remodelin or both for 24 h at 50 μM. n = 68, 69, 75 and 70 cells for NT, IAA, remodelin and remodelin + IAA, respectively, from three independent replicates. Statistical significance was determined by Kruskal–Wallis test (**P = 0.0018; ****P < 0.0001). h, Immunoblot showing p53 and p21 signals in CENP-AAID cells. The loading control was vinculin. i, Quantification of p53 signals from immunoblots of CENP-AAID cells, normalized to vinculin and NT (dashed line). n = 4 independent replicates. Statistical significance was determined by Kruskal–Wallis test (*P = 0.0220; **P = 0.0041). j, p21 immunofluorescence nuclear signals in CENP-AAID (circles) and CENP-CAID RPE-1 cells (squares). n = 3 (CENP-AAID) and 4 (CENP-CAID) independent replicates with 591, 553 and 545 cells for NT, IAA and remodelin + IAA, respectively. Statistical significance was determined by one-sample two-sided t-test (**P = 0.0019; ***P = 0.0001). In c–g, i and j, the error bars represent s.e.m. Each dot represents the mean of one experiment.
