Fig. 1: Chromosome mis-segregation triggers a p53–p21 response.
a, Schematic of the scope of the manuscript. A, CENP-A; C, CENP-C; ?, phenomenon sensed by p53/p21. b, Immunofluorescence images of p21 (red) activation and micronucleus formation upon IAA treatment (18 h) of CENP-CAID RPE-1 cells. Nuclei were counterstained with DAPI (grey). Scale bars, 5 μm. NT, not treated. c, Quantification of cells with micronuclei in untreated and IAA-treated CENP-AAID and CENP-CAID RPE-1 cells at the indicated times after release from G1 (palbociclib (Palbo) washout). n = 8 (untreated), 4 (IAA at 15 h) and 8 (IAA at 18 h) independent replicates, with 197, 59 and 159 CENP-AAID cells for these respective groups and 166, 61 and 160 CENP-CAID cells for these respective groups. Each dot represents the mean of one experiment. Statistical significance was determined by ordinary one-way analysis of variance (*P = 0.0229; ****P < 0.0001). d, Quantification of the normalized p21 immunofluorescence intensity in CENP-A/CAID or CENP-Ei+MPS1i RPE-1 cells after cell synchronization in G1, release and treatments performed according to the schematic to the right. Each dot represents the mean of one experiment. n = 6, 10 and 16 independent replicates with 60 cells per experiment for NT, IAA and CENP-Ei + MPS1i, respectively. Statistical significance was determined by one-sample two-sided t-test (**P = 0.0015; ***P = 0.0002). e, Immunoblot analysis with the indicated antibodies of CENP-AAID RPE-1 cells treated with IAA for 72 h where indicated. Two representative experiments are shown (#1 and #2). Vinculin served as the loading control. f, Quantification of IAA-induced aneuploidy relative to chromosomes 3, 6, 9 and X in CENP-AAID RPE-1 cells sorted by p21 negativity versus positivity, assessed by FISH in interphase. n = 150 nuclei counted per condition from one experiment. Statistical significance was determined by two-sided Fisher’s exact test (****P < 0.0001). g, Quantification of p21 signal during live-cell imaging of synchronized eGFP–p21 RPE-1 cells treated as indicated. T0 denotes telophase exit. The signals are normalized to T0 of the untreated group. n = 3 (background), 8 (NT) and 20 (CENP-Ei + MPS1i) cells from three independent replicates. Statistical significance was determined by Kruskal–Wallis test (****P < 0.0001), calculated at the last time point. h, Percentages of BrdU-negative and -positive CENP-AAID RPE-1 cells after cell cycle synchronization, release from G1 and treatments performed according to the schematic to the right. CENP-Ei and MPS1i were used at 0.5 and 1.0 μM, respectively. n = 2 independent replicates with 10,000 cells. In c, d, g and h, the error bars represent s.e.m.
