Fig. 2: Altered NE and chromatin architecture in chromosome mis-segregated cells.
a, Electron microscopy of untreated and IAA-treated (24 h) RPE-1 cells Scale bar, 5 μm. b, Lamin immunofluorescence of untreated and IAA-treated (24 h) CENP-AAID RPE-1 cells. Scale bar, 5 μm. c, Quantification of nuclei deformation (solidity). n = 23, 11, 14 and 7 independent replicates with 60 cells per experiment per condition for NT, CENP-AAID, CENP-CAID and CENP-Ei + MPS1i, respectively. Statistical significance was determined by Kruskal–Wallis test (****P < 0.0001). d, Imaging of IAA-treated RPE-1 cells from metaphase until G1. The arrows denote misaligned chromosomes causing nuclear deformations. The values in the bottom right corners represent the time (in min) since the beginning of the recording (metaphase). Scale bar, 5 μm. e, Nuclear solidity (arbitrary units) during CENP-AAID RPE-1 live-cell imaging in untreated cells or upon IAA treatment. n = 25 cells per condition. Statistical significance was determined by unpaired two-sided t-test (****P < 0.0001), calculated at the last time point. f, Left, lamin immunofluorescence of G1 RPE-1 cells. Right, lamin intensity variation in untreated versus IAA-treated (24 h post-palbociclib release) RPE-1 cells. n = 6 and 9 independent replicates with 570 and 627 cells for NT and IAA, respectively. Statistical significance was determined by unpaired two-sided t-test (**P = 0.0014; ***P = 0.0004). Scale bar, 5 μm. g, SIM of lamins in untreated versus IAA-treated (24 h) RPE-1 cells. Arrows denote irregular lamin patches and invaginations. Scale bar, 5 μm. h, Left, H3K9me3 and p21 immunofluorescence. The arrows denote cells with reduced H3K9me3 and increased p21. Right, relative H3K9me3, HP1α and H3K27me3 signals in untreated versus IAA-treated (24 h post-palbociclib release) RPE-1 cells. n = 3 (three NT groups), 7 (HP1α; IAA), 15 (H3K9me3; IAA) and 8 (H3K27me3; IAA) independent replicates with 50 cells per experiment per condition. Statistical significance was determined by unpaired two-sided t-test (*P = 0.02; **P = 0.002). Scale bar, 5 μm. i, Lamin B1 ChIP-qPCR of lamin-associated domains in IAA-treated (24 h post-G1 release) RPE-1 cells. Immunoglobulin G (IgG) was used as the isotype control. Enrichment was computed as the percentage of input normalized to the NT group. Each dot represents enrichment relative to a discrete LAD locus (7). n = 3 independent replicates. Statistical significance was determined by one-sample two-sided t-test (***P = 0.0002). j, Nuclear stiffness normalized to the NT or dimethyl sulfoxide (DMSO) treatment groups. n = 3 independent replicates with 235, 157, 173, 80 and 96 cells for NT, CENP-A + IAA, CENP-C + IAA, DMSO and CENP-Ei, respectively. Statistical significance was determined by Kruskal–Wallis test (****P < 0.0001). k, Nuclear stiffness post-G1 release (18 h). n = 107 and 100 cells for NT and IAA, respectively, from three independent replicates. Statistical significance was determined by unpaired two-sided t-test (****P < 0.0001). In c, e, f and h–k, the error bars represent s.e.m. In c, f, h, j and k, each dot represents the mean of one experiment.
