Extended Data Fig. 3: NE alterations are triggered by chromosome mis-segregation.
a. Solidity, circularity and EFC ratio of palbociclib-released RPE-1 cells. N = 85, 114, 98, 96, 67, 61, 34, 66 cells for the treatment groups shown from left to right. Unpaired two-sided t-test: ****p < 0.0001. b. Nuclear solidity of CENP-AAID RPE-1 cells. Each dot: one nucleus. N = 66, 53, 156, 139 cells for the treatment groups shown from left to right, from one experiment. Unpaired two-sided t-test ****p < 0.0001. c. Solidity and p21 immunofluorescence of CENP-AAID RPE-1 cells. n = 4 independent replicates with N = 60 cells/experiment/condition. d. RPE-1 cell immunoblot (IAA: 72 h). Loading control: Vinculin. e. Coefficient of variation of Lamins (IAA 24 h). N = 105, 113, 85 and 96 cells for the treatment groups shown from left to right, from one experiment. Unpaired two-sided t-test: ***p = 0.0008; ****p < 0.0001. f. H3K9me3 immunofluorescence of RPE-1 cells. N = 64, 100, 62, 59, 43, 52, 50, 83, 63, 42, 64 and 101 cells for the treatment groups shown from left to right. Unpaired two-sided t-test: **p = 0.0028, ***p = 0.0007; ****p < 0.0001. g. H3K9me3 immunofluorescence of U-2 OS cells. Each dot: mean of one experiment. n = 7 independent replicates with 469 and 486 cells for −/+IAA, respectively. One sample two-sided t-test: *p = 0.0273. h. Left, TEM of CENP-CAID RPE-1 cells. Right, heterochromatin thickness. Each dot: one nucleus (N = 29 and 33 cells for −/+IAA, respectively, from one experiment). Scale bar: 2 μm. Unpaired two-sided t-test: **p = 0.0012. i. Nuclear stiffness of CENP-A/CAID RPE-1 cells. N = 31, 41, 36 and 51 cells for the treatment groups shown from left to right, from one experiment. Unpaired two-sided t-test: ****p < 0.0001. j. Force-distance curve of an indentation experiment (Cytochalasin D: 200 nM, 15 min). k. RPE-1 cell nuclear stiffness (Cytochalasin D: 200 nM, 15 min). N = 54, 56, 53 and 64 cells for the treatment groups shown from left to right for 3 independent replicates. Unpaired two-sided t-test. l. Left, actin (phalloidin) and microtubule (α-tubulin) imaging in G1 CENP-AAID RPE-1 cells (24 h post-release). Right, average areas of actin stress fibers (SFs) and microtubules (MT) per cell (N = 10 cells/field of view, each dot is one field of view of one experiment). Scale bars: 25 μm. In a, e, f, i and k, the center lines are medians, the lower and upper bounds are 25th and 75th percentiles, the whiskers are the furthest observations within 1.5× the interquartile range and the dots outside the whiskers are outlier data points. In b, c, g, h and l, the error bars are s.e.m.
