Fig. 6: The NE stress checkpoint is a universal mechanism.
a, Inducible (i)-lamin A-GFP and progerin-GFP microscopy of doxycycline-treated RPE-1 cells. Scale bars, 5 μm. b, Lamin A-GFP and progerin-GFP immunoblot analysis. Vinculin was used as the loading control. Dox, doxycycline. c, Immunoblot quantification normalized to vinculin and the no-Dox condition. n = 3 independent replicates. Statistical significance was determined by unpaired two-sided t-test (*P = 0.0348). d, Colony formation assays after 7 d of doxycycline treatment (50 ng ml−1). e, AFM-measured nuclear stiffness. Doxycycline was administered for 72 h (10 ng ml−1 for lamin A and 50 ng ml−1 for progerin). Cytochalasin D was administered at 200 nM for 15 min. n = 97, 237, 90, 82, 162 and 107 cells for the indicated conditions (left to right in the graph), from three or four (Dox + cytochalasin D) independent replicates. Statistical significance was determined by Kruskal–Wallis test (*P = 0.0468 and 0.0396, from left to right; ****P < 0.0001). f, NE fluctuations following doxycycline treatment for 72 h. n = 311 and 268 cells for lamin A and progerin, respectively, from three independent replicates. Statistical significance was determined by two-sided Mann–Whitney test (****P < 0.0001). g, Workflow of the cell confinement experiment used to obtain the results shown in h and i. h, Percentages of cells with eGFP–FLAG–cGAS-E225A/D227A foci at the nuclear periphery in CENP-AAID RPE-1 cells left untreated or treated with IAA (24-48 h). n = 157 and 139 cells for NT and IAA, respectively, from three independent replicates represented by different symbols. Statistical significance was determined by two-sided Fisher’s exact test (*P = 0.0338). i, Numbers of eGFP–FLAG–cGAS-E225A/D227A foci at the nuclear periphery in CENP-AAID RPE-1 cells left untreated or treated with IAA (48 h). n = 157 and 139 cells for NT and IAA, respectively, from three independent replicates. Each dot represents a cell. Statistical significance was determined by two-sided Mann–Whitney test (****P < 0.0001). j, Crossing time through 4 and 2 μm microchannels (24 h imaging) of untreated or IAA-treated CENP-AAID RPE-1 cells. n = 120, 126, 72, 94, 83, 54, 28 and 47 cells for the indicated conditions (left to right in the graph), from two independent replicates. Statistical significance was determined by Mann–Whitney test (*P = 0.0416 and 0.0115, from left to right). k, Invasion path length through a collagen matrix of CENP-AAID RPE-1 cells. n = 234 and 568 cells for NT and IAA, respectively, from three independent replicates. Statistical significance was determined by two-sided Mann–Whitney test (****P < 0.0001). l, Model for the mechanosensitive NE checkpoint in response to chromosome mis-segregation. In f, j and k, the central lines represent median values, the lower and upper bounds represent 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 c, e and h, the error bars represent s.e.m. Each dot represents the mean of one experiment.
