Extended Data Fig. 6: AKT and ATR are activated upon nuclear envelope stress.
a. Left, phospho-AKT (Ser473) immunofluorescence signal in CENP-AAID RPE-1 cells. Scale bars: 5 μm. Right, quantification of phospho-AKT in CENP-A/CAID RPE-1 cells. Positive control: hypotonic buffer. N = 1,913, 1,484 and 1,651 cells for the treatment groups shown from left to right, from 4 independent replicates. Kruskal-Wallis test: ****p < 0.0001. b. Immunoblot showing mTOR activation. Loading control: Vinculin. c. p53/p21 immunoblot with AKTi. Repeated 5 times with similar results. Loading control: Vinculin. d. ATR immunofluorescence signal in CENP-AAID RPE-1 cells. Scale bar: 5 μm. e. Left, p53 immunoblot of CENP-AAID RPE-1 cells (ATRi: 10 μM). Loading control: Vinculin. Right, quantification of p53 levels, normalized to Vinculin and untreated (dashed line). n = 6 independent replicates. Unpaired two-sided t-test *p = 0.0229. f. p53 phosphorylation immunoblot. Loading control: Vinculin. Positive control: Etoposide 20 μM. Repeated 2 times with similar results. g. p53 phosphorylation immunoblot (72 h; INK-128: 200 nM, MK-2206: 5 μM, Ipatasertib: 5 μM). Loading control: H3. Positive control: Etoposide 20 μM. Repeated 3 times with similar results. h. ATR nuclear intensity in RPE-1 cells. N = 43, 37, 44, 46 and 47 cells for the indicated conditions (left to right in the graph), from one experiment. Kruskal-Wallis test: **p = 0.0068; ****p < 0.0001. i. Left, AKT phosphorylation immunoblot. Loading control: Vinculin. Right, quantification of AKT phosphorylation normalized to Vinculin and untreated (dashed line). n = 3 independent replicates. Kruskal-Wallis test: *p = 0.0459. j. p21 immunofluorescence intensity (INK-128: 200 nM, JIB-04: 1.5 μM, Remodelin: 50 μM). n = 8, 7, 5, 6 and 4 independent replicates with 616, 684, 451, 287 and 285 cells for the indicated conditions (left to right in the graph). One-sample two-sided t-test: *p = 0.0170; ***p = 0.0001; ****p < 0.0001. k. Left, p53/p21 immunoblot in BJ-hTERT1 (MPS1i 0.5 μM + CENP-Ei 0.2 μM; ATRi VE-821). Loading control: Vinculin. Right, quantification of p53 levels normalized to Vinculin and DMSO. N = 6 (DMSO/Mps1i+CENP-Ei) and 3 (all other conditions) independent replicates. One sample two-sided t test: *p = 0.0112; **p = 0.0058; ****p < 0.0001. In a and h, the center lines are the medians, the lower and upper bounds are the 25th and 75th percentiles, the whiskers are the furthest observations within 1.5× the interquartile range and the dots outside the whiskers are the outlier data points. In e, i, j and k, the error bars are s.e.m. In e and i, each dot represents one experiment. In j and k, each dot is the mean of one experiment.
