Figure 3

A high content siRNA screen accurately identifies factors influencing nuclear integrity and morphology. (a) Volcano plot of siRNA pools that significantly altered nuclear rupture frequency determined by proportion cells with RFP-Cyto Nuc:Cyto ratio over threshold compared to control wells. Labels = p < 0.05 by Bonferroni adjusted Barnards test. (b) Analysis of nucleus rupture frequency in fixed cells after single siRNA transfection against indicated target. *p < 0.05, Barnards test. Bar color indicates effect direction in screen. N = 4. (c) Volcano plot of siRNA pools that significantly altered rupture kinetics determined by proportion of RFP-Cyto positive nuclei where GFP-Nuc Cyto:Nuc ratio was also over threshold compared to control wells. Only conditions where >  = 175 RFP + nuclei were present in each replicate were analyzed. BANF1 is a positive control. Labels indicate control siRNAs and targets that increased GFP-Nuc mislocalization (p < 0.05, Barnards test). N = 4. (d) Validation of GFP-Nuc screen hits in fixed cells transfected with single siRNAs against indicated target. 1/1 hit from screen significantly increased GFP-Nuc mislocalization to cytoplasm. * = p < 0.05, Barnards test. N = 3–4. (e) Analysis of nuclear area from conditions in (a) with largest median difference from control median. (f) Analysis of nucleus solidity from conditions in (a) with largest median difference from control median. g. Median nucleus area did not correlate with increased rupture frequency across conditions tested in (a) (Pearson). (e–g): *p < 0.05, Wilcoxon test, N = 4. Cells: U2OS RuptR, Stats: Tables S3–S8.