The tumor cell–selective toxicity of E4orf4 maps to nuclear shape changes regulated by actomyosin contractility. (A) Deconvolved maximum intensity projections of confocal image stacks of representative RFP- or E4orf4-mCherry-expressing cells showing tumor cell–selective morphological phenotypes associated with cell death (e.g., cell blebbing, F-actin staining in green; nuclear condensation, Hoechst staining in magenta). Bars: 10 µm. The percentage of cell blebbing and nuclear condensation was scored relative to cells expressing RFP/E4orf4-mCherry 24–36 h after transduction, before and after blebbistatin treatment (50 µM, 2 h; means [n > 300 cells; n = 2] or means ± SEM [n > 350 cells; n ≥ 3]). See also Fig. S1, B and C. (B) Quantification of the nucleus/cytoplasmic ratio of E4orf4-mCherry, before or after treatment with blebbistatin 2 h (MCF10A: 75 µM; MDA-MB-468: 50 µM; means [n = 2] or means ± SEM [n = 3]). (C) Maximum intensity projections of confocal image stacks showing F-actin clusters (green) proximal to the nucleus (magenta) of a MDA-MB-468-E4orf4 cell, as highlighted on the x-z and y-z views (dotted lines). Bars: 20 µm. (D) Quantification of the nuclear circularity index (C.I.) for datasets acquired as in A (means ± SEM [n = 47, 17, 53, and 36 cells, respectively, from n = 3, 2, 3, and 3 experiments, respectively]). Maximum intensity projections of RFP- or E4orf4-expessing U2OS cells, showing an early decrease of the nuclear circularity index that matches the presence of lamina folds (lamin A/C staining; magenta arrows) and apical actin filaments at sites where E4orf4 signal is segregated. Bars: 10 µm. *, P < 0.05; ****, P < 0.0001; ns, not significant.