E4orf4-induced NE rupture is regulated by Par3 and SUN1/2. (A and C) Video stills taken from live-cell imaging of a representative HeLa cell (A) or U2OS cells (C) expressing E4orf4-mCherry and GFP-NLS. NB growing and transient NE rupture associated with cytoplasmic GFP-NLS signal are designated by filled and empty arrowheads, respectively; the green asterisks in C point to a cell displaying a prolonged loss of nuclear integrity associated with sustained E4orf4 cytoplasmic signal and cell blebbing. Bars: 10 µm. See also Videos 5 and 6. (B) The percentage of HeLa cells showing transient NE rupture or loss of NE integrity (no repair over >30 min), as scored relative to GFP-NLS-expressing cells (means ± SEM [n ≥ 95 cells; n = 4]). (D) Representative IB showing SUN1/2 depletion in U2OS cells using mixtures of two sequences targeting SUN1 or SUN2 (shSUN), as estimated by loading increasing amounts of control cell extracts (shCTL or siCTL, 1, 1/2, and 1/4); GAPDH levels, loading control. (E) The percentage of shRNA-treated U2OS cells expressing RFP or E4orf4-mCherry together with GFP-NLS as a marker of NE rupture, which showed transient NE rupture or loss of NE integrity (no repair over >30 min), as scored relative to GFP-NLS-expressing cells; data represent means (n ≥ 92 cells; n = 2) or means ± SEM (n ≥ 85 cells; n = 3). (F) Quantification of the nuclear circularity index for datasets represented in Fig. S4 C (means ± SEM [n ≥ 27 cells; n = 4, 2, 4, 4, 2, and 4, respectively]). (G) The percentage of U2OS cells showing cell blebbing and nuclear condensation, as scored relative to RFP- or E4orf4-mCherry-expressing cells for datasets represented in Fig. S4, B and C (means [n ≥ 200 cells, n = 2] or means ± SEM [n ≥ 363 cells; n = 3]). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not signifciant.