E4orf4 nuclear exit by nuclear bleb formation is inhibited by depletion of Par3 or SUN1/2. (A) Schematics of the protocols. (B) Quantification of the N/C ratio of E4orf4-mRFP in HeLa cells treated with blebbistatin 50 µM for 2 h and released for 1 h (means ± SEM [n ≥ 135 cells; n ≥ 3]). (C) The percentage of HeLa cells undergoing nuclear bleb (NB) formation (means ± SEM [n = 3]). (D) Video stills taken from live-cell imaging of siRNA-treated HeLa cells transfected with E4orf4-mRFP; filled arrowheads, NB formation; empty arrowheads, NB rupture in siCTL-cells (white, empty), as emphasized by a heatmap applied on the enlarged views, or NB resorption in siPar3-cells (green-empty). Bar: 20 µm. See also Video 1. (E and F) The percentage of HeLa cells showing at least 1 NB or more than 3 NB, and the percentage of resorbing NB over a 3 h-period, as scored relative to E4orf4-mRFP-expressing cells; means +/− SEM (n ≥ 207 cells, ≥ 82 blebs; n ≥ 3). (G and H) Video stills taken from live-cell imaging of E4orf4-mCherry-expressing MDA-MB-468 and U2OS cells; filled arrowheads, NB formation; empty arrowheads, NB rupture and E4orf4 nuclear exit as emphasized by applying a heatmap pseudo-color intensity scale. Bar: 20 µm. (H) The number of NBs per cell from one representative live-cell imaging of 12 h (MDA-MB-468 cells) or 4 h (U2OS cells; means ± 95% CI [n ≥ 25 cells]). See also Videos 3 and 4. (I) The percentage of shRNA-treated U2OS cells expressing GFP-NLS or E4orf4-mRFP that showed more than three NBs (means ± SEM [n ≥ 92 cells; n = 3]). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.