Binding to Par3 regulates E4orf4 toxicity in cancer cells. (A) Sequence alignments of the Par3-binding motif on E4orf4 in type 2 Ad and other serotypes (in magenta); conserved residues are highlighted in blue, and residue substitutions to generate the E4orf4 mutants are indicated in green. EV, empty vector. (B) Flag-E4orf4 IPs prepared from 293T cells transfected with Flag-E4orf4 constructs and HA-PP2A-Ba, followed by IB with the indicated antibodies; Input: total cell extracts; arrows: Par3 isoforms; n = 3. (C) Merged plane views from confocal image stacks of representative HeLa cells expressing E4orf4 (WT or GGG), showing PLA signal and HA-E4orf4 staining detected with anti-mouse-Alexa antibody; actin was stained using phalloidin. Bar: 20 µm. (D) Quantification of the number of PLA foci per cell; means ± SEM (n ≥ 58 cells; n = 3). (E) Quantification of the N/C ratio of E4orf4-mCherry for datasets represented in Fig. S3 C; means (n ≥ 51 cells; n = 2). (F) The percentage of cell blebbing and nuclear condensation was scored relative to U2OS or HeLa cells expressing RFP or E4orf4 (WT or GGG; means ± SEM [n ≥ 3]). (G) Quantification of the nuclear circularity index of cells fixed at 24–36 h after transduction, before and after blebbistatin treatment (50 µM, 2 h), for the datasets represented in Fig. S3 C (means ± SEM [n ≥ 67 cells; n = 3]). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. See also Figs. S2 and S3 C.