Tumorigenic cells are sensitized to E4orf4’s cytotoxicity. (A) The clonogenic activity of MCF10A cells and MDA-MB-231 cells transduced with varying PFUs/cell of Ad-Flag-E4orf4. The scatterplot represents the percentage of surviving cells relative to the total number of colonies obtained in cells transduced with GFP as control (means of three technical replicates for independent biological samples). Representative IB of Flag-E4orf4 levels analyzed 48 h after transduction. (B) Graphs showing proliferation and cell death rates of nontumorigenic cell lines (HaCaT and RPE-1) compared with tumorigenic cell lines (right) at various times after transduction of Ad-E4orf4-mCherry (75 PFUs/cell) or Ad-RFP (15 PFUs/cell), as measured by Trypan blue exclusion; measurements of cell proliferation consider only viable cells, while cell death rates represent the percentage of Trypan blue–stained cells relative to the total numbers of cells. Data are the means ± SEM (n ≥ 3); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (C) Representative IB of E4orf4-mCherry levels assessed 48 h after transduction. (D) The percentage of cells expressing E4orf4-mCherry relative to the total number of infected cells, as assessed by fluorescence microscopy 48 h after transduction. See also Fig. S1 A.