Large-scale HCS-based quantification of simultaneous induction of entosis and apoptosis upon TRAIL and TRAIL + CHX stimulation. (A–D) Overview of steps involved in HCS-based analysis of entotic structures. After seeding (7,500 cells/well), staining (Hoechst, LysoTracker, PI) and treatments (A), 36 random fields of view per treatment per experiment were recorded for 72 h (B). CellProfiler defined cell nuclei, lysosomes, and PI-positive cells, then overlaid images were used to detect entosis events (C). Entosis events detected by CellProfiler were manually verified using overlaid images of brightfield, Hoechst (blue), LysoTracker (magenta), PI (cyan), and Venus (yellow) by analyzing Hoechst-stained and PI-negative inner cells within another Hoechst-stained and PI-negative cell showing a crescent-shaped nuclear morphology (D). Both inner cells that do (arrows) and do not (dashed arrows) show LysoTracker accumulation were quantified as entotic. Scale bars: 25 μm. (E and F) TRAIL, but not TRAIL + CHX, promotes entosis in HCT116 cells. Quantification of PI-positive cells (E) and entotic structures (F) in HCT116 cells treated with or without CHX, TRAIL, and TRAIL + CHX for 72 h. Data are shown as individual values for each experiment as well as mean ± SEM (n = 3). Asterisks on top of individual bars indicate comparisons with control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****P < 0.0001, by one-way ANOVA followed by Tukey’s multiple comparison test.