Representative images and manual quantification of entotic structures in HCT116, MCF-7, and LS180 cells as well as HCT116 spheroids with or without TRAIL treatment. (A) Representative confocal microscopy images of Hoechst (blue) and β-catenin (red) staining and quantification of entotic structures in HCT116 cells treated with or without TRAIL in the absence or presence of z-VAD-fmk or Y-27632. More than 500 cells were manually quantified from at least two experiments. White and yellow arrowheads indicate inner and outer cells, respectively. Scale bar: 20 μm. (B) Quantification of entotic events in MCF-7 and LS180 cells treated with or without TRAIL. Representative confocal microscopy images of DIC, Hoechst (blue), and β-catenin (red) staining in MCF-7 cells treated with control or TRAIL are shown. Yellow arrows indicate entosis events. More than 500 cells were quantified from at least two experiments. Scale bar: 20 μm. (C) Quantification of entotic events in HCT116 spheroids treated with control or TRAIL (left).3D projections of light sheet fluorescence imaging of Venus (green), Hoechst (blue), and LysoTracker (red) in control and TRAIL-treated HCT116 spheroids. Three spheroids per treatment were quantified. Yellow arrows indicate LysoTracker-positive inner cells. Scale bar: 100 μm. Data are shown as mean ± SD. **, P < 0.01; ****, P < 0.0001 by unpaired two-tailed t test.