Deletion of Bax and Bak or inhibition of caspase activation alters inner cell fate toward release. (A) Schematic representation of inner cell fates and representative time-lapse images showing that the inner cell undergoes entotic cell death, the inner cell is released, and the inner cell completes cell division. Inner and outer cells are indicated as orange and white dashed lines, respectively. Scale bar: 10 μm. (B) Western blot images of Bax and Bak expressions in HCT116 WT and Bax^−/−Bak^−/− cells. β-Actin was used as loading control. (C) Quantification of inner cell fates in HCT116 WT and Bax^−/−Bak^−/− cells treated with or without TRAIL in the absence or presence of z-VAD-fmk. Data are shown as individual values for each experiment as well as mean ± SEM. n is the total number of cells analyzed over 48 h from three experiments. Asterisks inside individual bars indicate comparisons with control. ****, P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test. (D) Representative 3D confocal microscopy images of DIC, Hoechst (blue), LysoTracker, and fluorogenic cathepsin B substrate of late-stage entotic cells in WT and Bax^−/−Bak^−/− cells treated with or without TRAIL in the presence or absence of z-VAD-fmk. 3D colocalization of LysoTracker and cathepsin B substrate is visualized. Yellow arrows indicate inner cells. Scale bars: 10 μm. (E) Quantification of cathepsin B activity in late-stage entotic inner cells in WT and Bax^−/−Bak^−/− cells treated with or without TRAIL in the presence or absence of z-VAD-fmk. Data are shown as individual values for each cell as well as median and quartiles. Statistical significance was tested using unpaired two-tailed t test. n is the total number of cells analyzed. CatB, cathepsin B; IC, inner cell; MFI, mean fluorescence intensity; OC, outer cell.