Figure S1.
Long-term time-lapse microscopy verifies induction of entotic structures by TRAIL treatment.(A) Representative time-lapse images of Hoechst and LysoTracker staining in HCT116 cells showing formation of entotic structures in TRAIL treatment. Representative entosis events of an inner cell undergoing entotic cell death (1) and an inner cell being released (2) are highlighted. Formation and stages of depicted entosis events are shown. Inner cells and outer cells are highlighted with white arrows. (B) Quantification of relative cell movement of inner and outer cells in TRAIL-treated HCT116 cells. Thirteen entotic structures per group from three experiments were monitored by time-lapse microscopy. Data are shown as individual values for each cell as well as median and quartiles. ****, P < 0.0001 by unpaired two-tailed t test. (C) Representative time-lapse images of DIC, Venus, LysoTracker, and TMRM showing an inner cell being released (left) and inner cell undergoing entotic cell death (right). Accumulation of TMRM and LysoTracker coincides with the loss of Venus signal in the entotic inner cell (right). Solid and dashed lines indicate inner and outer cells, respectively. Scale bar: 20 μm. (D) Representative time-lapse images of DIC, Hoechst, TMRM, and LysoTracker showing the effects of FCCP treatment on inner cell TMRM signal during entotic cell death. Quantification of TMRM mean fluorescence intensity in the corresponding inner, outer, and neighboring cells. Yellow arrows indicate inner cell. Scale bar: 20 μm. (E) Representative 3D confocal microscopy images of Hoechst (blue) and cleaved caspase-3 (Cl-Casp3; red) staining showing Cl-Casp3–positive and Cl-Casp3–negative inner cells in control and TRAIL-treated cells. Quantification of inner cell Cl-Casp3 status in control and TRAIL-treated cells. White and black arrowheads indicate inner and outer cells, respectively. Scale bar: 20 μm. Data are shown as mean ± SEM from three experiments. *, P < 0.05 by unpaired two-tailed t test. IC, inner cell; Norm., normalized; OC, outer cell.

Long-term time-lapse microscopy verifies induction of entotic structures by TRAIL treatment. (A) Representative time-lapse images of Hoechst and LysoTracker staining in HCT116 cells showing formation of entotic structures in TRAIL treatment. Representative entosis events of an inner cell undergoing entotic cell death (1) and an inner cell being released (2) are highlighted. Formation and stages of depicted entosis events are shown. Inner cells and outer cells are highlighted with white arrows. (B) Quantification of relative cell movement of inner and outer cells in TRAIL-treated HCT116 cells. Thirteen entotic structures per group from three experiments were monitored by time-lapse microscopy. Data are shown as individual values for each cell as well as median and quartiles. ****, P < 0.0001 by unpaired two-tailed t test. (C) Representative time-lapse images of DIC, Venus, LysoTracker, and TMRM showing an inner cell being released (left) and inner cell undergoing entotic cell death (right). Accumulation of TMRM and LysoTracker coincides with the loss of Venus signal in the entotic inner cell (right). Solid and dashed lines indicate inner and outer cells, respectively. Scale bar: 20 μm. (D) Representative time-lapse images of DIC, Hoechst, TMRM, and LysoTracker showing the effects of FCCP treatment on inner cell TMRM signal during entotic cell death. Quantification of TMRM mean fluorescence intensity in the corresponding inner, outer, and neighboring cells. Yellow arrows indicate inner cell. Scale bar: 20 μm. (E) Representative 3D confocal microscopy images of Hoechst (blue) and cleaved caspase-3 (Cl-Casp3; red) staining showing Cl-Casp3–positive and Cl-Casp3–negative inner cells in control and TRAIL-treated cells. Quantification of inner cell Cl-Casp3 status in control and TRAIL-treated cells. White and black arrowheads indicate inner and outer cells, respectively. Scale bar: 20 μm. Data are shown as mean ± SEM from three experiments. *, P < 0.05 by unpaired two-tailed t test. IC, inner cell; Norm., normalized; OC, outer cell.

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