Figure 4.
Entosis induced by TRAIL requires death receptors and structural presence of caspase-8. (A–C) Internalization process during entosis is independent of caspase activity. (A) Inhibition of entosis does not affect the rate of TRAIL-induced apoptosis. Quantification of PI-positive cells in HCT116 cells treated with or without TRAIL in the absence or presence of z-VAD-fmk, z-IETD-fmk, or Y-27632. (B and C) Inhibition of caspase activity does not affect the rate of TRAIL-induced entosis. (B) Quantification of entotic structures in HCT116 cells treated with or without TRAIL in the absence or presence of z-VAD-fmk, z-IETD-fmk, or Y-27632. (C) Representative field of view from HCS-based entosis analysis is shown. Arrows indicate entotic events, four representative events are depicted and shown. Scale bar: 50 μm. (D–H) TRAIL death receptors are required for cell internalization during TRAIL-induced entosis. (D) Quantification of PI-positive cells in HCT116 WT and DR4−/− DR5−/− cells treated with or without TRAIL. (E) Western blot images of DR4 and DR5 expressions in HCT116 WT and DR4−/− DR5−/− cells. β-Actin was used as loading control. (F) Quantification of entotic structures in HCT116 WT and DR4−/− DR5−/− cells treated with or without TRAIL in the absence or presence of Y-27632. (G and H) DR4 and DR5 are required for inner cells during cell internalization. (G) HCT116 WT and DR4−/− DR5−/− cells were labeled with CellTracker Green and CellTracker Red, mixed in 1:1 ratio, and treated with or without TRAIL in the absence or presence of z-VAD-fmk. Images were recorded by HCS and analyzed using CellProfiler. Scale bar: 25 μm. Distribution of entosis events divided into four subcategories as indicated; absolute numbers are shown in parenthesis. Data are shown as mean from three experiments. n indicates number of events analyzed per group. (H) Representative images showing entotic structures formed between “wt in wt” (green) and “wt in −/−” (green and red, respectively) cells. Scale bar: 10 μm. (I–K) Induction of entosis by TRAIL requires structural presence of caspase-8. (I) Quantification of PI-positive cells in HCT116 WT and Casp8−/− cells treated with or without TRAIL. Data are shown as mean ± SEM from three experiments. (J) Western blot images of CASP8 expression in HCT116 WT, CASP8 C360A mutant, and CASP8−/− cells. β-Actin was used as loading control. (K) Quantification of entotic structures in CASP8−/− and CASP8 C360A mutant cells treated with or without TRAIL in the absence or presence of Y-27632. Data are shown as individual values for each experiment as well as mean ± SEM from three experiments except as noted in G and I. ****, P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test (A, B, D, F, G, I, and K).

Entosis induced by TRAIL requires death receptors and structural presence of caspase-8. (A–C) Internalization process during entosis is independent of caspase activity. (A) Inhibition of entosis does not affect the rate of TRAIL-induced apoptosis. Quantification of PI-positive cells in HCT116 cells treated with or without TRAIL in the absence or presence of z-VAD-fmk, z-IETD-fmk, or Y-27632. (B and C) Inhibition of caspase activity does not affect the rate of TRAIL-induced entosis. (B) Quantification of entotic structures in HCT116 cells treated with or without TRAIL in the absence or presence of z-VAD-fmk, z-IETD-fmk, or Y-27632. (C) Representative field of view from HCS-based entosis analysis is shown. Arrows indicate entotic events, four representative events are depicted and shown. Scale bar: 50 μm. (D–H) TRAIL death receptors are required for cell internalization during TRAIL-induced entosis. (D) Quantification of PI-positive cells in HCT116 WT and DR4−/− DR5−/− cells treated with or without TRAIL. (E) Western blot images of DR4 and DR5 expressions in HCT116 WT and DR4−/− DR5−/− cells. β-Actin was used as loading control. (F) Quantification of entotic structures in HCT116 WT and DR4−/− DR5−/− cells treated with or without TRAIL in the absence or presence of Y-27632. (G and H) DR4 and DR5 are required for inner cells during cell internalization. (G) HCT116 WT and DR4−/− DR5−/− cells were labeled with CellTracker Green and CellTracker Red, mixed in 1:1 ratio, and treated with or without TRAIL in the absence or presence of z-VAD-fmk. Images were recorded by HCS and analyzed using CellProfiler. Scale bar: 25 μm. Distribution of entosis events divided into four subcategories as indicated; absolute numbers are shown in parenthesis. Data are shown as mean from three experiments. n indicates number of events analyzed per group. (H) Representative images showing entotic structures formed between “wt in wt” (green) and “wt in −/−” (green and red, respectively) cells. Scale bar: 10 μm. (I–K) Induction of entosis by TRAIL requires structural presence of caspase-8. (I) Quantification of PI-positive cells in HCT116 WT and Casp8−/− cells treated with or without TRAIL. Data are shown as mean ± SEM from three experiments. (J) Western blot images of CASP8 expression in HCT116 WT, CASP8 C360A mutant, and CASP8−/− cells. β-Actin was used as loading control. (K) Quantification of entotic structures in CASP8−/− and CASP8 C360A mutant cells treated with or without TRAIL in the absence or presence of Y-27632. Data are shown as individual values for each experiment as well as mean ± SEM from three experiments except as noted in G and I. ****, P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test (A, B, D, F, G, I, and K).

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