Fig. 3: MLKL depletion-mediated sensitization was blocked by caspase inhibition.

a HeLa, H2009, and HCC4006 cells expressing MLKL shRNA, or a non-silencing control were pretreated with zVAD (20 μM) for 1 h and then treated with TRAIL for 24 h. Cell viability was analyzed by MTT assay. The results are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. b HT-29 and RIP3-expressing HeLa cells expressing MLKL shRNA, or a non-silencing control were pretreated with zVAD for 1 h and then treated with TRAIL for 48 or 12 h. Cell viability was analyzed by MTT assay. The results are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. c RIP3-expressing HeLa cells expressing MLKL shRNA, or a non-silencing control were pretreated with zVAD for 1 h and then treated with TRAIL (5 ng/ml) for 6 h. The cells lysates were analyzed by western blotting. d PI/annexin V staining to distinguish apoptosis and necroptosis in HT-29 shNC, shRIP3 and shMLKL cells. Cells were pretreated with SMAC mimetic + zVAD for 1 h and then treated with TNF (30 ng/ml) or treated with TRAIL (100 ng/ml) for indicated time points and stained with PI/annexin V then analyzed by FACS. e, f HT-29 (e) and RIP3-expressing HeLa (f) cells were pretreated with GSK’872 for 1 h and then treated with TRAIL (100 ng/ml or 5 ng/ml) and cell lysates were analyzed by western blotting. g Cell death analysis by PI/annexin V staining in HT-29 and MLKL-siRNA-silenced HT-29 cells inducibly expressing MLKL. these cells were pretreated with tamoxifen (1 μM) for 10 h and then treated with TRAIL (100 ng/ml) for 8 h.