Fig. 4: HS-1371 only rescues cells from RIP3-dependent necroptosis.
From: HS-1371, a novel kinase inhibitor of RIP3-mediated necroptosis

a Knockdown of RIP3 had no inhibitory effect on HS-1371 in necroptosis. HT-29 cells expressing RIP3 shRNA, MLKL shRNA, or non-silencing control were analyzed by western blotting (upper panel), and these cells were treated with TSZ (6 h for immunoblotting, 24 h for cell death assay). Cell lysates were analyzed by immunoblotting (middle panel), and cell viability was analyzed by MTT assay (bottom panel). The results are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. b HS-1371 protected cells not only from TNF-induced necroptosis but also from TRAIL-induced necroptosis. HT-29 cells were pretreated with indicated concentrations of HS-1371 and then treated with TRAIL + Smac + zVAD (6 h for immunoblotting, 24 h for cell death assay). Cell lysates were analyzed by immunoblotting, and cell viability was analyzed by MTT assay or phase-contrast microscopy. The results are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. c, d HS-1371 had no effect on TNF-induced apoptosis. HT-29 cells were pretreated with HS-1371 (5 μM) for 2 h and then treated with TC or TS (TNF + either cycloheximide or SMAC mimetic, hereafter referred to as TC or TS, 6 h for immunoblotting, 24 h (TC) or 36 h (TS) for cell death assay). Cell lysates were analyzed by immunoblotting, and cell viability was analyzed by MTT assay (TC) or CellTiter-Glo Luminescent Cell Viability Assay Kit (TS). The results are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001