Fig. 2. Novel inhibitors can block RIP3 S227 phosphorylation.

a Western blotting showing various kinase inhibitor effects on RIP3 S227 auto-phosphorylation. HT-29 cells were treated with four tested kinase inhibitors (1308, 1336, 1338, and 1371, 10 μM) for 9 h. As a control, KK5101 (Trk alpha inhibitor) and HS829 (IKK beta inhibitor) were also treated at a concentration of 10 μM for 9 h. Prototypical necroptosis stimuli (TNF + zVAD + either cycloheximide or SMAC mimetic; hereafter referred to as TCZ or TSZ) were applied for 6 h. Cell lysates were analyzed with a S227 specific p-RIP3 antibody. b Four tested kinase inhibitors displayed kinase inhibitory effects on basal levels of RIP3 auto-phosphorylation in a dose-dependent manner. HT-29 cells were treated with indicated concentrations for 9 h, and cell lysates were analyzed by immunoblotting. c Four tested kinase inhibitors showed small amounts of cytotoxicity in a dose-dependent manner. HT-29 cells were treated with 4 tested kinase inhibitors for 24 h, 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. d Various necroptosis inhibitors blocked TNF-induced RIP3 phosphorylation. HT-29 cells were pretreated with necrostatin-1 (Nec-1, 40 μM), dabrafenib (DAB, 10 μM) or necrosulfonamide (NSA, 1 μM) for 2 h and then treated with TSZ for 6 h. Cell lysates were analyzed by immunoblotting. e HS-1371 efficiently blocked TNF-induced RIP3 phosphorylation. HT-29 cells were pretreated with four tested kinase inhibitors for 2 h and then treated with TSZ for 6 h. Cell lysates were analyzed by immunoblotting. f HT-29 cells were pretreated with Nec-1 (40 μM), DAB (5 μM) or HS-1371 (5 μM) for 2 h and then treated with TSZ for 4 h. Cell lysates were immunoprecipitated with anti-RIP3 antibody. Immunoprecipitates and total lysates were analyzed by Western blotting