Fig. 3

Radiation-induced necroptosis depended on the RIP1/RIP3/MLKL signaling pathway. a Left panel, the level of pRIP1(S166), pRIP3(S227), pMLKL (T357/S358) and corresponding endogenous protein expression in RIP1 knockdown and vector-transfected HT29 cells at different time point post10 Gy-irradiation were evaluated by western blot. Right panel, the pRIP1/pRIP3/pMLKL and corresponding endogenous protein expression in RIP3 knockdown and vector-transfected HT29 cells treated like left panel. b Confocal immunofluorescence analysis showed increased pMLKL expression in 10 Gy irradiated HT29 cells than non-irradiated cells. Scale bar: 25 μm. c The pMLKL level in the HT29 xenograft tumor samples 48 h post 10Gy-irradiation or non-irradiation. Please note 30 μg and 100 μg were loaded on same blot. d LDH release were quantified in cell culture supernatants from HT29 cells pretreated with the inhibitor of RIP3 (left panel, GSK’ 872) or MLKL (right panel, NSA) for 24 h with 10 Gy irradiation or non-irradiation, one-way ANOVA, n = 3. e LDH release were quantified in cell culture supernatants from HT29 sh-NC cells, HT29 sh-RIP1 cells, HT29 sh-RIP3 cells and HT29 sh-MLKL cells treated with or without 10 Gy radiation, one-way ANOVA, n = 3. f Cell viability was performed through CCK-8 assay in HT29 cells pretreated with the inhibitor of RIP1 (Nec-1), RIP3 (GSK’ 872) or MLKL (NSA) and 6 days later after 10Gy irradiation or non-irradiation, one-way ANOVA, n = 3. g Cell viability was performed through CCK-8 assay 6 days later in HT29 sh-NC cells, HT29 sh-RIP1 cells, HT29 sh-RIP3 cells and HT29 sh-MLKL cells with or without 10 Gy irradiation, one-way ANOVA, n = 3. h The statistical analysis of Annexin V/PI stain cells by flow cytometry. Left panel, the percentage of PI positive in 10Gy-irradiated or non-irradiated HT29 cells which were pretreated with or without MLKL inhibitor (NSA). Right panel, the percentage of PI positive in HT29 sh-NC or sh-MLKL cells with non-irradiation or 10 Gy irradiation. Necroptotic cells were those decreased PI positive cells by Nec-1, one-way ANOVA, n = 3. n.s = not significant, * < p 0.05, ** p < 0.005, *** p < 0.001