Fig. 7.
Mitochondrial ROS was required for activation of t-BHPH-induced endothelial necroptosis. Endothelial cells were treated with t-BHP (50–500 μM) 1 h with or without Rot (20 μM), TTFA (10 μM), AA (5 μM) pretreatment for 1 h, ROS generation and LDH release were detected by flow cytometer (A and B) and LDH assay kit (C). After transfected with RIP1, RIP3, or MLKL siRNAs, cells were treated with t-BHPH for 15 min. The MMP and mitochondrial ROS generation were detected with JC-1 staining (D) and MitoSOX by fluorescence microscopy (E) and detected by FACScan flow cytometer (F). Cells were treated with t-BHPH for 1 h with or without Rot (20 μM), TTFA (10 μM), AA (5 μM) pretreatment for 1 h, RIP1-RIP3 interaction and MLKL phosphorylation were detected by immunoprecipitation (G) and Western blotting (H). The results were expressed as mean±SD of three independent experiments. AA, antimycin A; ALL, allopurinol; Cont, control group; DPI, diphenyleneiodonium; LDH, lactate dehydrogenase; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; Rot, rotenone; TTFA, 2-thenoyltrifluoroacetone.