Figure 6.

RIP3 and MLKL are required for Bak activation and mitochondrial perturbations during BV6/Dexa-induced necroptosis. Tanoue cells were transiently transfected with two distinct siRNAs targeting RIP3, MLKL or control siRNA, and treated for 6 h with 3 μM BV6 and 200 μM Dexa. MEFs were treated for 12 h with 5 μM BV6 and 200 μM Dexa in the presence of 20 μM zVAD.fmk. (a and b) Bak (a) or Bax (b) activation was determined by immunoprecipitation using active conformation-specific antibodies. Protein expression of Bak, Bax, RIP3 and MLKL were analyzed by western blotting. β-Actin served as loading control. (c and d) Loss of MMP was determined by TMRM staining and flow cytometry (c) or ImageXpress Micro XLS system (d). Mean and S.D. of three independent experiments performed in triplicate are shown; *P<0.05. (e and f) ROS production was determined by CellROX staining and flow cytometry (e) or ImageXpress Micro XLS system (f). Mean and S.D. of three independent experiments performed in triplicate are shown; *P<0.05. (g and h) Respiration was determined by the Oxygraph system. Mean and S.D. of three independent experiments performed in triplicate are shown; *P<0.05