TAK1 impairs ROS-mediated cell death triggered by catalytically active RIP1.

(A) Left panel TAK1 KO MEFs transfected with a control siRNA (siC) or with RIP1 targeting siRNAs were stimulated with TNFα and cell survival was analyzed by crystal violet staining (CV). The bar graphs depict mean values ± SEM %. Right panel Cell lysates were analyzed by western blotting (WB) with α-RIP1 and α-NEMO antibodies to control the efficiency of RIP1 down-regulation. (B) TAK1 KO MEFs were transfected as in (A) and stimulated with TNFα. Intracellular ROS accumulation was analyzed by flow cytometry. (C) TAK1 KO MEFs were treated with DMSO or Necrostatin-1 (Nec-1) for 1 hour and then stimulated with TNFα. Butylated hydroxyanisole (BHA) was added together with TNFα. Cell survival was analyzed by CV. The bar graphs depict mean values ± SEM %. (D) TAK1 KO MEFs were treated with DMSO, Nec-1 or BHA as in (C) and stimulated with TNFα for indicated time points. ROS accumulation was analyzed as in (B).