Smac mimetic and dexamethasone cooperate to trigger the formation of the ripoptosome, which is required for Smac mimetic-/dexamethasone-induced cell death. (A) Cells were treated for 6 hours with 300 µM dexamethasone and/or BV6 (Jurkat: 7 µM, Reh: 0.3 µM) in the presence of 20 µM zVAD.fmk. Caspase-8 was immunoprecipitated (IP) using an anti-RIP1 antibody (Jurkat) or an anti-caspase-8 antibody (Reh), and western blotting detected the indicated proteins. (B-E) Cells were transduced with control vector (shCtrl) or vector containing shRNA sequences against RIP1 (shRIP1#1, shRIP1#2). Expression of RIP1 was analyzed by western blotting (B). In (C), cells were treated for 6 hours (Jurkat) or 12 hours (Reh) with 300 µM dexamethasone and/or BV6 (Jurkat: 7 µM, Reh: 0.3 µM), and caspase activation was analyzed by western blotting, active cleavage fragments are indicated by arrowheads. In (D), cells were treated for 4 hours (Jurkat) or 8 hours (Reh) with 300 µM dexamethasone and/or BV6 (Jurkat: 7 µM, Reh: 0.3 µM), and ROS production was determined by CellROX staining and flow cytometry and is shown as fold increase. In (E), cells were treated for 24 hours (Jurkat) or 72 hours (Reh) with 300 µM dexamethasone and/or BV6 (Jurkat: 7 µM, Reh: 0.3 µM) and cell death was determined by forward/side scatter analysis and flow cytometry. In (D-E), mean and SD of 3 experiments performed in triplicate are shown; *P < .05; **P < .01.