FIGURE 3.

MLKL availability determines the cell death mode in response to MG132. A–D, Ripk3^−/− 3T3 cells were transduced with retrovirus expressing wild type HA-RIPK3-FLAG and lentivirus expressing non-silencing (NS) or MLKL shRNA. In A, knockdown of MLKL expression was confirmed by Western blot. In B and D, cell death was determined after 2 μm MG132 treatment for 16 h. In C, active caspase 3 was measured by Western blot after MG132 treatment for the indicated time. E–G, Ripk3^−/− 3T3 cells expressing wild type or T231A/S232A RIPK3 under the control of doxycycline-inducible promoter were used. Induction of RIPK3 expression was confirmed by Western blot after treatment with doxycycline (DOX) for 14 h (E). After induction of RIPK3 expression, cells were treated with MG132 for 4 h (F). The cells were treated with DOX and 10 μm zVAD for 1 h prior to treatment with 2 μm MG132 for 16 h (G). Cell death was determined by measuring loss of intracellular ATP (iATP) (B, D, G). *, p < 0.05.