Fig. 2: PP6 promotes TNFα-mediated RIPK1-independent cell death by regulating c-FLIPL.
A PP6+/+ or PP6−/− MEFs were pretreated with Nec-1s for 1 h and then treated with TNFα/CHX for indicated periods of time. B PP6-/- MEFs were reconstituted with control vector, WT-PP6 or phosphatase-inactive D84N-PP6. Cells were then treated with TNFα/CHX for 10 h. C WT or PPP6R1/2/3 triple-knockout MEFs were treated with TNFα/CHX for 10 h. D PP6+/+ or PP6−/− MEFs were pretreated with Nec-1s for 1 h and then treated with TNFα/CHX for indicated periods of time. Cells were lysed with RIPA buffer and analyzed by western blotting with indicated antibodies. E PP6+/+ or PP6−/− MEFs were treated with TNFα/CHX for indicated periods of time. Cells were lysed with RIPA buffer and analyzed by western blotting with indicated antibodies. F PP6+/+ or PP6−/− MEFs were pretreated with PS341 for 1 h and then treated with TNFα/CHX for indicated periods of time. Cells were lysed with RIPA buffer and analyzed by western blotting with indicated antibodies. G PP6 was removed in c-FLIP−/− MEFs. Cells were pretreated with Nec-1s for 1 h and then treated with TNFα for indicated periods of time. H c-FLIP−/− sgGFP or c-FLIP−/− sgPP6 MEFs were treated with TNFα for indicated periods of time. Cells were lysed with RIPA buffer and analyzed by western blotting with indicated antibodies. Concentrations of reagents used: TNFα (T), 50 ng/ml; CHX (C), 2 μg/ml; Nec-1s, 10 μM; PS341, 300 nM. The cell death in (A–C, G) was measured by CellTiter-Glo assay. Data represent mean ± SD of three independent experiments (Student’s t test **P < 0.01; ***P < 0.001; ****P < 0.0001, ns not significant).
