Fig 2: PP6 also promotes TNFa-mediated RIPK1-dependent cell death.A PP6+/+ or PP6-/- MEFs were pretreated with Nec-1s for 1 h and then treated with TNFa/5Z-7, TNFa/zVAD/5Z-7 or TNFa/zVAD/CHX for indicated periods of time. B PP6+/+ or PP6-/- MEFs were pretreated with Nec-1s for 1 h and then treated with TNFa/5Z-7 for indicated periods of time. Cells were lysed with RIPA buffer and analyzed by western blotting with indicated antibodies. C PP6+/+ or PP6-/- MEFs were pretreated with Nec-1s for 1 h and then treated with TNFa/zVAD/5Z-7 for 3 h or TNFa/zVAD/CHX for 6 h. Cells were stained with DAPI and PI. The panel below showed the quantification result of PI positive cells. D PP6-/- MEFs were reconstituted with control vector, WT-PP6 or phosphatase-inactive D84N-PP6. Cells were then treated with TNFa/5Z-7 and TNFa/zVAD/5Z-7 for 4 h or TNFa/zVAD/CHX for 8 h. E WT or PPP6R1/2/3 triple-knockout MEFs were treated with TNFa/5Z-7 and TNFa/zVAD/5Z-7 for 4 h or TNFa/zVAD/CHX for 8 h. F PP6 was removed in WT or PPP6R1/2/3 triple-knockout MEFs. Cells were treated with TNFa/5Z-7 and TNFa/zVAD/5Z-7 for 4 h or TNFa/zVAD/CHX for 8 h. Concentrations of reagents used: TNFa (T), 50 ng/ml; CHX (C), 2 µg/ml; Nec-1s, 10 µM; zVAD (Z), 50 µM; 5Z-7, 300 nM. The cell death in (A, D–F) 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).

Fig 3: PP6 promotes TNFa-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 TNFa/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 TNFa/CHX for 10 h. C WT or PPP6R1/2/3 triple-knockout MEFs were treated with TNFa/CHX for 10 h. D PP6+/+ or PP6-/- MEFs were pretreated with Nec-1s for 1 h and then treated with TNFa/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 TNFa/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 TNFa/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 TNFa for indicated periods of time. H c-FLIP-/- sgGFP or c-FLIP-/- sgPP6 MEFs were treated with TNFa for indicated periods of time. Cells were lysed with RIPA buffer and analyzed by western blotting with indicated antibodies. Concentrations of reagents used: TNFa (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).