Fig. 5

RIPK1 S321A mutation sensitizes cells to RIPK1-dependent cell death. a–c RIPK1 S321A(A/A) but not S321E(E/E) MEFs are more sensitive to RIPK1-dependent cell death. Immortalized MEFs generated from littermates of WT and RIPK1 S321A(A/A), or WT and S321E (E/E) were treated with TNFα (10 ng/ml), CHX (0.5 µg/ml), zVAD (20 µM) and Nec-1s (10 µM) as indicated. Cell death was measured by ToxiLight assay after 16 h treatment a or at indicated time-points b, c and normalized to TX-100-treated cells. d TNFα alone or TNFα/CHX induces RDA in RIPK1 S321A(A/A) mutant MEFs. WT and RIPK1 S321A(A/A) MEFs were treated with TNFα (10 ng/ml) or CHX (0.5 µg/ml) with or without Nec-1s (10 µM) for 24 h. After SYTOX Green staining, fluorescence intensity was quantified and normalized to TX-100-treated cells. e TNFα induces caspase-8 activation in RIPK1 S321A(A/A) mutant but not in WT and S321E(E/E) mutant MEFs. The MEFs were treated with TNFα (10 ng/ml) with or without Nec-1s (20 µM) for 24 h and caspase-8 activity was measured in the presence or absence of zVAD as described in Methods. Error bar, s.e.m. *, t-test P < 0.05; **P < 0.01, ***P < 0.001. Three independent repeats were included in each data point