a, b, RIPK1 cleavage site variants in MEFs promote cell death induced by TNF. Ripk1−/− MEFs were complemented with: GFP; wild-type RIPK1; or D325V or D325H mutant, and treated for 12 h (a) or as indicated (b). N, 20 μM Nec-1s; S, 20 nM SM-164; T, 50 ng ml−1 TNF; Z, 50 μM Z-VAD-FMK. Cell viability and cell death were measured by CellTiter-Glo assay (a) and ToxiLight assay (b), respectively. Data are mean ± s.e.m, n = 3. Circles correspond to each independent experiment. P values were determined by unpaired, two-tailed t-test (shown if P < 0.05). c, Western blots illustrating increased levels of p-S166-RIPK1, p-S345-MLKL and cleaved caspase-3 after stimulation with TNF and SM-164, which were inhibited by Nec-1s. Ripk1−/− MEFs complemented with: GFP; wild-type RIPK1; or D325V or D325H mutant, were treated as indicated. Cell lysates were analysed by immunoblotting using indicated antibodies. For gel source data, see Supplementary Fig. 1. Results are representative of three independent experiments. d, e, D325A knock-in Ripk1 mutation sensitizes Ripk3−/− MEFs to TNF-induced RIPK1-dependent apoptosis. Ripk1D325A/D325ARipk3−/− and Ripk1+/+Ripk3−/− MEFs were simulated with TNF only, TNF plus Nec-1s (d) or a combination of TNF, SM-164 and Nec-1s (e) as indicated (concentrations as in a). Cell death was measured by the SYTOX Green Nucleic Acid Stain assay. Data are mean ± s.e.m., n = 4. Circles correspond to each independent experiment. P values determined by unpaired two-tailed t-test, and indicate the comparison between Ripk1D325A/D325ARipk3−/− and Ripk1+/+Ripk3−/− MEFs after TNF or TNF plus SM-164 stimulation for indicated amount of time. f, RIPK1 cleavage site variants in MEFs sensitize TRAIL-induced cell death. Ripk1−/− MEFs complemented with: GFP, wild-type RIPK1, or D325V or D325H mutant were treated with TRAIL (100 ng ml−1) or TRAIL plus Nec-1s (20 μM) for 36 h. Data are mean ± s.e.m., n = 3. Circles correspond to each independent experiment. P values determined by unpaired two-tailed t-test. g, TRAIL stimulation of Ripk1−/− MEFs complemented with D325V or D325H mutant promotes RIPK1 activation, which was inhibited by Nec-1s (20 μM). TRAIL (100 ng ml−1); TNF (50 ng ml−1) for 12 h. For gel source data, see Supplementary Fig. 1. Results are representative of three independent experiments. h, Ripk1-knockout MEFs complemented with wild-type RIPK1, or D325V or D325H mutant plasmid showed similar responses to erastin- or RSL3-induced ferroptosis. Data are mean ± s.e.m., n = 3. Circles correspond to each independent experiment. i, Western blots of proteins involved in ferroptosis in Ripk1−/− MEFs complemented with wild-type RIPK1, or D325V or D325H mutant. Cells were treated with erastin for 5 or 10 h, followed by immunoblotting of cell lysates. For gel source data, see Supplementary Fig. 1. Results are representative of three independent experiments. j, RNA sequencing of Ripk1−/− MEFs complemented with wild-type RIPK1, D325V or D325H mutant indicated no difference in expression patterns of genes involved in ferroptosis and antioxidant. Analysis of each sample was performed in duplicate. k, GSH concentration of Ripk1−/− MEFs complemented with wild-type RIPK1, D325V or D325H mutant was similar both at baseline and after erastin or glutamate stimulation for 8 h. Data are mean ± s.e.m., n = 3. Circles correspond to each independent experiment. l, Immunofluorescence showed similar levels of cytosolic ROS after erastin stimulation in Ripk1−/− MEFs complemented with wild-type RIPK1, D325V or D325H mutant. Cells were treated by erastin for 8 h before incubation with the cytosolic ROS sensor carboxy-H 2 DCFDA. Green foci indicate cytosolic ROS. Scale bar, 150 μm. Results are representative of two independent experiments. Source Data