Fig 2: 3-BHA and TBHQ function as type III inhibitors for RIPK1.A Docking of Nec-1s (purple) and 3-BHA (yellow) into RIPK1 (PDB: 4ITH). Hydrogen bonds (2.7–3.0 Å) with residues V76 and S161 of RIPK1 are shown as red dashes. Hydrophobic residues M67, L70, and M92 of RIPK1 engaged in hydrophobic interaction are also indicated. B, H MEFs were pretreated for 30 min with indicated compounds before stimulation with 1 µg/ml FLAG-hTNF for the indicated duration. TNFR1 complex I was FLAG-immunoprecipitated and the IPs were treated with USP21 post-IP when indicated. The results are representative of at least two independent experiments. pRIPK1 refers to autophosphorylation of RIPK1 on S166/T169. C–E, I–J MEFs were pretreated for 30 min with indicated compounds (100 µM BHA, 100 µM 2-BHA, 100 µM 3-BHA, 100 µM TBHQ, 10 µM Nec-1s, 10 µM GSK8612 (TBK1i), 50 µM zVAD-fmk) before stimulation with 20 ng/ml hTNF (C–D, I–J) or 10 µM erastin (E) for the indicated duration. Cell death was measured over time by Sytox Green (SG+) positivity, and the results are presented as mean ± SEM of three independent experiments (n = 3). Statistical analysis on kinetic cell death assays with more than one timepoint is detailed in the Methods section. F Molecular structure models are presented for 2-, 3-BHA, BHT, and THBQ. G Kinase activity was quantitatively measured by ATP consumption using the ADP-Glo kinase assay, compounds were used at indicated concentrations. D is short for DMSO. Results are presented as a percentage relative to the activity of the kinase in absence of inhibitor and are the mean ± SEM of three independent kinase assays (n = 3). Statistical significance was determined via ordinary one-way ANOVA followed by a Tukey post hoc test. Significance between samples is indicated in the figures as follows: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Fig 3: JNK/IL-8 (CXCL8): a novel downstream axis of necroptosis in tumor repopulation. a Immunohistochemical staining of IL-8 in the HT29 xenograft tumors for 48 h from 10 Gy-irradiated and no-irradiated nude mice. Scale bar: 20 μm. b Left panel, IL-8 concentrations were measured in the supernatants of HT29 cells (pretreated with RIP1 inhibitor (Nec-1), RIP3 inhibitor (GSK’872) or MLKL inhibitor (NSA) for 24 h) with no radiation or after 10Gy radiation for 48 h by chemiluminescent immunoassays. Right panel, IL-8 concentrations were measured in the supernatants of HT29 sh-NC, HT29 sh-RIP1, HT29 sh-RIP3 and HT29 sh-MLKL cells with or without 10 Gy radiation for 48 h, one-way ANOVA, n = 3. c Western blot showed the protein level of phosphorylated JNK1/2 and pan-JNK in HT29 cells after 10Gy radiation. d Western blot showed expression of phosphorylated JNK1/2 and pan-JNK in MLKL knockdown and vector-transfected HT29 cells for 48 h after 10 Gy radiation. e IL-8 concentrations were measured in the supernatants of HT29 cells (pretreated with JNK inhibitor (SP600125)) with no radiation or after 10 Gy radiation for 48 h by chemiluminescent immunoassays, one-way ANOVA, n = 3. f Effect of dying HT29 cells on growth of HT29 Fluc after treatment of JNK inhibitor (SP600125), anti-IL-8 antibody and reparixin (an inhibitor of IL-8 receptor CXCR1 and CXCR2), one-way ANOVA, n = 3. g Effects of recombinant Human IL-8 on dying HT29 sh-MLKL cells induced growth of HT29 Fluc (left panel) and on growth of HT29 Fluc alone (right panel), one-way ANOVA, n = 3. h The Kaplan–Meier survival analysis in 71 colorectal cancer patients. i Representative images of positive (upper panel) and negative (lower panel) IL-8 staining in human colorectal cancer tissues. Scale bar: 50 μm. j Schematic representation of the proposed mechanisms for necroptosis–mediated tumor cell repopulation. n.s = not significant, * < p 0.05, ** p < 0.005, *** p < 0.001

Fig 4: ZJU-37 promotes OPC proliferation and enhances remyelination by inhibiting RIPK1 kinase activity.A Schematic diagram of the experimental design. B Representative fluorescence images of the proliferating OPCs within the demyelination region at 5 dpl. Scale bars, 50 μm. C–E Quantification of (C) the oligodendrocyte lineage cell numbers, (D) the proliferating OPCs numbers and (E) the proportion of proliferating OPCs in all oligodendrocyte lineage cells within the lesion region at 5 dpl (one-way ANOVA, Tukey’s post hoc test, n = 5 mice in the WT group, n = 4 mice in the RIPK1D138N group, n = 7 mice in the RIPK1D138N vehicle and RIPK1D138N ZJU-37 group). F Representative transmission electron micrographs within the demyelination region at 21 dpl. Scale bars, 1 μm (upper row), 500 nm (lower row). G–J Quantification of (G) the remyelinated axons (one-way ANOVA, Tukey’s post hoc test), (H) G-Ratio (one-way ANOVA, Tukey’s post hoc test), (I) individual G-Ratio distribution (linear regression) and (J) the myelin pathology level (two-way repeated ANOVA, Sidak’s post hoc test) within the demyelination region at 21 dpl (n = 5 mice per group). All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. no significance.

Fig 5: RIPK1 kinase inhibition attenuates clinical disease progression and suppresses microglial and astrocyte signaling pathways in a murine model of MS(A) Mean disease scores for vehicle (0.5% methylcellulose, twice a day [BID]) and RIPK1 kinase inhibitor groups (RIPK1inh-1, 30 or 60 mg/kg BID) in C57BL/6 EAE mice (n = 12 mice/group).(B) Cumulative disease scores for each mouse are depicted.(C) Neurofilament heavy levels measured in the plasma of naive or EAE-induced mice at sacrifice (n = 11 naive and vehicle, n = 12 RIPK1inh-1).(D–H) RNA-seq data in microglia (D) and astrocytes (G) isolated from naive or EAE-induced mice depicting upregulated and downregulated genes (cutoff |FC| 1.5, p < 0.05; n = 4 naive, n = 6 EAE-vehicle [20% Captisol], and n = 5 EAE-RIPK1inh-1 [50 mg/kg BID] mice). Hierarchical heatmap clustering of RIPK1-kinasedependent genes in microglia (E) and astrocytes (H). Top pathways predicted from IPA for RIPK1-kinase-dependent genes in microglia (F) and astrocytes (I).(J–M) Gene expression levels in RNA-sequenced microglia (J) and astrocytes (K and M); Ccl2 p = 0.072 (EAE-vehicle versus EAE-RIPK1inh-1), all other genes p < 0.05. (L) Diagram of genes and sterol intermediates in the cholesterol biosynthesis pathway.(N) Heatmap of cholesterol biosynthesis genes from RNA-sequenced astrocytes, normalized to naive mice, with significant changes for naive versus EAE-vehicle and EAE-vehicle versus EAE-RIPK1inh-1 shown.Error bars represent mean ± SEM. One-way ANOVA with Tukey post hoc test (B and C) or two-way ANOVA with Dunnett test (A) was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S6.