Figure S1 A B Figure S1. SPATA2 is required for TNFα or zVAD.fmk induced necroptosis in L929 cells. (A) L929 cells were transfected with a pool of four Spata2-targeting siRNAs, treated with human TNFα or zVAD.fmk in the presence or absence of Nec-1s overnight. Cell viability was examined by CellTiterGlo assay. The knockdown efficiency was demonstrated by western blotting. (B) L929 cells were infected with shRNA targeting Spata2 3’UTR and stable cell lines were derived. The cells were infected with pMIG-Spata2 expressing vector. The cells were treated with human TNFα or zVAD.fmk overnight. Cell viability was examined by CellTiterGlo assay. The efficiency of knockdown and transfection were demonstrated by RT-PCR. Error bars represent SEM from three simultaneous technical replicates.

Figure S2 A C B E D Figure S2. The expression pattern of SPATA2 in mouse and the generation of Spata2–/– mice. (A) Western blotting was used to examine the levels of SPATA2 protein in different mouse tissues. (B) Schematic diagram of wild-type (top) and targeted (bottom) Spata2 genomic locus. PCR primers for genotyping were indicated below. (C) PCR genotyping using total genomic DNA isolated from primary MEFs with indicated genotypes. (D) MEFs and BMDMs of indicated genotypes were collected and the expression of Spata2 was examined by western blotting. (E) Spata2 +/+, Spata2 -/- MEFs and Spata2+ MEFs were harvested for western blot.

Figure S3 Figure S3. SPATA2 promotes necroptosis induced by TNFα/zVAD.fmk. Immortalized Spata2-/- MEFs and Spata2+ MEFs were planted at E-plate, pretreated with or without 100 nM SM-164 for 30 min, then treated with 10 ng/mL human TNFa, 20 μM zVAD.fmk for 24 h. Time-dependent cell death was measured by xCELLigence assay. Error bars represent SEM from three technical replicates.

Figure S4 A B C Gene LFQ Intensity DMSO (T+S+Z)-3h (T+S+Z+N)-3h RIPK1 2.79e8 Caspase-8 4.93e7 cFLIP 5.19e6 FADD 1.03e7 TRADD 4.38e6 RIPK3 4.39e6 D Gene LFQ Intensity Spata2-/- Spata2+ DMSO (T+Z)-3h RIPK1 7.00e7 Caspase-8 1.52e8 FADD 2.55e7 Figure S4. p-S166 RIPK1 antibody specifically recognizes phosphorylated RIPK1. (A) RGC5 cells were pretreated with or without 20 μM Nec-1s, then treated with 100 ng/mL hTNFα, 20 μM zVAD.fmk and 100 nM SM-164 for 3 h. The cell lysates were analyzed by immunoprecipitation using anti-p-S166 RIPK1, then analyzed by western blotting. (B) 293T cells were transfected with HA-mRIPK1-WT or mutant S166A RIPK1 plasmids for 24 h, treated with or without Nec-1s (20 μM) for the last 6 h. The cell lysates were analyzed by immunoprecipitation using anti-p-S166 RIPK1, then analyzed by western blot. (C-D) The summary tables of the top proteins identified in anti-pS166 RIPK1 immunoprecipitation in RGC5 (C) and in Spata2-/-, Spata2+ MEF cells (D) by LC-MS/MS analysis. The protein intensity was quantified by the module of label free quantification in MaxQuant.

Figure S5 A B C Figure S5. SPATA2 promotes CYLD cleavage and recruitment into Complex-IIb. (A) Primary Spata2-/-, Spata2+/+ and Spata2+/- MEFs were treated with 100 ng/mL human TNFα for 2h, and the cell lysates were analyzed by western blotting using indicated abs. Actin is a loading control. (B) WT and D138N MEFs were treated with 100 ng/mL human TNFα, with or without zVAD.fmk (50 μM) and Nec-1s (30 μM) for 2h, and the cell lysates were analyzed by western blotting using indicated abs.(C) Spata2+/+ MEFs and Spata2-/- MEFs were treated with 100 ng/mL human TNF plus zVAD.fmk (50 μM) for indicated periods of time. The cell lysates were subjected to immunoprecipitation using anti-FADD antibody, then analyzed by western blotting using indicated abs.

Figure S6 Figure S6. Spata2-/- MEFs were partially protected against TNFα plus CHX induced apoptosis. Spata2+/+ MEFs and Spata2-/- MEFs were treated with 1μg/mL CHX alone or with 10 ng/ml hTNFα for indicated periods of time. Cell viability was measured by CellTiterGlo assay.

Figure S7 A B Figure S7. SPATA2 is required for CYLD to interact with HOIP and TNF-RSC. (A) Spata2-/- MEFs and Spata2+ MEFs were stimulated by TNFα (100 ng/mL) for indicated periods of time. The cell lysates were immunoprecipitated using anti-TNFR1. The immunocomplexes were analyzed by western blotting using indicated abs. (B) Spata2-/- MEFs and Spata2+ MEFs were stimulated by TNFα (100 ng/mL) for indicated periods of time. The cell lysates were analyzed by immunoprecipitation using anti-CYLD. The immunocomplexes were then analyzed by western blotting using indicated abs.

Figure S8 A Figure S8 SPATA2 knockout increases M1 ubiquitination of RIPK1 with LPS stimulation. Spata2+/+ iBMDMs and Spata2-/- iBMDMs were treated with 100 ng/mL LPS for indicated periods of time. The cell lysates were subjected to immunoprecipitation using anti-RIPK1 antibody, then analyzed by western blotting.

Figure S9 A B Figure S9. HOIP overexpression increases the activation of JNK kinases and inflammatory cytokine production. (A) BV2 cells expressing GFP or HA-HOIP were treated with LPS for indicated periods of times and analyzed by western blotting using indicated abs. (B) BV2 cells expressing GFP or HA-HOIP were treated with LPS for indicated periods of times. The mRNA levels of the indicated genes were measured by quantitative RT-PCR. The fold of induction was calculated based on the levels in untreated cells. Error bars represent SEM from three simultaneous technical replicates. GAPDH was used as loading controls.

Figure S10 A B C Figure S10. Loss of Spata2 sensitizes mice to LPS-Induced SIRS in vivo. (A) Survival curve of Spata2+/+ and Spata2-/- mice injected with LPS at 25 mg/kg i.p. (n=7), ***p<0.001. Spata2+/+ is marked in black and Spata2-/- is marked in red. (B, C) Sera from treated mice were collected at indicated time points and IL-6 or TNFα levels were measured using ELISA. Error bars represent SEM from three technical replicates, *p<0.05.