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1 S shrna S Viility, % of NT sirna trnsfete ells 1 1 Srmle NT sirna Csp-8 sirna RIP1 Atin L929 shrna sirna: Csp8 Atin L929: shrna Csp8 e Viility, % of NT sirna trnsfete ells NT sirna Csp-8 sirna M45 M45mutRHIM IP: my f L929 M45-my M45mutRHIM-my M36 Viility, % of DMSO trete ontrol ells zvad+ne-1 zvad Srmle Viility, % of DMSO trete ontrol ells zvad+ne-1 zvad M45 M45mutRHIM % ell eth M36 + M36 + Ne-1 + M36 + M45 M36 + Srmle shrna M36 + shrna % GFP+ ells (48h post-trnsfetion) Srmle shrna shrna Supplementry Figure 1. Csp8 suppresses progrmme nerosis. () Viility (left) n, RIP1, Csp8, n β-tin immunolots (right) of L929 ells expressing ontrol srmle shrna or -seifi shrna following trnsfetion with non-trgeting (NT) or Csp8-speifi sirna. Cell viility ws etermine 96 h post-trnsfetion y mesuring intrellulr ATP levels (Cell Titer-Glo Luminesent Cell Viility Assy kit, Promeg). () Viility (left) n M45 immunolot (right) of L929 ells trnsue with empty vetor (), M45-my, or M45mutRHIMmy t 96 h post-trnsfetion with NT or Csp8 sirna. () Viility of L929 ells expressing ontrol srmle shrna or -speifi shrna following tretment for 18 h with zvad-fmk (25 µm) in the sene or presene of the RIP1 kinse inhiitor Ne-1 (3 µm). () Viility of L929 ells trnsue with empty vetor (), M45-my, or M45mutRHIM-my following tretment s esrie in. (e) Perent ell eth, lulte s the ifferene etween the inite tretment n the -trnsfete ell viility, in L929 ells expressing n or M45-my (left), or ontrol srmle shrna or -speifi shrna (right), onstruts t 96 h post-trnsfetion with plsmi enoing M36 n ulture in the presene of Ne-1 (3µM) or vehile DMSO for 12 h. Vilility ssys (-e) represent the men n SD from three inepenent mesurements. (f) L929 ells rrying ontrol srmle shrna or -speifi shrna following otrnsfetion with M36 or ontrol plsmis together with GFP expression plsmi. The perent GFP positive ells for representtive smple ws lulte y ompring to ontrol -trnsfete ells etermine y flow ytometry t 48 h post-trnsfetion. 1

2 E11.5; Csp8+/-+/- E11.5; Csp8-/-+/- CD41 Dpi (Merge) Csp8+/-+/- Csp8+/-+/- PECAM Dpi Merge E9.5 Csp8-/--/- Csp8-/-+/- Csp8-/-+/- E11.5 PECAM Supplementry Figure 2. Emryoni lethlity n etetion in Csp8-efiient mie. () Photomirogrphs of E11.5 yolk s n emryo with the inite genotype, highlighting the region of hyperemi (rrow in right pnel). () PECAM (re), (green) n DAPI (lue) fluoresent stining of representtive Csp8 +/- Rip3 +/- n Csp8 -/- Rip3 +/- E1.5 yolk ss (X originl mgnifition). () (re), CD41 (green) n DAPI (lue) fluoresent stining of representtive Csp8 +/- Rip3 +/- yolk ss (63X originl mgnifition). () PECAM-1 (CD31) stining of whole-mount E9.5 n E11.5 yolk s from representtive Csp8 -/- Rip3 +/- (left pnels) n Csp8 -/- Rip3 -/- (right pnels) emryo (X). 2

3 Csp8 p p 7 p 5 p Csp8+/--/- Csp8+/++/+ Csp8+/+-/- Csp8+/--/- Csp8-/--/- ge: 4 months Csp8-/--/- Supplementry Figure 3. Csp8 -/- Rip3 -/- mie re vile. () PCR onfirmtion of genotype on til DNA from the inite Csp8 +/- Rip3 +/- interross progeny to etet wil-type (upper ns) n mutnt (lower ns) Csp8 n Rip3 lleles. () Photogrph of 4-month-ol Csp8 -/- Rip3 -/- mouse re from DKO ross long sie Csp8 +/- Rip3 -/- mie re from n interross. 3

4 LPS-GlN Perent survivl Csp8-/--/- Csp8+/++/+ Csp8+/+-/- Csp8-/--/- TRIF lps2/lps2 1 3 Hours LPS/GlN (8h) Csp8+/++/+ Csp8+/+-/- TRIF lps2/lps2 Supplementry Figure 4. Suseptiility to LPS+GlN inue heptitis in Csp8 +/+ Rip3 +/+, Csp8 +/+ Rip3 -/-, Csp8 -/- Rip3 -/- n ontrol TRIF-efiient (lps2/lps2) mie. () Kpln-Meier survivl plot of inite strins of mie following intrperitonel inoultion with LPS ( ng) n GlN ( mg). 6 nimls per Csp8 +/+ Rip3 +/+ genotype n 5 nimls per Csp8 -/- Rip3 -/-, Csp8 +/+ Rip3 -/-, n TRIF Lps2/Lps2 genotype were nlyze. () Histology of liver setions from inite strins of mie 8h following injetion with LPS/GlN. 4

5 Csp8-/--/- Csp8+/--/- months: Axil LN Csp8+/--/- Csp8-/--/- ervil LN weight (mg) Csp8+/++/+ f Csp8+/--/- Csp8-/--/- Thymus Lymph Noe CD CD8 3 Cell Count (x1 8 ) Csp8+/++/ p = e Cell Count (x1 8 ) g % of CD3+ Cells Csp8+/+ +/+ B Cells T Cells Lymph Noe: CD4-CD8-B p =.14 p =.51 Csp8+/+ +/+ Csp8+/ - -/- Csp8 -/- -/ Csp8+/--/- Csp8-/--/- Csp8+/++/+ Csp8+/--/- Csp8-/--/- Csp8+/- -/- Csp8-/- -/- Supplementry Figure 5. DKO mie umulte errnt T-ells. () Photogrphs of sixmonth-ol Csp8 +/- Rip3 -/- n Csp8 -/- Rip3 -/- mie. The rrow inites enlrge ervil LN present in the DKO mouse. () Imges of spleen n xil LNs from Csp8 +/- Rip3 -/- n Csp8 -/- Rip3 -/- mie of the inite ges. () Grph of weights of spleen from mie of the inite genotype. Horizontl lines represent the men. () The numers of ells n (e) numers of B n T ells reovere from spleens from mie with the inite genotype. Sttistil nlyses were performe pplying two-tile unpire Stuent s t-test. (f) CD4 vs. CD8 expression of CD3 + T ells in spleen (top pnels), LN (mile pnels) n thymus (ottom pnels) ientifie s in C, in representtive wil-type (left pnels), Csp8 +/- -/- (mile pnels) n Csp8 -/- -/- (right pnels) mie. (g) Frequeny (grph) n level of CD3 + CD4 - CD8 - B2 + T ells from LN of mie. For (), (e), n (g) the verge n SD for three wil-type, four Csp8 +/- -/- littermte ontrol n three DKO mie re shown. 5