PMC

a. Expected and observed frequency of caspase-8 status in offspring from crosses of mice with the indicated genotypes. “Fl” indicates an allele of caspase-8 that is present but flanked with LoxP sites. (p<0.0001 left, p=0.6733 right) b. Effect of tail vein injection of 15µg per animal of the CD95-activating antibody Jo2 on mice of the indicated genotypes. (n=6 RIPK3^−/−:Casp8^+/−, 8 DKO)

a. Lymphoid organs removed from 15 week old littermate mice of the indicated genotypes. LN is Lymph Node. Scale bar is 1cm. b. Percentage of total blood cells (following red blood cell lysis) that are B220⁺CD3⁺ in mice of the indicated genotypes and ages. (Error bars are s.d., n=3 each genotype). Both a and b are representative of similar results obtained from all sampled mice of the indicated genotypes.

a. Inhibitory effect of CrmA on FKBP-Caspase-8 homodimers or caspase-8^DA-FLIP heterodimers induced by dimerizer treatment of purified recombinant protein. b. Cell death (PI uptake) of RIPK3-deficient (NIH) or RIPK3 expressing (SA) 3T3 cells treated as indicated for 12h. (Error bars are s.d., n=3) c, d, e. Cell death (PI uptake) of 3T3-SA cells stably expressing vector (c) or CrmA (d), or anti-apoptotic Bcl-XL (e) following transfection with the indicated siRNA and treatment with TNF and the RIPK1 inhibitor Nec-1 as indicated for 24h. (Error bars are s.d., n=3) f. 3T3-SA cells expressing Bcl-XL were subjected to immunoprecipitation of FADD following transfection of siRNAs to caspase-8 and FLIP and treatment with TNF for 90 minutes as indicated. Immune complexes were resolved by western blotting with the indicated antibodies. The data presented are representative of results obtained with either of 2 separate siRNAs to both caspase-8 and FLIP.

a. Fluorogenic substrate cleavage activity of recombinant purified FKBP-caspase-8^WT or non-cleavable FKBP-caspase-8^DA in the presence of recombinant purified FRB-FLIP, a compound that induces FKBP-FRB heterodimers (heterodimerizer), or FRB-FLIP and heterodimerizer. ND indicates none detected. (Error bars are s.d., n=3) b. Western blot analysis of RIPK3^−/−:Caspase-8^−/− (DKO) MEF stably expressing the indicated mutants of caspase-8 and RIPK3. Caspase-8^CA indicates catalytically inactive caspase-8. c, d. Cell death assessed by propidium iodide (PI) uptake of DKO MEF expressing the indicated caspase-8 mutants in the absence (c) or presence (d) of stably expressed RIPK3, following transfection with scramble or FLIP-targeted siRNA, and 12 hours TNF treatment. (Error bars are s.d., n=3) e, f. Cell death (PI uptake) of SVEC 4–10 cells stably expressing RIPK3 specific (e) or scramble (f) shRNA, transfected with the indicated siRNAs and treated with TNF for 12 hours. (Graph is mean of 2 independent experiments, error bars indicate range) g. Cell death (PI uptake) of L929 cells expressing scramble or RIPK3-specific shRNA, transfected with siRNAs specific for caspase-8 and/or FLIP as indicated. Death was assessed 48h post-transfection. (Error bars are s.d., n=3.) The data presented are representative of results obtained with either of 2 separate siRNAs to both caspase-8 and FLIP.