1. Survival Function of the FADD-CASPASE-8-cFLIPL Complex
Christopher P. Dillon, Andrew Oberst, Ricardo Weinlich, Laura J. Janke, Tae-Bong Kang, Tehila Ben-Moshe, Tak W. Mak, David Wallach, Douglas R. Green 
Cell Reports 
Volume 1, Issue 5, Pages (May 2012)
DOI: /j.celrep
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2. Cell Reports 2012 1, 401-407DOI: (10.1016/j.celrep.2012.03.010)
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3. Figure 1
FADD−/−, RIPK3−/− Mice Are Viable and Overtly Normal, Functionally Deficient for FADD, and Display Severe Progressive Lymphoaccumulation
(A) Expected and observed frequency of FADD status in offspring from crosses of FADD+/−, RIPK3−/− animals. The resulting offspring were genotyped at weaning.
(B) Plot of weight of littermate FADD+/+, RIPK3−/−, FADD+/−, RIPK3−/−, and FADD−/−, RIPK3−/− animals.
(C) Effect of anti-CD95 in vivo. A total of 15 μg of agonist anti-CD95 antibody Jo2 was injected intravenously into FADD+/−, RIPK3−/− or FADD−/−, RIPK3−/− animals. Animals were monitored and euthanized when moribund.
(D) Lymphoid organs removed from young (4 weeks) and old (20 weeks) littermate mice of the indicated genotypes. Scale bar represents 1 cm.
See also Figure S1.
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4. Figure 2 FLIP−/−, RIPK3−/− Mice Are Embryonic Lethal
(A) Expected and observed frequency of FLIP status in offspring from crosses of FLIP+/−, RIPK3−/− animals. The resulting offspring were genotyped at the indicated developmental time points or at weaning. Asterisks reflect malformed embryos.
(B) Embryos (left) and hematoxylin and eosin-stained sections of fixed embryos (right) of the indicated genotypes at the indicated time points. Representative images are presented (n ≥ 3 for each genotype). Scale bars for images on left are 1 mm and for sections on right are 500 μm.
(C) Embryos of the indicated genotypes at E13.5 (left and middle) showing vascularization of the embryos and yolk sacs. Left scale bar is 1 mm, and middle scale bar is 333 μm. Right panels show PECAM-1 immunostaining on sections from E11.5 embryos of the indicated genotypes. Representative images are presented (n ≥ 3 for each genotype). Scale bar represents 250 μm.
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5. Figure 3
FLIP-Deficient Cells and Embryos Undergo Apoptosis in the Absence of RIPK3
(A) Immunoprecipitation of a FADD-containing complex from SVEC4-10 cells treated with or without 20 ng/ml TNF and 50 μM zVAD-fmk. NIH 3T3 cells, with (B) or without (C) stably expressed RIPK3, were transfected with the indicated siRNAs for 48 hr, followed by treatment with TNF for 9 hr. At harvest, cultures were split, and cell death was assessed by AnnexinV-APC and PI staining (with AnnexinV+, PI− as apoptotic [pink] and AnnexinV+, PI+ as necrotic or late apoptotic [blue]). The presence of cleaved caspase-3 (green) was assessed by intracellular staining.
(D) Cleaved caspase-3 immunostaining in sections from E9.5–E10 embryos of the indicated genotypes. Arrowheads mark areas of focal cleaved caspase-3 staining. Scale bars are 500 μm.
(E) Cleaved caspase-3 immunostaining in heart section from an E9.5 FLIP−/−, RIPK3−/− embryo. Scale bar represents 100 μm.
For (D) and (E), representative images are presented (n ≥ 3 for each genotype).
See also Figure S2.
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6. Figure 4
FLIP−/−, FADD−/−, RIPK3−/− Mice Are Viable with Overtly Normal Development but Display Severe Progressive Lymphoaccumulation
(A) Expected and observed frequency of FLIP and FADD status in offspring from crosses of FLIP+/−, FADD−/−, RIPK3−/− animals. The resulting offspring were genotyped for FLIP status at weaning.
(B) Plot of weight of littermate animals of the indicated genotypes at different ages.
(C) Lymphoid organs removed from young (6 weeks) and old (16 weeks) littermate mice of the indicated genotypes. Scale bars represent 1cm.
(D) Expected and observed frequency of caspase-8 status in offspring from crossing of αMyHC-Cre+, Casp8+/− with Casp8flox/flox animals.
See also Figures S3 and S4.
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7. Figure S1
Characterization of FADD−/−, RIPK3−/− Mice, Related to Figure 1
(A) FADD+/−, RIPK3−/− and FADD−/−, RIPK3−/− littermates at 6 weeks.
(B) Effects of agonist anti-CD95 injection in indicated genotypes 3 hr post-injection as shown by 10X and 40X magnified sections of livers stained with hematoxylin and eosin (H&E). Scale bars are 250 and 50 μm, respectively. Serum levels of alanine aminotransferase (ALT) (p = ) (C) and aspartate aminotransferase (AST) (p = ) (D) in indicated genotypes pre- and post-(3 hr)injection of anti-CD95.
(E) Percentage of B220+CD3+ cells among peripheral blood mononuclear cells (following red blood cell lysis) in mice of the indicated genotypes and ages.
(F) Proliferation of anti-CD3 plus anti-CD28 activated splenic T cells of the indicated genotypes. Cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and proliferation was assessed at 72 hr.
(G) Proliferation of splenic B cells of the indicated genotypes following the indicated treatments. Cells were stained with CFSE and proliferation was assessed at 72 hr.
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8. Figure S2
Lack of Cleaved Caspase-3 Staining in Embryos of Selected Genotypes, Related to Figure 3
(A and B) Western blot analysis of cells from Figures 3B and 3C.
(C) Cleaved caspase-3 immunostaining of an E10 FADD−/−, RIPK3−/− embryo. Scale bar is 500 μm.
(D) Cleaved caspase-3 immunostaining in heart sections from E embryos of the indicated genotypes. Scale bars are 100 μm. For C and D, representative images are presented, n ≥ 3 for each genotype.
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9. Figure S3
Characterization of FLIP−/−, FADD−/−, RIPK3−/− Mice, Related to Figure 4
(A) PCR-based genotyping analysis of two litters from crosses of FLIP+/−, FADD−/−, RIPK3−/− males and FLIP+/−, FADD+/−, RIPK3−/− females. For FLIP, lower band is wt, upper is knockout. For FADD, lower band is knockout, upper is wt. Note animals #5 and #8 are FLIP−/−, FADD−/−, RIPK3−/− TKO animals.
(B) Photograph of FLIP−/−, FADD−/−, RIPK3−/− and FLIP+/−, FADD+/−, RIPK3−/− littermates at 6 weeks.
(C) Western blot analysis of splenic T cells from animals of the indicated genotypes.
(D) Expected and observed frequency of FLIP and FADD status in offspring from crosses of FLIP+/−, FADD−/−, RIPK3−/− males and FLIP+/−, FADD+/−, RIPK3−/− females.
(E) Proliferation of anti-CD3 plus anti-CD28 activated splenic T cells of the indicated genotypes. Cells were stained with CFSE and proliferation was assessed at 72 hr.
(F) Western blot analysis of T cells from animals of the indicated genotypes harvested at the indicated times post anti-CD3 plus anti-CD28 stimulation.
(G) Percentage of B220+CD3+ cells among peripheral blood mononuclear cells, spleen, or lymph node (following red blood cell lysis) in mice of the indicated genotypes and ages.
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10. Figure S4
Model of the Survival Function of the FADD-Caspase-8-FLIP Complex, Related to Figure 4
(A) TNF, or other poorly defined upstream signals, cause the formation of a complex containing RIPK1, RIPK3, FADD, FLIP, and caspase-8. In this context, there is no RIPK3 activation and no caspase-8 homodimer formation, so cells survive and development is normal. In the absence of FADD or caspase-8, RIPK3 becomes activated leading to necrosis and embryonic lethality. However, if both FLIP and RIPK3 are absent, caspase-8 homodimer can form, driving apoptosis and causing embryonic lethality. Ablation of FADD together with FLIP and RIPK3 prevents both necrosis and apoptosis, so cells survive and development is normal.
(B) Assessment of cre-mediated recombination in αMyHC-Cre+, Casp8flox/flox mice via PCR-based genotyping.
Cell Reports 2012 1, DOI: ( /j.celrep )
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