1. RIP3 Induces Apoptosis Independent of Pronecrotic Kinase Activity
Pratyusha Mandal, Scott B. Berger, Sirika Pillay, Kenta Moriwaki, Chunzi Huang, Hongyan Guo, John D. Lich, Joshua Finger, Viera Kasparcova, Bart Votta, Michael Ouellette, Bryan W. King, David Wisnoski, Ami S. Lakdawala, Michael P. DeMartino, Linda N. Casillas, Pamela A. Haile, Clark A. Sehon, Robert W. Marquis, Jason Upton, Lisa P. Daley-Bauer, Linda Roback, Nancy Ramia, Cole M. Dovey, Jan E. Carette, Francis Ka-Ming Chan, John Bertin, Peter J. Gough, Edward S. Mocarski, William J. Kaiser 
Molecular Cell 
Volume 56, Issue 4, Pages (November 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 56, 481-495DOI: (10.1016/j.molcel.2014.10.021)
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3. Figure 1 Small-Molecule RIP3 Inhibitors Block Programmed Necrosis
(A) Chemical structure of three distinct RIP3 kinase inhibitor (RIP3i) compounds, GSK’840, GSK’843, and GSK’872.
(B) Dose response of RIP3i compound binding to recombinant human RIP3 kinase (aa 1-328) assessed by FP. The IC50 was calculated from % maximum binding (mean ± SEM).
(C) Dose response of RIP3i on recombinant human RIP3 kinase activity assessed by ADP-Glo assay. The IC50 was calculated from the response to increasing concentrations of compound (mean ± range of 4, 4, and 2 replicates of GSK’840, GSK’843, and GSK’872, respectively).
(D) Relative viability of human HT-29 cells 24 hr posttreatment (hpt) with TNF (10 ng/ml), zVAD-fmk (zVAD; 20 μM), and SMAC007 (100 nM) in the presence of increasing concentrations of RIP3i, assessed by determining ATP levels (mean ± range is shown) compared to cells treated with vehicle (DMSO) alone.
(E) Relative viability of murine PECs treated and graphed as described in (D).
(F) Relative viability of necrosis-sensitive murine L929 cells treated for 18 hr with TNF (25 ng/ml) plus zVAD (25 μM) (left) or primed with interferon (IFN)β (50 U/ml) for 18 hr followed by stimulation with poly(I:C) (50 μg/ml) plus indicated concentrations of GSK’872 ± zVAD (right) and assessed by determining ATP levels (mean ± SEM) compared to cells treated with vehicle (DMSO) alone.
(G) Relative viability of the murine endothelial cell line SVEC4-10 (SVEC) 18 hpt with TNF (25 ng/ml) plus zVAD (25 μM) in increasing concentrations of GSK’872 (left) or 18 hpi with WT or M45mutRHIM MCMV (MOI = 10) ± zVAD (25 μM) in increasing concentrations of GSK’872 (right), assessed as described in Figure 1F. See also Figure S1 and Table S1.
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4. Figure 2 RIP3i-Induced Apoptosis
(A) Relative viability of SVEC, L929, 3T3SA, and mouse embryo fibroblast (MEF) 18 hpt with increasing concentrations of GSK’872 (black bars) or GSK’843 (gray bars) ± zVAD (25 μM), assessed as described in Figure 1F.
(B) Time course cell viability analysis of 3T3SA cells measuring permeability to the nucleic acid detecting stain SYTOX Green (50 nM) using an IncuCyte instrument. Cells were treated with GSK’872 (10 μM) in the ± zVAD or with TNF (25 ng/ml) plus cyclohexamide (CHX; 5 μg/ml) (left), or with TNF and zVAD ± GSK’872 (right).
(C) Immunoblot (IB) for Casp3 and cleaved Casp3 (Cl-Casp3) products in 3T3SA cells at the indicated hpt with GSK’872 ± zVAD.
(D) Time course of Casp3/Casp7 proteolytic activity (DEVDase) in 3T3SA cells treated with GSK’872 ± zVAD.
(E) Transmission electron microscopy images of 3T3SA cells treated with DMSO or GSK’872 for 2.5 hr. See also Figure S2 and Movies S1, S2, and S3.
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5. Figure 3
Concentration-Dependent Apoptosis of GSK’840, GSK’843, and GSK’872 Requires RIP3
(A) Relative viability of NIH 3T3 cells (left) 18 hpt with increasing concentrations of GSK’872 (black bars) or GSK’843 (gray bars) or 3T3SA cells (right) in 10 μM RIP3i compounds, ± zVAD, assessed as described in Figure 1F.
(B) IB showing RIP3 and β-actin levels in NIH 3T3 and 3T3SA cells.
(C) Relative viability at 18 hpt with GSK’872 (10 μM) in 3T3SA, SVEC, and L929 cells, transduced with nontargeting (NT) shRNA (black bars) or RIP3-specific (gray bars) shRNA.
(D) Analysis of Casp3/Casp7 proteolytic activity (DEVDase) in transduced 3T3SA cells at 4 hpt with GSK’843, GSK’872, or TNF plus CHX.
(E) Relative viability comparing WT (black bars) and Rip3−/− (gray bars) MEF at 18 hpt with GSK’872 ± zVAD.
(F) Relative viability comparing 3T3SA cells transfected with NT or MLKL-specific siRNA 18 hpt with GSK’872 ± zVAD or with TNF plus zVAD ± GSK’872. An IB inset (right) shows the levels of MLKL prior to any other treatment.
(G) Relative viability comparing Rip3−/− MEF alone and after transduction with human hRIP3 and hRIP3mutRHIM 18 hpt with GSK’840, GSK’843, or GSK’872 or with a combination of TNF (25 ng/ml), zVAD (25 μM), and BV6 (0.5 μM).
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6. Figure 4
Haploid Genetic Screen for Genes Involved in RIP3-Mediated Cell Death
(A) Haploid screen results depicting each gene as a bubble, where size corresponds to the number of independent gene trap insertions (also indicated in parentheses); the significance of enrichment is plotted on the y axis. The top 25 most significantly enriched genes are labeled, colored, and horizontally grouped by function (other genes are gray and in arbitrary position along the x axis).
(B) IB of the indicated components from anti-FADD immunoprecipitation of Triton X-100-solubilized 3T3SA cell supernatants (IP: FADD; top section), together with total supernatant (middle section) and total pellet (bottom section) fractions. Cells were assessed without treatment (untreated; lane 1), as well as 2.5 hpt with vehicle alone (DMSO; lane 2), zVAD (25 μM) alone (lane 3), and varying concentrations (10, 3, 1 and 0.3 μM) of GSK’872 in the presence (lanes 4 through 7) or absence (lanes 8 through 11) of zVAD. Single asterisk indicates modification of RIP1 in the presence of GSK’872 when caspases are active (lane 8), and double asterisks indicate slower migrating forms of RIP3 in the pellet fraction (lanes 4, 5, and 8). See also Figure S3.
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7. Figure 5
Requirement for Casp8, FADD, and RIP1 in RIP3-Initiated Apoptosis
(A) Relative viability of 3T3SA cells 18 hpt with GSK’872 in the absence (DMSO) or presence of caspase inhibitors (zVAD, zYVAD zLEHD, and zIETD; 25 μM), an inhibitor of autophagy, 3-methyladenine (3-MA; 5mM), or an inhibitor of reactive oxygen species, butylated hydroxyanisole (BHA; 25 μM), assessed as described in Figure 1F.
(B) Analysis of Casp8 proteolytic activity (IETDase) in 3T3SA cells treated with GSK’872 for the indicated times ± zVAD.
(C) IB of Cl-Casp8 in 3T3SA cells treated with GSK’872 for the indicated times ± zVAD.
(D) Relative viability of 3T3SA cells transfected with NT (black bars) or Casp8-specific (gray bars) siRNA and treated as described in (C). An IB inset on the right shows the level of Casp8 knockdown.
(E) Relative viability of 3T3SA cells transduced with EV (black bars) or FADD-DN-expressing retrovirus vector (gray bars) 18 hpt with GSK’872, zVAD and/or TNF, as indicated.
(F) Relative viability of 3T3SA cells transfected with NT (black bars) or RIP1-specific siRNA (gray bars) 18 hpt with GSK’872, zVAD and/or TNF. An IB inset on the right shows level of RIP1 knockdown.
(G) Relative viability of WT (black bars) or Rip1K45A/K45A mutant MEF (gray bars) 18 hpt with GSK’872 ± zVAD.
(H) Relative viability of different MEF genotypes 18 hpt with GSK’872.
(I) Relative viability of primary WT or cFLIP−/− MEF treated with GSK’872, TNF, zVAD, and/or BV6.
(J) Relative viability of cFLIP−/− MEF transduced with EV or cFLIPL-expressing retrovirus 18 hpt with GSK’872 or TNF.
(K) Analysis of Casp8 proteolytic activity (IETDase) in cFLIPL-deficient MEFs 4 hpt with GSK’872 and TNF.
(L) Relative viability in SVEC cells transduced with EV (white bar) or M45-expressing retrovirus (black bars), or M45mutRHIM-expressing retrovirus (gray bars) 18 hpt with GSK’872 alone or TNF plus zVAD. See also Figure S4.
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8. Figure 6 Characterization of RIP3 Kinase Domain Mutants
(A) Table summarizing microscopic assessment of Rip3−/− MEF survival 3 days after transduction with WT RIP3 or indicated mutants ± zVAD.
(B) Relative viability of transduced cells 18 hr posttreatment with TNF, zVAD, and GSK’872, as indicated and assessed as described in Figure 1F.
(C) IB for FLAG-RIP3 levels in transduced cells.
(D) Relative viability of RIP3shRNA-transduced 3T3SA cells following transfection with shRNA-resistant RIP3 or indicated RIP3 mutants for 16 hr and treated with GSK’872, zVAD, and TNF.
(E) IB of the indicated components from RIP3shRNA-transduced 3T3SA cells showing anti-FLAG IP, supernatant, and pellet fractions 8 hr posttransfection with RIP3 or mutants (K51A and D161N) ± GSK’872 for 2 hr prior to harvesting.
(F) IB of the indicated components in unmanipulated supernatants and pellets of RIP3shRNA transduced 3T3SA cells 16 hr after transfection with shRNA RIP3 or mutants (K51A and D161N) ± GSK’872 without or with zVAD for 3 hr prior to harvesting. See also Figure S5.
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9. Figure 7 Characterization of RIP3 Kinase-Inactive Mice
(A) Schematic representation of the WT, recombined, cre-mediated excised, and Flp-mediated excised RIP3 alleles with the relevant restriction sites for the DNA blot analysis.
(B) Photograph of age-matched WT, Rip3K51A/K51A, and Rip3K51A/K51ACasp8−/− mice.
(C) Relative viability of WT and Rip3K51A/K51A MEF 18 hpt with GSK’872, TNF, zVAD, and BV6, as indicated and as assessed in Figure 1F.
(D) Relative viability of WT and Rip3K51A/K51A mutant BMDM 18 hpt with GSK’872, TNF, zVAD, BV6, poly (I:C)/zVAD, or LPS/zVAD for 18 hr.
(E) Relative viability of WT (left) and Rip3K51A/K51A (right) MEF transfected with either NT siRNA, RIP1 siRNA, or a combination of RIP1 and RIP3 siRNA for 48 hr and then 48 hpt with IFNβ (100 U/ml), zVAD, and GSK’872, as indicated.
(F) MCMV titers in spleen (left) and liver (right) from mice of indicated genotypes 3 days postinfection.
(G) Recall response to MCMV infection. Total numbers (left) or percentages (right) of splenic CD8+ T cells producing IFNγ or IFNγ and TNFα following stimulation with M45-specific peptide 4 days postchallenge with RM427 virus 14 days postinfection with MCMV-M45mutRHIM in mice of indicated genotypes. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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