TNF-α Induces Two Distinct Caspase-8 Activation Pathways Lai Wang, Fenghe Du, Xiaodong Wang  Cell  Volume 133, Issue 4, Pages 693-703 (May 2008) DOI: 10.1016/j.cell.2008.03.036 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Smac Mimetic Induces TNF-α-Dependent Cell Death without Affecting NF-κB Activation (A) Panc-1 cells were treated with indicated increasing concentration of TNF-α in the presence or absence of 100 nM Smac mimetic for 48 hr. Cell survival was determined by measuring ATP levels using Cell Titer-Glo kit. Data were represented as mean ± standard deviation of duplicates. (B) Panc-1 cells were treated with 100 ng/ml TNF-α in the presence or absence of 100 nM Smac mimetic. Identical concentrations were used in later experiments unless otherwise stated. Cell lysates were prepared using freezing and thawing method as described in Experimental Procedures. Caspase activity was determined using a caspase-3 flurogenic substrate. Data were represented as mean + standard deviation of three independent experiments. (C) Panc-1 cells were treated as indicated and later were harvested in lysis buffer as described in Experimental Procedures. Changes in IκB levels were measured by western blot analysis. β-actin was used as loading control. T: TNF-α; S: Smac mimetic. (D) NF-κB activation luciferase reporter assay in T98G cells. T98G cells were transfected and treated as described in experimental procedures. Fold activation was calculated by normalizing firefly luciferase activity/renilla luciferase activity of each treatment to that of z-VAD treatment. Data were represented as mean + standard deviation of duplicates. All experiments were repeated at least three times with similar results. Cell 2008 133, 693-703DOI: (10.1016/j.cell.2008.03.036) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 c-FLIP Differentially Regulates Smac Mimetic or Cycloheximide-Induced TNF-α-Dependent Apoptosis (A) After Smac mimetic and/or TNF-α treatment, Panc-1 cells were harvested and an aliquot of cell lysate was subjected to western blot analysis of c-FLIP and β-actin levels. (B) To determine the effect of cycloheximide on c-FLIP, Panc-1 cells were treated with cycloheximide or PBS vehicle for 1 hr prior to the treatment of TNF-α. The time point of TNF-α addition was designated as 0 hr. Four microgram per ml of cycloheximide was used to treat the cells. Identical concentration was used in later experiments unless otherwise stated. At the indicated time points, cell lysates were collected and subjected to western blot analysis of c-FLIP and β-actin levels. (C) Parental or Flag-c-FLIP stably overexpressing Panc-1 cells (Clone 2–9 and Clone 2–8) were treated for 48 hr and cell viability was determined by measuring ATP levels. Cell survival rate was normalized to that of the corresponding treatment without TNF-α. Data were represented as mean ± standard deviation of duplicates. (D) Cell lysates from parental Panc-1 cells and Flag-c-FLIP overexpressing stable cells were collected and subjected to western blot analysis of c-FLIP and β-actin levels. To be noted, the c-FLIP encoded by the transgene migrated slightly bigger on the SDS-PAGE because of a FLAG tag at its N-terminus. All experiments were repeated at least three times with similar results. Cell 2008 133, 693-703DOI: (10.1016/j.cell.2008.03.036) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 RIPK1 Is Essential for Smac Mimetic, but Not for Cycloheximide-Induced TNF-α -Dependent Cell Death (A and B) Panc-1 cells were transfected with the indicated siRNAs to determine RIPK1 dependence. Forty-eight hours post-transfection, cells were treated as indicated. After another 24 hr, cell viability was determined by measuring ATP levels. Data were represented as mean + standard deviation of duplicates. Similar results were observed using individual oligoes from siRNA pools (data not shown). (C) The RNAi efficiency of RIPK1 siRNA. Forty-eight hours post transfection, cell lysates from cells transfected with luciferase (Luc) or RIPK1 siRNA were collected and subjected to western blot analysis of RIPK1 and β-actin levels. Western blot analysis of caspase-8 and c-FLIP RNAi efficiency is shown in Figures S4B and S5D. (D–H) H2009 RIPK1-shRNA, RIPK1 wild-type (WT) with shRNA target Scramble (Scr), or RIPK1 kinase dead mutant (K45A) Scr stable cells generated as described in the Experimental Procedures were treated with or without 0.03 μg/ml tetracycline for 72 hr prior to any additional treatment. (D) RIPK1-shRNA cells were treated with or without TNF-α plus Smac mimetic for additional 72 hr. Cell viability was determined by measuring ATP levels. Data were represented as mean + standard deviation of duplicates. (E) RIPK1-shRNA cells were treated with cycloheximide (10 μg/ml) or PBS vehicle for 1 hr prior to the treatment of TNF-α or PBS vehicle for additional 24 hr. Cell viability was determined by measuring ATP levels. Data were represented as mean + standard deviation of duplicates. (F) After 72 hr treatment with or without tetracycline, cell lysates were collected and subjected to western blot analysis of RIPK1 and β-actin levels. (G) H2009 RIPK1-WT Scr or RIPK1-K45A Scr stable cells were treated with or without TNF-α plus Smac mimetic for additional 48 hr. Cell viability was determined by measuring ATP levels. Data were represented as mean + standard deviation of duplicates. (H) After 72 hr treatment with or without tetracycline, cell lysates were collected and subjected to western blot analysis of RIPK1 and β-actin levels. To be noted, the RIPK1 encoded by the transgene migrated slightly bigger on the SDS-PAGE because of a FLAG tag at its C terminus. All experiments were repeated at least three times with similar results. Cell 2008 133, 693-703DOI: (10.1016/j.cell.2008.03.036) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 FADD and TRADD Play Opposite Roles in Smac Mimetic-Induced TNF-α-Dependent Apoptosis (A and B) Panc-1 cells were transfected with the indicated siRNAs for 48 hr prior to any treatment. (A) Cells were treated with TNF-α plus Smac mimetic for the indicated time. Cell lysates were collected for western blot analysis of PARP, FADD, TRADD, and β-actin levels. ∗ indicates a cross reactive band. (B) Cells were treated with or without TNF-α plus Smac mimetic for 24 hr. Cell viability was determined by measuring ATP levels. Data were represented as mean + standard deviation of duplicates. The effect of TRADD RNAi was verified using individual oligoes from siRNA pool (data not shown). (C) Parental or stably expressing dominant negative FADD Panc-1 cells were treated as indicated for 48 hr and cell viability was determined by measuring ATP levels. Cell survival rate was normalized to that of the corresponding treatment without TNF-α. Data were represented as mean ± standard deviation of duplicates. (D) Cell lysates from parental Panc-1 cells and FADD dominant negative cells were collected and subjected to western blot analysis of FADD and β-actin levels. All experiments were repeated at least twice with similar results. Cell 2008 133, 693-703DOI: (10.1016/j.cell.2008.03.036) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Smac Mimetic Plus TNF-α Induces the Formation of a Large Caspase-8 Activating Protein Complex Consisting of RIPK1 and FADD Panc-1 (A, D, E, and F) or H2009 stable cells (B and C) were treated as indicated and lysates were immunoprecipitated with a caspase-8 antibody as described in Experimental Procedures. Twenty micro-molar of Pan-caspase inhibitor z-VAD (Z) was added to prevent full activation of caspase-8 and subsequent dissociation from the complex. Identical concentration was used in later experiments unless otherwise stated. The caspase-8 immunocomplex was eluted by acid (A, B, and C) or caspase-8 peptide (D, E, and F). RIPK1, FADD, and caspase-8 levels were measured by western blot analysis. (A) Time course of RIPK1/FADD/caspase-8 complex formation. H2009 RIPK1-shRNA (B) or H2009 RIPK1-WT Scr and H2009 RIPK1-K45A Scr cells (C) were treated with or without 0.03 μg/ml tetracycline for 72 hr and cells were then treated as indicated for additional 6 hr. D: DMSO. ∗ indicates a cross reactive band of IgG. (D and E) Panc-1 cells were treated for 3 hr. (D) RIPK1, caspase-8, and FADD protein levels in the cell lysates (Input) were compared with those in the caspase-8 immunocomplex. One percent of input and the whole caspase-8 peptide elution were used for western blot analysis. (E) One eighth of the caspase-8 peptide elution (1st elution) was saved for Western blot analysis. The rest of elution was diluted in lysis buffer and further immunoprecipitated using an anti-FADD antibody. The entire acid elution of FADD immunocomplex (2nd elution) was used for Western blot analysis. ∗ indicates a cross reactive band of IgG. (F) Fifty microliters of caspase-8 peptide elution was loaded on to Superose 6 3.2/30 column. One hundred microliters per fraction was collected and half of it was analyzed for RIPK1, caspase-8 and FADD levels. All experiments were repeated at least three times with similar results. Cell 2008 133, 693-703DOI: (10.1016/j.cell.2008.03.036) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 cIAP Degradation Is Required for Smac Mimetic-Induced RIPK1/FADD/Caspase-8 Complex Formation (A) Panc-1 cells were treated as indicated. cIAP1, cIAP2, XIAP, and β-actin levels were measured by western blot analysis. (B and C) Prior to Smac mimetic and/or TNF-α treatment, cells were treated with 100 μM MG-132 or DMSO vehicle control for 1 hr. (B) Cells were treated for additional 1 hr and harvested for western blot analysis of cIAP1, cIAP2, XIAP, and β-actin levels. Pan cIAP antibody used here detected both cIAP1 and cIAP2 proteins, but with much higher affinity for cIAP2. (C) Cells were treated for additional 3 hr and harvested for caspase-8 immunoprecipitation. The levels of RIPK1, FADD, and caspase-8 in the immunocomplex were determined by western blot analysis. (D) Panc-1 cells were transfected with luciferase (Luc), cIAP1, and/or cIAP2 siRNA. Forty-eight hours post transfection, cells were treated as indicated for 4 hr. Cells were then harvested and analyzed for the caspase-8 immunocomplex as described in Experimental Procedures. Similar results were observed using another set of siRNA oligoes against cIAP1 or cIAP2 (data not shown). (E) The RNAi efficiency of cIAP1/2 siRNAs. Forty-eight hours post transfection, cell lysates from cells transfected with respective siRNA(s) were collected and subjected to western blot analysis of cIAP1, cIAP2, and β-actin levels. All experiments were repeated at least three times with similar results. Cell 2008 133, 693-703DOI: (10.1016/j.cell.2008.03.036) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Smac Mimetic Triggers RIPK1 Release from the Receptor and Deubiquitination of RIPK1 Is Required for RIPK1-Dependent Caspase-8 Activation (A) After the indicated treatment, receptor complex was captured with GST pull-down as described in the Experimental Procedures. TNF-RI, RIPK1, and cIAP1/2 levels in the receptor complex were measured by western blot analysis. (B) HEK293T cells were transfected with RIPK1 and/or cIAP expression plasmids. Forty-eight hours post transfection, cells were harvested for immunoprecipitation with HA agarose beads as described in experimental procedures. Flag, HA, and β-actin antibodies were used for western blot analysis. (C) Mapping of RIPK1 or Smac mimetic interaction sites on cIAP1. Experiments are described in details in Figure S12. (D and E) Panc-1 cells were transfected with the indicated siRNAs. Forty-eight hours post transfection, cells were treated for additional 24 hr (D) or 3 hr (E). (D) Cell viability was determined by measuring ATP levels. Data were represented as mean + standard deviation of duplicates. Similar result was obtained with another set of siRNA oligo against CYLD (data not shown). (E) Cells were harvested and analyzed for caspase-8 immunocomplex as described in Experimental Procedures. (F) RNAi efficiency of CYLD siRNA. Forty-eight hours post transfection, cell lysates from cells transfected with respective siRNA were collected and subjected to western blot analysis of CYLD and β-actin levels. All experiments were repeated at least three times with similar results. Cell 2008 133, 693-703DOI: (10.1016/j.cell.2008.03.036) Copyright © 2008 Elsevier Inc. Terms and Conditions