A JNK-Dependent Pathway Is Required for TNFα-Induced Apoptosis Yibin Deng, Xiaoyang Ren, Lin Yang, Yahong Lin, Xiangwei Wu  Cell  Volume 115, Issue 1, Pages 61-70 (October 2003) DOI: 10.1016/S0092-8674(03)00757-8

Figure 1 Mitochondria Pathway Is Critical for TNFα-Mediated Caspase 8 Activation and Apoptosis (A) HeLa cells were transfected with either m-iκB or m-iκB and Bcl-xl (HA-tagged) for 48 hr. A green fluorescent protein (GFP) expressing plasmid (pEGFP-C1, Clontech) was cotransfected to monitor the transfection efficiency and to mark the transfected cells. The cells were treated with TNFα (50 ng/ml) for 8 hr and pictures were taken. (B) HeLa cell extracts were prepared from cells described in (A) and analyzed with antibodies specifically recognizing cleaved human caspase 8 (Casp 8) and cleaved PARP (C-PARP), respectively. Transfected Bcl-xl was detected by anti-HA antibody. Endogenous iκB and transfected m-iκB were detected by an anti-iκB antibody. Expression of tubulin was used as loading controls. (C) p65−/− MEFs were transfected with either control vector or Bcl-xl and stable cell lines were established. Expression of Bcl-xl was analyzed by Western blots using an anti Bcl-xl antibody and tubulin was used as loading control. (D) p65−/− and p65−/−/Bcl-xl cells were treated by TNFα (10 ng/ml) for indicted time points. The apoptotic cells were counted by trypan blue exclusion and percentages of cell death were plotted. The data are derived from two independent experiments. (E) Western blot analysis of procaspase 8 expression in TNFα-treated cells using an anticaspase 8 antibody. Three independent cell lines were tested and similar results were obtained. Cell 2003 115, 61-70DOI: (10.1016/S0092-8674(03)00757-8)

Figure 2 Inhibition of JNK Blocks TNFα-Mediated Caspase 8 Activation and Apoptosis (A) MKK7siRNA blocks TNFα-mediated JNK activation. HeLa cells stably expressing m-iκB were transfected with either vector pSUPER or MKK7siRNA. Cells were treated with TNFα (50 ng/ml) for time indicated after 48 hr transfection and expression of MKK7; total JNK (T-JNK1/2) and phosphorylated JNK (P-JNK1/2) were analyzed by Western blots. (B) HeLa-m-iκB cells were transfected with either GFP or MKK7siRNA-GFP for 48 hr and treated by TNFα (50 ng/ml). Pictures were taken 12 hr after TNFα treatment. (C) Western analysis of MKK7 and cleaved caspase 8 in HeLa-m-iκB cells transfected with GFP and MKK7siRNA-GFP. (D) p65−/− and p65−/− stably expressing MKK7siRNA were treated with TNFα (10 ng/ml) for time indicated. Whole-cell extracts were analyzed by Western blots for MKK7, P-JNK1/2, and T-JNK1/2. (E) The control p65−/− and p65−/− stably expressing MKK7siRNA were treated with TNFα. The pictures were taken 8 hr after TNFα treatment. (F) Apoptotic cells were counted at multiple fields and percentages of survival cells were plotted. The data are the average of two independent experiments. (G) TNFα treated cells were analyzed for procaspase 8 expression by Western blots. Tubulin was used as loading control. Three independent cell lines were tested and similar results were obtained. Cell 2003 115, 61-70DOI: (10.1016/S0092-8674(03)00757-8)

Figure 3 TNFα-Induced Mitochondria Smac Release Requires JNK Activation (A) p65−/−FADD-DN cells were treated for 4 hr with or without TNFα (10 ng/ml). The cells were stained for either cytochrome c or Smac proteins. DNA was visualized by staining with Hoechst 3342. (B) Mitochondria (Mito) and cytosolic (Cyto) extracts of TNFα-treated (50 ng/ml) and untreated HCT116-m-iκB-FADD-DN cells were blotted for Smac and cytochrome c expression. Mitochondria CoxIV and cytosolic actin were used as subcellular fraction controls. (C) HeLa-m-iκB-FADD-DN cells were transiently transfected with plasmids pSUPER and Smac-GFP, or Smac-GFP and MKK7siRNA. Distribution of Smac-GFP was viewed under a fluorescent microscope before and after TNFα treatment for 12 hr (50 ng/ml). (D) GFP positive cells were counted at multiple fields and percentages of cytosolic GFP cells were plotted. The data are derived from three independent experiments. (E) JNK-MKK7-wt and JNK-MKK7-kd (Myc-tagged) were transiently transfected into HeLa-m-iκB-FADD-DN cells along with either Smac-GFP or a Cyto C-GFP. Expression of JNK-MKK7 proteins was analyzed using anti-Myc antibody. (F) GFP positive cells were counted at multiple fields and percentages of cytosolic GFP cells were plotted. The data are derived from three independent experiments. Cell 2003 115, 61-70DOI: (10.1016/S0092-8674(03)00757-8)

Figure 4 Caspase 8-Independent and JNK-Dependent Bid Cleavage in TNFα Signaling (A) p65−/− and p65−/− stable cell lines expressing MKK7siRNA or FADD-DN were treated with TNFα (10 ng/ml) for time indicated. Expression of full-length Bid was analyzed using a mouse specific anti-Bid antibody. (B) HeLa-m-iκB-FADD-DN cells were transiently transfected with either pSUPER vector or MKK7siRNA. After 48 hr, the transfected cells were treated with TNFα (50 ng/ml) for 12 hr. HeLa cells were treated with TRAIL (100 ng/ml) for 6 hr. Expression of Bid and MKK7 was examined by Western blots. (C) HeLa-m-iκB-FADD-DN cells were transiently transfected with either Bid-GFP or Bid-GFP and MKK7siRNA. The transfected cells were treated with TNFα (50 ng/ml) for 12 hr. Mitochondrial GFP fluorescence was counted and plotted and pictures were taken. The data represents three independent experiments. Cell 2003 115, 61-70DOI: (10.1016/S0092-8674(03)00757-8)

Figure 5 Identification and Characterization of JNK-Mediated Bid Cleavage (A) In vitro JNK-mediated Bid cleavage. In vitro translated Bid protein labeled with 35S-methionine was incubated with cytosol extracts prepared from either TNFα-treated or nontreated HeLa-m-iκB-FADD-DN and HeLa-m-iκB-FADD-DN cell expressing MKK7siRNA. The reaction mix was separated on a 12% SDS-PAGE and subject to autoradiography. In vitro translated tBid protein labeled with 35S-methionine was used as size comparison. (B) In vivo JNK-mediated Bid cleavage. HeLa-m-iκB and HeLa-m-iκB-FADD-DN cells were transfected with Bid-GFP and treated with TNFα (50 ng/ml) for 8 hr. Transfected Bid proteins were detected by an anti-GFP monoclonal antibody (Santa Cruz). HeLa cells transfected with tBid-GFP were used as size comparison. (C) Detection of endogenous jBid. HeLa-m-iκB-FADD cells were transfected with either MKK7siRNA or pSUPER vector. After 48 hr, cells were treated with or without TNFα (50 ng/ml) for 6 hr. Expression of Bid and MKK7 were detected by Western blots. Tubulin was used as loading control. The experiments were repeated multiple times and representative results were presented. (D) Bid-GFP or GFP fused deletion mutants of Bid was transfected into HeLa cells. Subcellular localization of transfected proteins was observed using GFP fluorescence and mitochondria localization was verified by colocalizing with MitoTracker-Red (Molecular Probes). Apoptosis were plotted as described in Figure 1D. Data represents the average of two independent experiments. (E) Size comparison of jBid with Bid deletion mutants. Extracts from HeLa cells transfected with either BidΔ12-GFP, or BidΔ25-GFP, or BidΔ30-GFP, or BidΔ40-GFP, and extract from TNFα treated HeLa-m-iκB-FADD-DN cells transfected with Bid-GFP were separated on a 7% SDS-PAGE. The transfected proteins were detected by an anti-GFP monoclonal antibody. (F) In vitro cleavage of Bid mutants. Wild-type full-length Bid and alanine scanning mutants of Bid for amino acid 23–27 were transcripted and translated and labeled with 35S-methionine (top image) and subject to in vitro Bid cleavage assay using extracts of TNFa-treated HeLa-m-iκB-FADD-DN cells as described in Figure 5A (bottom image). The nonspecific band generated by in vitro translation was indicated by an *. (G) BidΔ25-GFP was transfected into HeLa cells and cells were stained for either cytochrome c or Smac proteins. DNA was visualized by staining with Hoechst 3342. (H) Bid-GFP, BidΔ25-GFP, and tBid-GFP were separately transfected into HeLa cells. Smac and Cyto C localization were examined by immunostaining. Data from three independent experiments were plotted. Cell 2003 115, 61-70DOI: (10.1016/S0092-8674(03)00757-8)

Figure 6 Requirement of Bid for TNFα-Mediated Caspase 8 Activation and Apoptosis (A) GFP-m-iκB was transiently transfected into Bid−/− and wild-type MEFs. The cells were treated with TNFα (10 ng/ml) for 12 hr and expression of Bid; GFP-m-iκB was examined by Western blots. (B) Morphology of GFP positive cells as described in (A). (C) HeLa-m-iκB cells were transfected with either GFP or BidsiRNA-GFP for 48 hr. The cells were treated with TNFα (50 ng/ml) for 16 hr. Expression of Bid and cleaved caspase 8 were examined by Western blots. (D) Cells were counted at multiple fields and percentages of apoptotic cells were plotted. (E) JNK-mediated cleavage of wild-type and L25A mutant Bid in cells. Bid-GFP or BidL25A-GFP was transfected into HeLa-m-iκB-FADD-DN cells and Western analysis was performed as in Figure 5B. (F) Rescue of TNFα-mediated apoptosis in Bid−/− cells. Bid−/− MEFs were transfected with either Bid-GFP or BidL25A-GFP along with m-iκB. The transfected cells were treated with TNFa and percentages of apoptosis were plotted. Cell 2003 115, 61-70DOI: (10.1016/S0092-8674(03)00757-8)

Figure 7 Smac Is Involved in TNFα-Mediated Caspase 8 Activation and Apoptosis (A) HeLa cells were transfected with GFP and either Ub-Smac or Ub-ΔAVPI-Smac (HA-tagged), and cells were treated with TNFα (50 ng/ml) for 16 hr. Expression of Smac and cleaved caspase 8 were detected using anti-HA and anti-human cleaved caspase 8 antibodies, respectively. (B) Quantitation of apoptosis of GFP positive cells in (A) was plotted. (C) HeLa-m-iκB (m-iκB) and HeLa-m-iκB-MKK7siRNA (m-iκB-MKK7siRNA) cells were treated with Smac peptide N7 (50μM), control peptideN7-NP (50μM), and TNFα (50 ng/ml) in various combinations as indicated and cell extracts were analyzed for cleaved caspase 8 by Western blots. (D) GFP and SmacsiRNA-GFP were transfected into HeLa-m-iκB cells for 48 hr. Cells were treated with TNFα (50 ng/ml) for 16 hr. Expression of Smac and cleaved caspase 8 were analyzed by Western blotting. (E) Percentage of apoptosis in GFP positive cells described in (D) was plotted. Three independent experiments were performed. (F) N7 leads to TRAF2-cIAP1 dissociation. HeLa cells were treated for 1 hr with either TNFα (50 ng/ml) or TNFα and N7 (50μM). Whole-cell extracts were immunoprecipitated with an antibody against TRAF2 and blotted with anti-cIAP1 and anti-TRAF2 antibodies respectively. Ig: immunoglobulin heavy chain. Cell 2003 115, 61-70DOI: (10.1016/S0092-8674(03)00757-8)