Fig 2: Role of TRAIL receptors and TRAIL-receptor-binding proteins in the triggering of apoptosis. (a) Percentage of cells with high levels of cleaved PARP in populations transfected with pooled siRNAs targeting DR4 (DR4), DR5 (DR5), DcR2 (DcR2) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged with TRAIL (6 h). (b) Percentage of cells with high levels of cleaved PARP in naïve (control) or TRAIL-challenged (6 h) DR5 knockout (DR5 KO) or DcR2 knockout (DcR2 KO) cells as compared with controls (ctrol). (c) Contour plot for cleaved PARP immunolabelling in DcR2 knockout (DcR2 KO) cells either unchallenged (control) or treated 6 h with TRAIL (TRAIL). Subpopulations displaying low (survivors, '1') and high (apoptotic, '2') levels of PARP cleavage in treated samples are depicted. Image shows one representative experiment out of three independent biological replicates. (d) Percentage of cells with high levels of cleaved PARP in control and DcR2 KO naïve cells (control) or control and DcR2 KO cells challenged with TRAIL for 16 h (TRAIL). (e) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting FADD (FADD), caspase-8 (casp-8), RIPK1 (RIPK1), TRAF2 (TRAF2) or cFlip (cFlip) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged for 6 h with TRAIL (TRAIL). (f) Percentage of cells with high levels of cleaved PARP in populations transfected with siRNAs targeting RIPK1 (RIPK1), TRAF2 (TRAF2) or RIPK1 and TRAF2 (RIPK1+TRAF2) mRNAs or non-targeting scramble siRNAs at concentrations corresponding to single (30 nM; scr 30) or double (60 nM; scr 60) transfections, further left either untreated (control) or challenged during 6 h with TRAIL (TRAIL). Results obtained in conditions of pan-caspase inhibition (control+ zVAD and TRAIL+zVAD) as compared with basal conditions of cell response (control and TRAIL) are shown. (g) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting RIPK1 (RIPK1), TRAF2 (TRAF2), DR5 (DR5), RIPK1 and DR5 (RIPK1+DR5), TRAF2 and DR5 (TRAF2+DR5) mRNAs or non-targeting scramble siRNAs ('scr 30' and 'scr 60') further left either untreated (control) or challenged 6 h with TRAIL (TRAIL). (a, b and d–g) Mean±standard deviation (S.D.) from three independent biological replicates is shown. ***P-value <0.0005, **P-value <0.005, *P-value <0.05. NS, not significant

Fig 3: Apoptotic and pro-survival TRAIL-induced signaling complex(es) are assembled at the plasma membrane. (a) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DcR2, FADD, caspase-8, cFlip) as well as RIPK1 and TRAF2 with Death Receptor 5 (DR5 immunoprecipitation: DR5 IP). (b and c) Western blots assessing the co-immunoprecipitation of DR5, DR4, DcR2, FADD, cFlip and TRAF2 (b) as well as caspase-8 (c) with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP). (d) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DR5, DcR2, FADD, caspase-8, cFlip) as well as RIPK1 with TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP). (e) Western blots assessing the co-immunoprecipitation of DR5, RIPK1 and TRAF2 with FADD (FADD immunoprecipitation: FADD IP). (a–e) Immunoprecipitations were performed from the purified plasma membrane (plasma membrane) obtained from either naïve cells (-) or cells treated with TRAIL (30 min '+' unless indicated otherwise in c). Immunoprecipitation using isotypic IgG1 (IgG) was used as a background control. Protein levels of indicated proteins at the plasma membrane are depicted as 'inputs'. Same blots are depicted as inputs for (d) and (e) as TRAF2 and FADD immunoprecipitation experiments were performed in parallel using the same protein input. Depicted images correspond to one representative experiment out of at least two independent biological replicates. N-Cadherin (N-Cdh) is shown as a plasma membrane marker. Controls for plasma membrane purification are depicted as Supplementary Figure S5. ND, not detected

Fig 4: Role of TRAIL receptors in the triggering of non-apoptotic and pro-survival pathways. (a and b) Western blots displaying total and phosphorylated protein levels of Erk1/2, Akt, p38 and I?Ba in clonal populations of DcR2 knockout (DcR2 KO, a), DR5 knockout (DR5 KO, b) and control BJELR cells in basal conditions (0) or after treatment with TRAIL for the indicated time points. PARP cleavage is shown as a control for TRAIL treatment whereas DcR2 (a) or DR5 (b) is depicted as knockout controls. Images correspond to one representative experiment out of two independent biological replicates. (c) Western blots displaying total and phosphorylated protein levels of Erk1/2, Akt, p38 and I?Ba in BJELR cells challenged with DR5-selective TRAIL-mimetic peptide M1D (10 µM) as compared with controls (0). (d) Western blots displaying total protein and phosphorylation levels of Erk1/2, Akt, p38 and I?Ba in BJELR cells transfected either with siRNAs targeting DR4 and DcR2 (siDR4+siDcR2) mRNAs or non-targeting scramble siRNAs (scr) further left untreated (0) or challenged for 6 h with TRAIL. Uncropped image is shown as Supplementary Figure S4b. Images correspond to one representative experiment out of at least two independent biological replicates. a-Tubulin, loading control

Fig 5: Role of TRAIL receptors in the formation of TRAIL-induced signaling platforms. (a) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR4, DcR2, FADD, caspase-8, cFlip) with Death receptor 5 (DR5 immunoprecipitation: DR5 IP) in plasma membrane fractions obtained from DcR2 knockout (DcR2 KO) and control BJELR cells either left untreated (-) or challenged with TRAIL (30 min '+'). (b) Western blots assessing the co-immunoprecipitation of DR5 with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP) or TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP) in DcR2 knockout (DcR2 KO) and control BJELR cells either left untreated (-) or challenged with TRAIL (30 min '+'). (c) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR5, DcR2, FADD, caspase-8) with Death receptor 4 (DR4 immunoprecipitation: DR4 IP) in plasma membrane fractions obtained from DR5 knockout (DR5 KO) and control BJELR cells either left untreated (-) or challenged with TRAIL (30 min '+'). (d) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR5, DR4, DcR2, FADD) with RIPK1 (RIPK1 immunoprecipitation: RIPK1 IP) or TRAF2 (TRAF2 immunoprecipitation: TRAF2 IP) in DR5 knockout (DR5 KO) and control BJELR cells either left untreated (-) or challenged with TRAIL (30 min '+'). (e) Western blots assessing the co-immunoprecipitation of canonical DISC components (DR5, DR4, DcR2, FADD, cFlip) with caspase-8 (caspase-8 immunoprecipitation: C8 IP) in whole-cell lysates from DR5 knockout (DR5 KO) and control BJELR cells either left untreated (-) or challenged with TRAIL (30 min '+'). (a–c and e) Controls for plasma membrane purification are depicted as Supplementary Figure S5. ND, not detected; NA, not analyzed