Fig 2: Cancer cell cytotoxicity of TRAIL-ATNCs via TRAIL receptors. (A) Cell viability following treatment with TRAIL, TRAIL-ATNC, or TRAIL-ATNCIL4rP. MDA-MB-231 cells were incubated with proteins for 24 h and then measured by EZ-count assay. Data represent means ± SEM. (B) The apoptosis inducing ability of TRAIL or TRAIL-ATNC was analyzed with Annexin V and propidium iodide (PI) double staining using flow cytometry. The percentage of annexin V/PI double-negative (viable) cells was plotted. (C) Apoptosis inducing ability of TRAIL or TRAIL-ATNC was blocked by anti-DR4/DR5 antibodies. MDA-MB-231 cells were pre-incubated with DR4/DR5 blocking antibody or control IgG for 1 h and were incubated with TRAIL or TRAIL-ATNC for 3 h. Cell viability was measured with Annexin V and PI double staining using flow cytometry.

Fig 3: 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

Fig 4: 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 5: TRAIL induces the assembly of apoptotic and non-apoptotic signaling complex(es). (a) Surface levels of Death Receptor 5 (anti-DR5-488), Death Receptor 4 (anti-DR4 PE), Decoy Receptor 1 (anti-DcR1 PE) and Decoy Receptor 2 (anti-DcR2-488) in BJELR cells. Surface levels of Death Receptor 4 in Hela cells and surface level of Decoy Receptor 1 in BJ cells were used as a positive control for DR4 and DcR1 labeling, respectively. Isotypic IgG1PE or Alexa 488 or IgG2B Alexa 488 labeling was used as a control for background fluorescence. (b) Western blots assessing the co-immunoprecipitation of indicated proteins (canonical DISC components) with Death Receptor 5 (immunoprecipitation target; DR5 IP), Death Receptor 4 (immunoprecipitation target; DR4 IP) or Decoy Receptor 2 (immunoprecipitation target; DcR2 IP) from whole-cell lysates obtained from naïve cells (-) or cells treated with TRAIL for 30 min (+). NF, not detected. (c) Western blots assessing the co-immunoprecipitation of RIPK1 and TRAF2 as well as canonical DISC components (internal control) with Death Receptor 5 (DR5 IP) from whole-cell lysates obtained from naïve cells (-) or cells treated with 1 µg/ml TRAIL for 30 min (+). (d) Western blots assessing the co-immunoprecipitation of RIPK1 and TRAF2 as well as canonical DISC components (internal control) with caspase-8 (immunoprecipitation target; C8 IP) from whole-cell lysates obtained from naïve cells (-) or cells treated with TRAIL for 30 min (+). (b–d) Depicted images correspond to one representative experiment out of three independent biological replicates. Immunoprecipitation using isotypic IgG1 (IgG) was used as a background control