Fig 2: TRAF2 overexpression‐induced cell death requires both the RING domain and caspase‐2 shCasp2 #1 cells were transfected with indicated caspase‐2 construct (wild type or 3KR mutant) containing shCasp2‐resistant sequence. Cells were treated with 10 μM cisplatin for 24 h, followed by IB. Relative band intensity of cleaved caspase‐2 was quantified and indicated below each lane (signal was normalized to cisplatin treatment of WT) (top). Transfected cells were treated with 5 μM cisplatin for 24 h, and then analyzed by annexin V staining and flow cytometry. n = 4 independent experiments (means + s.e.m.) (bottom).HeLa cells were transfected with indicated Myc‐TRAF2 construct (wild type or RING domain mutant) for 48 h. Apoptosis was analyzed by annexin V staining and flow cytometry. n = 3 independent experiments (means + s.e.m.) (left). Expression of Myc‐TRAF2 was confirmed by IB (right).HeLa shNT, shCasp2 #1, or shCasp2 #2 cells were transfected as in (B), and apoptosis was analyzed by annexin V staining and flow cytometry. n = 3 independent experiments (means + s.e.m.). Source data are available online for this figure.

Fig 3: TRAF2 interacts directly with caspase‐2 to induce activation A diagram of the caspase‐2 protein showing the major domains and the location of a putative TRAF‐interacting motif (TIM).Casp2(C320A)‐Flag plasmids (wild type or TIM mutant) were expressed in HeLa cells for 24 h followed by anti‐Flag IP and IB.Stably transfected, catalytically active caspase‐2 (wild type or TIM mutant) was induced in HeLa Tet‐On cells with 0.1 μg/ml doxycycline for 24 h, followed by propidium iodide (PI) staining and flow cytometry. n = 3 independent experiments (means + s.e.m.). *P < 0.05 by unpaired two‐tailed t‐test (top). Casp2‐Flag expression was confirmed by IB (bottom).Indicated amounts of Casp2(C320A) BiFC constructs were transiently transfected, and cells were cultured for 24 h followed by 20 μM cisplatin treatment in the presence of 10 μM Q‐VD(OMe)‐OPh for 24 h, and then, BiFC signal was detected by flow cytometry. n = 3 independent experiments (means + s.e.m.). *P < 0.05 by unpaired two‐tailed t‐test. Source data are available online for this figure.

Fig 4: Mutation of the TRAF‐interacting motif of caspase‐2 does not disrupt RAIDD binding, while the caspase‐2 prodomain is also important for binding TRAF2 HEK293T cells were co‐transfected with indicated caspase‐2(C320A)‐Flag (wild type or TIM mutant) and HA‐RAIDD constructs. After 48‐h culture for expression, lysates were prepared, followed by anti‐Flag IP and IB.HEK293T cells were transfected with siRNA targeting caspase‐2 3′‐UTR to deplete endogenous caspase‐2. 24 h post‐siRNA transfection, cells were then transfected with indicated Casp2‐mVenus constructs (full‐length C320A, prodomain, or Δ1–169 C320A (Δprodomain)). Casp2‐mVenus variants were immunoprecipitated by GFP‐Trap, followed by IB for co‐immunoprecipitated TRAF2. Source data are available online for this figure.

Fig 5: TRAF2 shifts active, dimerized caspase‐2 to a detergent‐insoluble fraction in a RING domain‐dependent manner Representative confocal images. HeLa cells were co‐transfected with Casp2(C320A)‐mVenus and Myc‐TRAF2 (wild type or RING domain mutant (ΔRING)) and then allowed to express for 48 h before imaging to detect caspase‐2 localization. Nuclei were visualized by co‐transfection of H2A‐mCherry. Scale bar indicates 10 μm. The percentage of cells with caspase‐2 punctate signals was quantified and listed below each image. At least 40 cells were counted from n = 5 independent experiments.HeLa cells were co‐transfected with Casp2(C320A)‐mVenus and the indicated Myc‐TRAF2 constructs for 48 h and then biochemically fractionated (S: soluble, I: insoluble fraction) to monitor caspase‐2 localization in 0.1% NP‐40‐soluble or ‐insoluble fractions.Casp2pro BiFC cells were treated with 20 μM cisplatin in the presence of 10 μM Q‐VD(OMe)‐OPh for 24 h and then biochemically fractionated as in (B) or lysed as whole cell lysate (WCL). Lysates were subjected to GFP‐Trap IP and analyzed by IB.HeLa cells were co‐transfected with Casp2(C320A)‐mVenus (wild type or 3KR mutant) and Myc‐TRAF2, and cultured for 48 h. Then, cells were biochemically fractionated as in (B) and analyzed by IB to monitor Casp2(320A)‐mVenus localization (top). Casp2(320A)‐mVenus protein level was quantified and plotted. n = 4 independent experiments (means + s.e.m.). *P < 0.05 by unpaired two‐tailed t‐test (bottom).HeLa cells were treated with or without 20 μM cisplatin in the presence of 10 μM Q‐VD(OMe)‐OPh for 24 h and then biochemically fractionated as in (B) to monitor endogenous caspase‐2 localization (top). Caspase‐2 protein level was quantified and plotted. n = 4 independent experiments (means + s.e.m.). *P < 0.01 by unpaired two‐tailed t‐test (bottom). Source data are available online for this figure.