Extended Data Fig. 5: The protection of RDA by ICCB-19/Apt-1 requires TRADD.

a, b, Mass spectrometry analysis shown in Fig. 2b using ICCB-19 (a) or Apt-1 (b) were confirmed by immunoprecipitation-immunoblotting using indicated antibodies, quantified on the right (n = 1), representative of 3 independent experiments. c, MEFs were treated with Flag-mTNF (50 ng/ml) in the presence of Apt-1 (10 μM) for indicated time. The complex I was isolated by anti-Flag beads and denatured in 6 M urea. The complex I was further analysed by immunoprecipitation using anti-M1 (6 M urea) or K63 (3 M urea) ubiquitin antibody under denatured condition. The levels of RIPK1 ubiquitination were analysed by immunoblotting. d, WT and Traf2^−/− MEFs were stimulated by mTNF (1 ng/ml) and 5Z-7-Oxozeaenol (0.5 μM) in the presence of indicated compounds for 8 h. Cell survival was determined by CellTiter-Glo assay. Mean ± s.d. of n = 4 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA, post hoc Bonferroni’s tests. ***P = 0.0001. e, WT and cIap1/2^−/− MEFs were stimulated by mTNF (10 ng/ml) in the presence of vehicle, ICCB-19 (10 μM) or Nec-1 s (10 μM) for indicated time. Cell death was determined by CellTiter-Glo assay. Mean ± s.d. of n = 4 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA. f, MEFs were pretreated with SM-164 (50 nM) for 1 h, then stimulated by mTNF (10 ng/ml) in the presence of vehicle, ICCB-19 (10 μM) or Nec-1 s (10 μM) for indicated time. Cell death was determined by SYTOX Green assay. Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA. ***P = 4 × 10⁻⁵; n.s. not significant, (P = 0.1772). g, cIap1/2^−/− MEFs were stimulated with Flag-TNF (50 ng/ml) for indicated minutes in the presence of vehicle or ICCB-19 (10 μM) and the complex I was pulled down using anti-Flag beads. The levels of activated RIPK1 and total RIPK1 were determined by immunoblotting. h, i, cIap1/2^−/− MEFs (h) and cIAP1-reconstituted cIAP1/2 DKO MEFs (i) were stimulated with Flag-TNF (50 ng/ml) for indicated minutes in the presence of vehicle or Apt-1 (10 μM) and the complex I was pulled down using anti-Flag beads. TRADD recruitment to complex I was determined by immunoblotting, quantified on the right (n = 1), representative of 3 independent experiments. j, k, Fadd-deficient and Ripk1-deficient Jurkat cells were treated with Velcade (50nM) in the presence of ICCB-19 (10 μM), Nec-1 s (10 μM), NAC (100 μM), or zVAD.fmk (20 μM). The activation of caspase-8, PARP cleavage (j), and activation of caspase-3 (k) were determined by immunoblotting. For gel source data, see Supplementary Fig. 1.

Source data