Extended Data Fig. 4: ICCB-19/Apt-1 block Velcade-induced apoptosis, RIPK1-dependent apoptosis and necroptosis.

a, b, Jurkat (a) or SH-SY5Y (b) cells were stimulated by Velcade (50 nM) in the presence of Apt-1 (10 μM), Nec-1 s (10 μM), or zVAD (20 μM) for 12 h and 24 h. The levels of cleaved caspase-3 were determined by immunoblotting. c, Tak1^−/− MEFs were treated with 1 ng/ml mTNF in the presence of indicated compounds for 3 h. Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments. One-way ANOVA, post hoc Dunnett’s tests. ***P = 1 × 10⁻¹⁵ (left to right); n.s. not significant, (P = 0.7989). d, Tak1^−/− MEFs were treated as in (a), the cell lysates were analysed by immunoblotting using indicated antibodies. e, MEFs were treated with mTNF (1 ng/ml) and 5Z-7-Oxozeaenol (0.5 μM) in the presence of indicated compounds for 1 h and 2 h and the cell lysates were analysed by immunoblotting using indicated antibodies. f, g, ICCB-19/Apt-1 inhibit RDA, including complex IIa formation (f) and caspase-8 activation (g). MEFs treated as in (e) were lysed with IP buffer and FADD was immunoprecipitated by anti-FADD antibody. Total lysates and IP samples were analysed by immunoblotting to determine the recruitment of RIPK1 to FADD in complex IIa (f). MEFs were treated with mTNF (1 ng/ml) and 5Z-7-Oxozeaenol (0.5 μM) in the presence of ICCB-19 (10 μM), Apt-1 (10 μM), Nec-1 s (10 μM), or zVAD.fmk (20 μM) for 4 h and the activity of caspase-8 was determined using Caspase-Glo 8 Assay Systems (g). Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments. One-way ANOVA, post hoc Dunnett’s tests. ***P = 1 × 10⁻¹⁵ (g). h-k, RDA was induced in Tbk1^−/− MEFs (h, i), Nemo^−/− MEFs (j, k) by the treatment with mTNF (10 ng/ml) together with ICCB-19 (10 μM) and Nec-1 s (10 μM) at indicated times and cell death was determined by SYTOX Green (h, j) and caspase-3 cleavage (CC3) immunoblotting (i, k). Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments (h, j).). Two-way ANOVA. ***P = 5 × 10⁻⁷ (h), 4 × 10⁻⁷ (j). l, RDA was induced in WT MEFs by the treatment with mTNF (10 ng/ml) and IKK inhibitor TPCA-1 (5 μM) in the presence of ICCB-19 (10 μM) or Nec-1 s (10 μM) for indicated times and cell death was determined by SYTOX Green. Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA. ***P = 1 × 10⁻⁵. m, Recombinant active caspase-8 was incubated with vehicle, ICCB-19 (10 μM), Apt-1 (10 μM), or zVAD.fmk (20 μM) for 1h and the activity of caspase-8 was determined using Caspase-Glo 8 Assay Systems. Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments. One-way ANOVA, post hoc Dunnett’s tests. n.s. not significant, (P = 0.9931, 0.9215 (left to right)). n, WT MEFs were treated with mTNF (1 ng/ml) and cycloheximide (CHX, 1 μg/mL) to induce RIA in the presence or absence of ICCB-19 (10 μM) or Nec-1 s (10 μM) for indicated time and cell survival was determined by CellTiter-Glo assay. Mean ± s.d. of n = 8 biologically independent samples, representative of 3 independent experiments. One-way ANOVA, post hoc Dunnett’s tests. n.s. not significant, (P = 0.9962). o, p65/p50 DKO MEFs were treated with mTNF (1 ng/ml) together with ICCB-19 (10 μM) for indicated times and cell survival was determined by CellTiter-Glo assay. Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments. Two-way ANOVA. n.s. not significant, (P = 0.1895). p, MEFs were treated as indicated and the cell survival was measured by CellTiter-Glo assay. The concentrations of reagents used: mTNF: 1 ng/mL; (5Z)-7-oxozeaenol: 0.5 μM; zVAD: 20 μM; ICCB-19: 10 μM; Apt-1: 10 μM; Nec-1 s: 10 μM. Mean ± s.d. of n = 3 biologically independent samples, representative of 3 independent experiments. q, Necroptosis of MEFs was induced by the treatment with TNF/5z7/zVAD in the presence of indicated compounds for indicated hours and the activation of RIPK1(p-S166), RIPK3(p-T231/S232), and MLKL(p-S345) was determined by immunoblotting. r, HEK293T cells were transfected with Flag-RIPK1 expression construct for 12 h in the presence of Nec-1 s (10 μM), ICCB-19 (10 μM), or Apt-1 (10 μM). The activation of RIPK1 was determined by immunoblotting using p-RIPK1(S166) antibody. For gel source data, see Supplementary Fig. 1.

Source data