Extended Data Fig. 5. ZBP1-dependent cell death induced by CBL0137.

a, Mlkl^−/− or Mlkl^−/−Casp8^−/− MEFs were treated or untreated with CBL0137 (5 μM) in the presence or absence of zVAD (50 mM) and viability was examined at 18 hrs post treatment. b, Z-DNA formation in primary early-passage (p<5) Zbp1^+/+ and littermate control Zbp1^−/− MEFs treated with CBL0137 (5 μM). c, Quantification of fluorescence intensity of Z-DNA signals in b. d, Zbp1^+/+ and Zbp1^−/− MEFs were treated with CBL0137 (5 μM) in the presence or absence of zVAD (50 mM) and RIPK3 inhibitor (R3i) GSK’843 (5 μM) and viability was examined at 18 hrs post treatment. e, Immunoblot analysis of ZBP1-dependent MLKL activation in primary MEFs. f, Immunoblots showing levels of ZBP1, RIPK3 and MLKL in the human fibroblast cell line HS68 in the presence or absence of hIFNβ (100 ng/mL, 6 hrs). g, Z-DNA formation in HS68 cells treated with CBL0137 (5 μM, 12 hrs). h, Quantification of fluorescence intensity of Z-DNA signals in g. i, hIFNβ pretreated (100 ng/mL, 6 hrs) HS68 cells were exposed to CBL0137 in the presence or absence of zVAD (50 mM) and RIPK3 inhibitor (R3i, GSK’872, 5 μM) and viability was examined at 36 hrs post treatment. j, MLKL activation in hIFNβ pretreated (100 ng/mL, 6 hrs) HS68 cells treated with CBL0137 in the presence or absence of zVAD (50 mM) and RIPK3 inhibitor (R3i, GSK’872, 5 μM) was examined by immunoblot analysis 30 hrs post-CBL0137 treatment. k, CBL0137-induced cell death kinetics in Zbp1^−/− MEFs stably reconstituted with empty vector (Vec), FLAG-ZBP1, or its mutants. l, Immunoblot analysis of MLKL activation in Zbp1^−/− MEFs reconstituted with empty vector (Vec), FLAG-ZBP1, or FLAG-ZBP1 mutants after CBL0137 treatment. m, Primary Zbp1^+/+ and littermate-matched Zbp1^−/− BMDMs were treated with CBL0137 (5 μM, 18 hrs) and stained for Z-DNA (red) and the macrophage marker F4/80 (green). n, Primary Zbp1^+/+ and Zbp1^−/− BMDMs were treated with CBL0137 (5 μM) and viability was examined at 24 hrs post treatment. o, Distribution of FLAG-enriched peaks following treatment with CBL0137 (5μM) for 14 hrs. p, Immortalized Zbp1^−/− MEFs stably reconstituted with either FLAG-ZBP1 or FLAG- ΔZα mutant were treated with CBL0137 (1.5 μM, 14 hrs), and anti-FLAG immunoprecipitates were examined for FLAG. Whole-cell extract (5% input) was examined in parallel for FLAG. GAPDH was used as a loading control. Data are mean ± s.d. (n = 4 in a, d, i, k, n or n = 20 in c, h per group). Two-tailed unpaired t-test with Welch’s correction (a, c, n), one-way ANOVA test (d, i) or two-way ANOVA test (k). ***P < 0.0005 (P = 0.0002 in a, P < 0.0001 in c, d, i, k, n). Data are representative of at least three independent experiments.