Extended Data Fig. 10. Myc overexpression and p53 depletion stimulate Z-RNA/Z-DNA accumulation and ZBP1-dependent necroptosis in pMMECs.

a, Representative images of Z-RNA/Z-DNA immunocytochemistry in sgControl pMMECs. Scale bar, 10 μm. b, Quantification of cells with Z-RNA/Z-DNA aggregation percentage. n = 2 independent experiments; 600 cells for each cell lines. NT vs Trp53^fl/fl; p = 0.0043, NT vs Myc^OE;Trp53^fl/fl; p < 0.0001. c, Western blot confirmation of ZBP, pMLKL and Actin in pMMECs 8 days after sgControl lentiviral infection, demonstrating consistent findings across three independent replicates. d, Western blot analysis of untransduced or Myc^OEp53^−/− pMMECs expressing the indicated sgRNA, 6 h after treatment with DMSO control or 25 µM 2′3′-cGAMP. e, ICC analysis for pMLKL intensity 6 h after treatment with DMSO control or 25 µM 2′3′-cGAMP in Myc^OEp53^−/− pMMECs expressing the indicated sgRNA. n = 2 independent experiments; 10 cells for each cell lines. sgMre11 vs sgMre11+cGAMP; p < 0.011, sgcGas vssgcGas+cGAMP; p = 0.0001. f, Western blot confirming inhibition of Mre11, cGAS, and ZBP1 proteins in Myc^OEp53^−/− pMMECs expressing the indicated sgRNA, in support of the tumorigenesis experiment shown in Fig. 4g. Data are mean ± SEM. Unless otherwise specified, statistical analyses were determined by one-way ANOVA followed by Sidak’s multiple comparison post-test using a two-tailed test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

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