Extended Data Fig. 9: Supporting data regarding TNF/IFNγ-induced cell death signalling in control and Tbk1-null cells.

a, heatmap of % cytochrome C (cyt C) release for in vitro BH3 profiling of unstimulated control (sg1 and sg2) and Tbk1-null (sg1 and sg2) B16 cells. Mean values shown; n=3 biologically independent samples; 2-way ANOVA, Dunnett’s multiple comparisons test. b, heatmap of % cytochrome C (cyt C) release for in vitro BH3 profiling of control sgRNA and Tbk1 sgRNA B16 cells. Mean values shown; n = 3 biologically independent samples; 2-way ANOVA, Tukey’s multiple comparisons test. No statistically significant differences observed between control sgRNA and Tbk1 sgRNA B16 cells at any time point. c, Viability assessment (Cell Titer Glo) of B16 cells in standard 2D culture after 24 h treatment with indicated concentrations of staurosporine (STS) in control and Tbk1-null B16 cells. Means (bars) and individual values (open circles) are shown (n = 6, 2 independent experiments, 2-way ANOVA, Sidak’s multiple comparisons test). d, Viability assessment (Hoechst/propidium iodide) of B16 tumour spheroids (lacking immune cells) in 3D microfluidic culture after 48 h treatment indicated concentrations of staurosporine (STS) compared to unstimulated cells Means (bars) and individual values (open circles) are shown (n = 6, 2 independent experiments, 1-way ANOVA, Holm-Sidak’s multiple comparisons test). e, Western blot for STING, IRF3, TBK1, and β-actin in B16 cells with single CRISPR cell lines with single-guide RNAs targeting Tmem173, Irf3, and Tbk1 compared to control sgRNA. Data are representative of three independent experiments. f, Western blot for STING, IRF3, TBK1, and β-actin in double CRISPR B16 cells with indicated sgRNA pairs. Data are representative of three independent experiments. g, Viability assessment (Cell Titer Glo) of indicated sgRNA B16 cells after 48 h treatment with TNF (160 ng ml⁻¹) + IFNγ (40 ng ml⁻¹) compared to unstimulated cells. Means (bars) and individual values (open circles) are shown (n = 4 biological replicates, 2-way ANOVA, Sidak’s multiple comparisons test, **P < 0.01; **** P < 0.0001; ns, not significant). h, PDOTS viability assessment from patients (n = 2) with cutaneous melanoma with indicated treatments. Means (bars) and individual values (open circles) are shown (n = 6 biological replicates, 2 independent specimens; one-way ANOVA with Dunn’s multiple comparisons test, **P < 0.01; **** P < 0.0001; ns, not significant). i, heatmap of secreted cytokine profiles (L2FC) of conditioned media from PDOTS in response to indicated treatments (n = 2). Mean values shown. **P < 0.01; **** P < 0.0001; ns, not significant.