Extended Data Fig. 6: Supporting data that loss/inhibition of TBK1 sensitizes tumour cells to TNF/IFNγ.

a, volcano plot depicting relative sgRNAs gene depletion/enrichment. Top 5 depleted sgRNAs indicated. b, scatter plot of gene essentiality from in vitro CRISPR screen (control and Tbk1-null B16 cells). c, TBK1 expression and cell viability (control vs. TNF/IFNγ;) for single cell clones derived from polyclonal control and Tbk1-null B16 cells. Western blot is representative of three independent experiments. Means (bars) and individual values (open circles) are shown (n = 6 across two independent experiments, 2-way ANOVA, Sidak’s multiple comparisons test; **** P < 0.0001; ns, not significant). d, TBK1 indel spectrum from control sgRNA and Tbk1 sgRNA B16 single cell clones. e, Viability assessment (Cell Titer Glo) of B16-ova cells in standard 2D culture after 24 h treatment with TNF (160 ng ml⁻¹) + IFNγ (40 ng ml⁻¹) compared to unstimulated cells (n = 6, 2 independent experiments, 1-way ANOVA, Holm-Sidak’s multiple comparisons test). f, Viability assessment (Hoechst/propidium iodide) of B16 tumour spheroids (lacking immune cells) in 3D microfluidic culture after 96 h treatment with TNF (10 ng ml⁻¹) + IFNγ (10 ng ml⁻¹) compared to unstimulated cells (n = 6, 2 independent experiments, 1-way ANOVA, Holm-Sidak’s multiple comparisons test). g, Cell viability assessment of B16 cells after 24 h treatment with TNF (200 ng ml⁻¹) + IFNγ (40 ng ml⁻¹) compared to unstimulated cells treated with increasing concentrations of MRT67307 (n = 9, 3 independent experiments 2-way ANOVA, Sidak’s multiple comparisons test). h, Cell viability assessment of B16 cells in standard 2D culture after 24 h treatment with TNF (200 ng ml⁻¹) + IFNγ (40 ng ml⁻¹) compared to unstimulated cells treated with increasing concentrations of GSK8612 (n = 3, 1 independent experiment, 2-way ANOVA, Sidak’s multiple comparisons test). i, Cell viability assessment of B16 cells in standard 2D culture after 24 h treatment with TNF (200 ng ml⁻¹) + IFNγ (40 ng ml⁻¹) with increasing concentrations of TBK1 PROTAC 3i (n = 6, 2 independent experiments 2-way ANOVA, Sidak’s multiple comparisons test). **** P < 0.0001; ns, not significant.