Extended Date Figure 10|. Supporting data that IFNγ sensing is essential for effector cytokine-induced death in TBK1-null cells.

a, Frequency histograms of enrichment (z-score) for all sgRNAs per target in a Tbk1-null B16 cells +/− in vitro stimulation with TNFα (10ng/mL) and IFNγ (10ng/mL). b, scatter plot depicting relative depletion of sgRNAs targeting 19,674 genes in a Cas9+ B16 control and Tbk1 sgRNA cell line +/− in vitro stimulation with TNFα (10ng/mL) and IFNγ (10ng/mL). c, Western blot of control sgRNA and Tbk1-null B16 cells treated with TNFα (160 ng/mL) and IFNγ (40 ng/mL) for the indicates times. Data are representative of three independent experiments. d, cell viability assessment in parental B16 cells pre-treated with TBK1i (1μM) +/− JAK 1/2 inhibitor (ruxolitinib, 0.5 μM) +/− TNFα/IFNγ for 48 hours compared to unstimulated controls. Means (bars) and individual values (open circles) are shown (n=3, 1 independent experiment; 2-way ANOVA, Dunnett’s multiple comparisons test; *P < 0.05; ***P < 0.001; **** P < 0.0001; ns, not significant). e, Western blot of indicated proteins in Tbk1-null B16 cell lysates following 2-hour pre-treatment with vehicle control (0.1%DMSO), ruxolitinib (1 μM), Q-VD-OPh (20μM), Nec-1s (10 μM), or Q-VD-OPh plus Nec-1s followed by 10-hour treatment with TNFα (160 ng/mL) and IFNγ (40 ng/mL) or unstimulated (PBS) control. Data are representative of three independent experiments. f, GR values for 9-point inhibitor titration of ruxolitinib (JAK1/2i) in parental, control sgRNA (monoclonal), and Tbk1 sgRNA (monoclonal) B16 cells (2 independent experiments; representative data from single experiment with 6 technical replicates per condition). Means (solid circles) are shown +/− s.e.m (error bars).