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. 2024 Feb 20;15:1532. doi: 10.1038/s41467-024-46048-7

Fig. 6. Irf6 promotes susceptibility to T cell killing by enhancing TNF-induced apoptosis.

Fig. 6

a Normalized viability of 4662 parental EV, Esc EV, and Esc Irf6 tumors with varying concentrations of TNF-α in the presence of IFN-γ (0.2 μg/ml) plus cycloheximide (1 μg/ml) for 48 h (n = 4). IC50 values are 0.03472 ug/ml for parental EV, 0.6494 ug/ml for Esc Irf6, and not determined for Esc EV tumors. Mean fluorescence intensities (MFIs) or percentages of cleaved caspase-3 in 4662 parental EV, Esc EV, and Esc Irf6 tumors treated with or without TNF-α (0.5 μg/ml) plus IFN-γ in the presence of cycloheximide by flow cytometry (n = 3) (b) or in s.c. implanted YFP+ those tumors with or without immunotherapy by IF staining. Each dot represents biological replicates (c). d Normalized viability of 4662 Esc EV and Esc Irf6 tumors, treated with vehicle or z-VAD (20 μM), in response to TNF-α plus IFN-γ in the presence of cycloheximide (n = 3). e Left, immunoblots of IRF6 and TNF-related cell death mediators in 4662 Esc EV and Esc Irf6 cells with or without ablation of each gene. Right, normalized viability of 4662 Esc EV and Esc Irf6 tumors with or without indicated gene ablation in response to TNF-α plus IFN-γ in the presence of cycloheximide (n = 3). The percentages of 7-AAD+ cells among OVA-tdTomato+ 4662 Esc EV and Esc Irf6 cells with or without the indicated gene ablation (f), or the same cells with or without TNF-α neutralizing Ab (5 μg/ml) (g), or OVA-tdTomato+ 4662 parental and Esc cells in the presence of vehicle or birinapant (5 μM) (h), co-cultured with or without activated OT-I for 2 d. n = 3 for tumor alone and 4 for co-cultures. Tumor growth (i) and survival (j) of mice bearing 4662 parental EV and Casp8 KO tumors treated with control IgG or FCP (arrow) (n = 10). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 by Student’s t test (b–d), one-way ANOVA (e–h), and log-rank (Mantel-Cox) test (j). Data represent two independent experiments. Source data and exact P value are provided as a Source Data file.