IFNγ induces CYLD and caspase-8 expression via the JAK1/JAK2/STAT1/IRF1 signaling. (A) HeLa cells (parental, IFNGR1 KO, JAK1 KO, JAK2 KO, STAT1 KO, and IRF1 KO) were treated with IFNγ for 24 h. The expression of indicated proteins was analyzed by immunoblotting (n = 4). (B) Indicated cells were treated with TNF/IFNγ for 48 h (n = 3). Cell viability was determined by ATP levels. One-way ANOVA was performed: ****P < 0.0001. (C) Indicated cells were treated with TNF/IFNγ for 36 h. The protein expression of full-length caspase-8 was analyzed by immunoblotting (n = 3). (D and E) Indicated cells were treated with TNF/IFNγ in the presence or absence of the JAK inhibitors PF-004965842 (3 μM) or Upadacitinib (3 μM) (n = 3). The protein expression of cleaved caspase-8 was analyzed by immunoblotting (D). Cell viability was determined by ATP levels (E). One-way ANOVA was performed: ****P < 0.0001. (F–I) HT-29 (F), U2OS (G), U937 (H), and THP-1 (I) cells were treated with IFNγ in the presence or absence of PF-004965842 (3 μM) or Upadacitinib (3 μM) for 24 h (n = 3). The expression of indicated proteins was analyzed by immunoblotting. Data represent the mean value ± SD from n biological replicates, or a representative immunoblot from n independent experiments. Source data are available for this figure: SourceData F5.