IFNγ induces CYLD levels to synergize with TNF to induce cell death. (A) EMT6 cells were infected with lentivirus with control (shcontrol) or Cyld shRNA (shCyld) and treated with IFNγ for 24 h. The protein expression of CYLD and IRF1 was analyzed by immunoblotting (n = 3). (B) EMT6 shcontrol and shCyld cells were treated with TNF, IFNγ, or TNF/IFNγ for 48 h (n = 3). Cell death was measured by PI staining. Two-tailed t test was performed: ****P < 0.0001. (C) Murine BMDMs were transfected with control or Cyld siRNAs (#1 and #2 targeting different regions of Cyld) for 48 h, after which cells were treated with IFNγ for 24 h. The protein expression of CYLD was analyzed by immunoblotting (n = 3). (D) BMDMs (Control and Cyld knockdown) were treated with TNF/IFNγ for 48 h (n = 3). PI staining was used to show dead cells. Scale bar, 275 μm. (E and F) HeLa cells (parental, mutated ISRE-1 knock-in clones #1 and #2) were treated with IFNγ for 24 h (n = 3). The mRNA levels of IRF1 (E) and CYLD (F) were determined by qPCR. One-way ANOVA was performed: NS (E); NS, *P = 0.0133 (F). (G and H) HeLa cells (parental, mutated ISRE-2 knock-in clones #1 and #2) were treated with IFNγ for 24 h (n = 3). The mRNA levels of IRF1 (G) and CYLD (H) were determined by qPCR. One-way ANOVA was performed: NS. (I and J) HeLa cells (parental, CYLD KI-4 clones #1 and #2) were treated with IFNγ or vehicle control for 24 h (n = 3). IRF1 (I) and CASP8 (J) mRNA levels were determined by qPCR (n = 3). One-way ANOVA was performed: NS (I); NS (J). 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 FS4.