Figure 6.
IRF1 binds to the promoters of CYLD/CASP8 and activates their transcription. (A and B) HeLa parental (A) and STAT1 KO cells (B) were infected with lentivirus-expressed IRF1, IRF1 W11R mutant variant, or the empty vector. Cells were then treated with TNF, IFNγ, or TNF/IFNγ for 48 h in the presence or absence of dox (n = 3). The expression of indicated proteins from these cells in the presence or absence of dox was analyzed by immunoblotting (n = 3). Cell viability was determined by ATP levels. Two-tailed t test was performed: NS, ***P = 0.0001, ****P < 0.0001 (A); NS, ****P < 0.0001 (B). (C and D) ChIP-qPCR analysis of IRF1 (C) or IRF1 W11R mutant variant (D) stably expressed in HeLa IRF1 KO cells binding to the promoter region of the indicated genes using an anti-IRF1 antibody (n = 3). Values are a percentage of the input. Two-tailed t test was performed: NS, ***P = 0.000488, 0.000172, 0.000173 (from left to right), ****P < 0.0001 (C); NS (D). (E) ChIP-qPCR analysis of IRF1-Flag binding to the promoter region of indicated genes in HeLa IRF1 KO cells stably expressed IRF1-Flag using an anti-Flag antibody (n = 3). Two-tailed t test was performed: NS, ***P = 0.000166, 0.000138 (from left to right), ****P < 0.0001. (F–I) HeLa cells stably expressed IRF1, IRF1 W11R mutant variant, or an empty vector were transfected with STAT1 (F), CASP8 (G), CYLD (H), and NF-κB (I) promoter region containing TA-Firefly luciferase reporter plasmids and Renilla luciferase plasmid. After 24 h transfection, dox was added to induce protein expression for 12 h (n = 2 or 3). The reporter activities were determined by firefly luciferase signals normalized to the Renilla luciferase signal. Two-tailed t test was performed: NS, **P = 0.001097 (F); NS, ****P < 0.0001 (G); NS, ****P < 0.0001 (H). One-way ANOVA was performed: ****P < 0.0001 (I). (J) Schematic of the biochemical mechanism of TNF/IFNγ-induced cell death in cancer cells. IFNγ induces the expression of CYLD/CASP8/CASP7 by JAK1/JAK2/STAT1/IRF1 pathway to promote TNF/IFNγ-induced cell death. 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 F6.

IRF1 binds to the promoters of CYLD/CASP8 and activates their transcription. (A and B) HeLa parental (A) and STAT1 KO cells (B) were infected with lentivirus-expressed IRF1, IRF1 W11R mutant variant, or the empty vector. Cells were then treated with TNF, IFNγ, or TNF/IFNγ for 48 h in the presence or absence of dox (n = 3). The expression of indicated proteins from these cells in the presence or absence of dox was analyzed by immunoblotting (n = 3). Cell viability was determined by ATP levels. Two-tailed t test was performed: NS, ***P = 0.0001, ****P < 0.0001 (A); NS, ****P < 0.0001 (B). (C and D) ChIP-qPCR analysis of IRF1 (C) or IRF1 W11R mutant variant (D) stably expressed in HeLa IRF1 KO cells binding to the promoter region of the indicated genes using an anti-IRF1 antibody (n = 3). Values are a percentage of the input. Two-tailed t test was performed: NS, ***P = 0.000488, 0.000172, 0.000173 (from left to right), ****P < 0.0001 (C); NS (D). (E) ChIP-qPCR analysis of IRF1-Flag binding to the promoter region of indicated genes in HeLa IRF1 KO cells stably expressed IRF1-Flag using an anti-Flag antibody (n = 3). Two-tailed t test was performed: NS, ***P = 0.000166, 0.000138 (from left to right), ****P < 0.0001. (F–I) HeLa cells stably expressed IRF1, IRF1 W11R mutant variant, or an empty vector were transfected with STAT1 (F), CASP8 (G), CYLD (H), and NF-κB (I) promoter region containing TA-Firefly luciferase reporter plasmids and Renilla luciferase plasmid. After 24 h transfection, dox was added to induce protein expression for 12 h (n = 2 or 3). The reporter activities were determined by firefly luciferase signals normalized to the Renilla luciferase signal. Two-tailed t test was performed: NS, **P = 0.001097 (F); NS, ****P < 0.0001 (G); NS, ****P < 0.0001 (H). One-way ANOVA was performed: ****P < 0.0001 (I). (J) Schematic of the biochemical mechanism of TNF/IFNγ-induced cell death in cancer cells. IFNγ induces the expression of CYLD/CASP8/CASP7 by JAK1/JAK2/STAT1/IRF1 pathway to promote TNF/IFNγ-induced cell death. 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 F6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal