Figure 2.
IFNγ induces CYLD/caspase-8 expression in cancer cells. (A) Schematic of the CRISPR-Cas9 screen strategy to identify regulators in TNF/IFNγ-induced cell death. (B) Schematic of the RNA-seq strategy to identify genes’ response to IFNγ treatment. (C) HeLa-Cas 9 cells were transfected with gRNA library and treated with TNF/IFNγ for 48 h, followed by DNA isolation and sequencing. Log2-transformed average fold change (FC) for enrichment of sgRNAs in HeLa-Cas 9 cells treated with TNF/IFNγ versus control cells is shown by volcano plot. Significant enriched genes identified in this paper are labeled. (D) HeLa cells were treated with IFNγ for 12 h, followed by RNA isolation and 3′mRNA-sequencing. Differentially expressed genes (DEGs) that are up- or downregulated in IFNγ-treated HeLa cells compared with untreated HeLa cells are shown by a volcano plot. Adjusted P < 0.05, cut-off values log2(FC) > 1 or log2(FC) < −1. Labeled genes were the top elevated genes involved in TNF/IFNγ signaling pathway. (E) HeLa cells (parental and STAT1 KO) were treated with IFNγ for 24 h, followed by RNA isolation and 3′mRNA-sequencing. The effect of IFNγ treatment on indicated genes was assessed by a heatmap. (F–H) HeLa cells were treated with IFNγ at the indicated concentration for 24 h. The mRNA expression of CYLD (F), CASP8 (G), and CASP7 (H) was measured by qPCR (n = 3). One-way ANOVA was performed: **P = 0.0084, ****P < 0.0001 (F); *P = 0.0298, **P = 0.0011, ****P < 0.0001 (G); NS, ***P = 0.0004, ****P < 0.0001 (H). (I) HeLa cells were treated with the indicated concentration of IFNγ for 24 h. The expression of indicated proteins involved in the IFNγ and TNF signaling pathways was analyzed by immunoblotting (n = 4). (J) ME-180 cells were treated with TNF, IFNγ, or TNF/IFNγ for 24 h. The protein expression of CYLD, caspase-8, and iNOS was analyzed by immunoblotting (n = 3). (K and L) ME-180 cells were treated with TNF, IFNγ, or TNF/IFNγ for 24 h (n = 3). The mRNA level of CYLD (K) and CASP8 (L) was measured by qPCR. One-way ANOVA was performed: ***P = 0.0005, ****P < 0.0001 (K); *P = 0.0276, **P = 0.0042 (L). 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 F2.

IFNγ induces CYLD/caspase-8 expression in cancer cells. (A) Schematic of the CRISPR-Cas9 screen strategy to identify regulators in TNF/IFNγ-induced cell death. (B) Schematic of the RNA-seq strategy to identify genes’ response to IFNγ treatment. (C) HeLa-Cas 9 cells were transfected with gRNA library and treated with TNF/IFNγ for 48 h, followed by DNA isolation and sequencing. Log2-transformed average fold change (FC) for enrichment of sgRNAs in HeLa-Cas 9 cells treated with TNF/IFNγ versus control cells is shown by volcano plot. Significant enriched genes identified in this paper are labeled. (D) HeLa cells were treated with IFNγ for 12 h, followed by RNA isolation and 3′mRNA-sequencing. Differentially expressed genes (DEGs) that are up- or downregulated in IFNγ-treated HeLa cells compared with untreated HeLa cells are shown by a volcano plot. Adjusted P < 0.05, cut-off values log2(FC) > 1 or log2(FC) < −1. Labeled genes were the top elevated genes involved in TNF/IFNγ signaling pathway. (E) HeLa cells (parental and STAT1 KO) were treated with IFNγ for 24 h, followed by RNA isolation and 3′mRNA-sequencing. The effect of IFNγ treatment on indicated genes was assessed by a heatmap. (F–H) HeLa cells were treated with IFNγ at the indicated concentration for 24 h. The mRNA expression of CYLD (F), CASP8 (G), and CASP7 (H) was measured by qPCR (n = 3). One-way ANOVA was performed: **P = 0.0084, ****P < 0.0001 (F); *P = 0.0298, **P = 0.0011, ****P < 0.0001 (G); NS, ***P = 0.0004, ****P < 0.0001 (H). (I) HeLa cells were treated with the indicated concentration of IFNγ for 24 h. The expression of indicated proteins involved in the IFNγ and TNF signaling pathways was analyzed by immunoblotting (n = 4). (J) ME-180 cells were treated with TNF, IFNγ, or TNF/IFNγ for 24 h. The protein expression of CYLD, caspase-8, and iNOS was analyzed by immunoblotting (n = 3). (K and L) ME-180 cells were treated with TNF, IFNγ, or TNF/IFNγ for 24 h (n = 3). The mRNA level of CYLD (K) and CASP8 (L) was measured by qPCR. One-way ANOVA was performed: ***P = 0.0005, ****P < 0.0001 (K); *P = 0.0276, **P = 0.0042 (L). 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 F2.

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