Figure 4.
Upregulation of caspase-8 and CYLD levels is required for TNF/IFNγ-induced cell death. (A) Homology-directed repair facilitates mutating endogenous predicted ISRE motif 1 and 2 (ISRE-1 and ISRE-2) in CASP8 promoter region. The designed mutant bases are underlined. Sanger sequencing confirms the successful knockin of mutated ISRE-1 and ISRE-2. (B) HeLa cells (parental, mutated ISRE-1 knock-in clones #1 and #2) were treated with IFNγ for 24 h (n = 3). The CASP8 mRNA level was determined by qPCR. One-way ANOVA was performed: NS. (C) HeLa cells (parental, mutated ISRE-1 knock-in clones #1 and #2) were treated with IFNγ for 24 h. The protein expression of caspase-8 and CYLD was analyzed by immunoblotting (n = 2). (D) HeLa cells (parental, mutated ISRE-1 knock-in clones #1 and #2) were treated with TNF/IFNγ (n = 3). Cell viability was determined by ATP levels. One-way ANOVA was performed: NS. (E) HeLa cells (parental, mutated ISRE-2 knock-in clones #1 and #2) were treated with IFNγ for 24 h (n = 3). The CASP8 mRNA level was determined by qPCR. One-way ANOVA was performed: *P = 0.0208; **P = 0.0014, 0.002 (left to right); ***P = 0.001. (F) HeLa cells (parental, mutated ISRE-2 knock-in clones #1 and #2) were treated with IFNγ for 24 h. The protein expression of caspase-8 and CYLD was analyzed by immunoblotting (n = 4). (G) HeLa cells (parental, mutated ISRE-2 knock-in clones #1 and #2) were treated with TNF/IFNγ (n = 3). Cell viability was determined by ATP levels. One-way ANOVA was performed: ****P < 0.0001. (H) The sequences of predicted ISREs at CYLD promoter region. (I) Schematic diagram of the reporter constructs. (J) The GFP fluorescence intensity derived from the indicated CYLD promoter reporters in the presence or absence of IFNγ. The GFP fluorescence intensity was quantified by flow cytometry (n = 3), and the values in the groups without IFNγ stimulation are set as 1.0. Two-tailed t test was performed: ****P < 0.0001. (K) Homology-directed repair facilitates mutating ISRE #4 in CYLD promoter region. Representative sequencing results from CYLD KI-4 clones confirm the success in mutation. (L) HeLa cells (parental, CYLD KI-4 clones #1 and #2) were treated with IFNγ or vehicle control for 24 h. CYLD mRNA levels were determined by qPCR (n = 3). One-way ANOVA was performed: NS, ***P = 0.0001, 0.0002 (from left to right). (M) HeLa cells (parental, CYLD KI-4 clones #1 and #2) were treated as indicated for 24 h. The protein expression of caspase-8 and CYLD were analyzed by immunoblotting (n = 3). (N and O) HeLa cells (parental, CYLD KI-4 clones #1 and #2) were treated as indicated for 36 h. Cell viability was determined by ATP levels (N) (n = 3). Cell death was measured by PI staining followed by flow cytometry (O) (n = 3). One-way ANOVA was performed: ****P < 0.0001. 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 F4.

Upregulation of caspase-8 and CYLD levels is required for TNF/IFNγ-induced cell death. (A) Homology-directed repair facilitates mutating endogenous predicted ISRE motif 1 and 2 (ISRE-1 and ISRE-2) in CASP8 promoter region. The designed mutant bases are underlined. Sanger sequencing confirms the successful knockin of mutated ISRE-1 and ISRE-2. (B) HeLa cells (parental, mutated ISRE-1 knock-in clones #1 and #2) were treated with IFNγ for 24 h (n = 3). The CASP8 mRNA level was determined by qPCR. One-way ANOVA was performed: NS. (C) HeLa cells (parental, mutated ISRE-1 knock-in clones #1 and #2) were treated with IFNγ for 24 h. The protein expression of caspase-8 and CYLD was analyzed by immunoblotting (n = 2). (D) HeLa cells (parental, mutated ISRE-1 knock-in clones #1 and #2) were treated with TNF/IFNγ (n = 3). Cell viability was determined by ATP levels. One-way ANOVA was performed: NS. (E) HeLa cells (parental, mutated ISRE-2 knock-in clones #1 and #2) were treated with IFNγ for 24 h (n = 3). The CASP8 mRNA level was determined by qPCR. One-way ANOVA was performed: *P = 0.0208; **P = 0.0014, 0.002 (left to right); ***P = 0.001. (F) HeLa cells (parental, mutated ISRE-2 knock-in clones #1 and #2) were treated with IFNγ for 24 h. The protein expression of caspase-8 and CYLD was analyzed by immunoblotting (n = 4). (G) HeLa cells (parental, mutated ISRE-2 knock-in clones #1 and #2) were treated with TNF/IFNγ (n = 3). Cell viability was determined by ATP levels. One-way ANOVA was performed: ****P < 0.0001. (H) The sequences of predicted ISREs at CYLD promoter region. (I) Schematic diagram of the reporter constructs. (J) The GFP fluorescence intensity derived from the indicated CYLD promoter reporters in the presence or absence of IFNγ. The GFP fluorescence intensity was quantified by flow cytometry (n = 3), and the values in the groups without IFNγ stimulation are set as 1.0. Two-tailed t test was performed: ****P < 0.0001. (K) Homology-directed repair facilitates mutating ISRE #4 in CYLD promoter region. Representative sequencing results from CYLD KI-4 clones confirm the success in mutation. (L) HeLa cells (parental, CYLD KI-4 clones #1 and #2) were treated with IFNγ or vehicle control for 24 h. CYLD mRNA levels were determined by qPCR (n = 3). One-way ANOVA was performed: NS, ***P = 0.0001, 0.0002 (from left to right). (M) HeLa cells (parental, CYLD KI-4 clones #1 and #2) were treated as indicated for 24 h. The protein expression of caspase-8 and CYLD were analyzed by immunoblotting (n = 3). (N and O) HeLa cells (parental, CYLD KI-4 clones #1 and #2) were treated as indicated for 36 h. Cell viability was determined by ATP levels (N) (n = 3). Cell death was measured by PI staining followed by flow cytometry (O) (n = 3). One-way ANOVA was performed: ****P < 0.0001. 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 F4.

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