Figure 2.
DNA-PKcs dampens 2′3′-cGAMP signaling. (A) T98G cells were treated or not with 2 µM NU7441 DNA-PKcs inhibitor for 1 h prior to transfection or not of 10 µg/ml 2′3′-cGAMP for 6 h and analysis of WCE by WB using indicated antibodies. Representative WB of three independent experiments. (B) As in A, except that gene expression analysis was conducted. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) As in A, except that IFNβ levels were measured by ELISA 6 h after treatment. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) T98G cells were transfected for DNA-PKcs– or KU70-targeting siRNA or a control nontargeting siRNA prior to transfection or not for 2′3′-cGAMP for 6 h and gene expression analyses. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) Control and DNA-PKcs knockout T98G cells were transfected with 2′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control, cGAS−/−, and STING−/− THP-1 cells were treated or not with 2 µM of NU7441 for 1 h prior to transfection with 10 µg/ml 2′3′-cGAMP for 6 h and gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) As in F, except that CXCL10 levels were measured by ELISA 6 h after treatment. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) Control and DNA-PKcs knockout THP-1 cells were transfected with 2′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (I) T98G expressing cGAS (T98GcGAS) were transfected with dsDNA in presence or absence of NU7441. Gene expression analysis was conducted at 3, 6, 16, 24, and 48 h after transfection. Graphs present mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98GcGAS was transfected or not with dsDNA in presence or absence of NU7441 for 24 h prior to analysis of cytokines in media using cytokine arrays. Heatmap (representative of n = 2 independent experiments) represents fold change in spot intensity in NU7441-treated samples versus vehicle. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see Fig. S2. Source data are available for this figure: SourceData F2. WCE, whole-cell extracts. Refer to the image caption for details. The image contains multiple bar graphs and a heatmap. Panel A shows western blot of DNA-PKcs signaling proteins; Panel B shows vertical bar graphs of IFN gene expression; Panel C shows vertical bar graph of IFN beta secretion; Panel D shows vertical bar graphs of cytokine gene expression; Panel E shows vertical bar graphs of IFNB1 and CXCL1 0 expression; Panel F shows multiple vertical bar graphs of antiviral genes; Panel G shows vertical bar graphs of CXCL10 secretion; Panel H shows vertical bar graphs of cytokine gene expression; Panel I shows time-course vertical bar graphs of gene induction; Panel J shows heatmap of cytokine array expression changes.

DNA-PKcs dampens 2′3′-cGAMP signaling. (A) T98G cells were treated or not with 2 µM NU7441 DNA-PKcs inhibitor for 1 h prior to transfection or not of 10 µg/ml 2′3′-cGAMP for 6 h and analysis of WCE by WB using indicated antibodies. Representative WB of three independent experiments. (B) As in A, except that gene expression analysis was conducted. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) As in A, except that IFNβ levels were measured by ELISA 6 h after treatment. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) T98G cells were transfected for DNA-PKcs– or KU70-targeting siRNA or a control nontargeting siRNA prior to transfection or not for 2′3′-cGAMP for 6 h and gene expression analyses. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) Control and DNA-PKcs knockout T98G cells were transfected with 2′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control, cGAS−/−, and STING−/− THP-1 cells were treated or not with 2 µM of NU7441 for 1 h prior to transfection with 10 µg/ml 2′3′-cGAMP for 6 h and gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) As in F, except that CXCL10 levels were measured by ELISA 6 h after treatment. Graphs represent mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) Control and DNA-PKcs knockout THP-1 cells were transfected with 2′3′-cGAMP for 6 h prior to gene expression analysis. Graphs present mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (I) T98G expressing cGAS (T98GcGAS) were transfected with dsDNA in presence or absence of NU7441. Gene expression analysis was conducted at 3, 6, 16, 24, and 48 h after transfection. Graphs present mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98GcGAS was transfected or not with dsDNA in presence or absence of NU7441 for 24 h prior to analysis of cytokines in media using cytokine arrays. Heatmap (representative of n = 2 independent experiments) represents fold change in spot intensity in NU7441-treated samples versus vehicle. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see Fig. S2. Source data are available for this figure: SourceData F2. WCE, whole-cell extracts.

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