Fig 2: DNA-PKcs inhibits 3′3′-cGAMP- and agonist-associated STING activation. (A) ATP hydrolysis by DNA-PK was measured in vitro in the presence of increasing doses (0.8–2,500 µM) of 3′3′-cGAMP or c-di-AMP. Graphs present the mean of three independent experiments. Statistical significance was calculated by one-way ANOVA. (B) Recombinant DNA-PKcs was immunoprecipitated using either mock IgG or a DNA-PKcs–specific antibody prior to incubation with 3′3′-cGAMP or c-di-AMP and ELISA-based measurement of bound CDNs. Graph presents mean (±SEM) 3′3′-cGAMP and c-diAMP levels as measured in mock and DNA-PKcs–specific IP in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (C) THP-1 cells were treated or not with 2 μM NU7441 in combination or not with 10 µg/ml fluorinated 3′3′-cGAMP for 6 h prior to WB analysis using the indicated antibodies. Representative WB of three independent experiments. (D) As in C, except that gene expression analyses were conducted. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (E) As in C, except that IFNβ, CXCL10, and CCL5 levels were measured by ELISA. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (F) Control and DNA-PKcs knockout THP-1 cells were treated with 3′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. (G) Human primary monocytes were isolated from buffy coats prior to treatment or not with 2 µM NU7441 for 1 h, followed by administration of 10 µg/ml fluorinated 3′3′-cGAMP for 6 h and gene expression analysis. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (H) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of E7766 or ADU-S100. Statistical significance was calculated by one-way ANOVA. (I) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 1 µM of E7766 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 50 µM of ADU-S100 STING agonist for 3 h and gene expression analysis. Graphs present the mean (±SEM), n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (K) T98G cells were treated or not with 2 µM of NU7441 prior to addition or not of 10 µM of diABZI for 3 h and gene expression analysis. Graphs present the mean (±SEM); n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see Fig. S3. IP, immunoprecipitation.

Fig 3: DNA-PKcs regulates antiviral responses towards DNA and RNA virus infection. (A) Experimental scheme: T98G cells were treated with 10 µM diABZI, 1 µM E7766, or 10 µg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441. Cells were subsequently infected or not with HSV-1 for 48 h prior to DAPI nuclear staining and image acquisition. (B) Image acquisition of T98G cells treated as in A; GFP (yellow) indicates HSV-1 viral load. Images are representative of three independent experiments. Scale bars, 500 μm. (C) Quantification of HSV-1 infected signal in experiments conducted as in A. Graph presents the mean (±SEM) of three independent experiments. Statistical significance was calculated by Student's t test. (D) As in C, except that treated cells were infected with MPXV clade 2b strain S2626 for 48 h. Graph presents the mean (±SEM) of three independent experiments. Statistical significance was calculated by Student's t test. (E) As in C, except that treated cells were infected with VSV for 16 h. Graph presents the mean (±SEM) of three independent experiments. Statistical significance was calculated by Student's t test. (F) Human primary macrophages were treated with 10 µg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441 prior to infection with VSV for 48 h prior to DAPI nuclear staining and image acquisition. Images are representative of three independent experiments. (G) Graph presents the mean (±SEM) VSV infected cells when treated as in F. Statistical significance was calculated by Student's t test. (H) Experimental scheme. T98G cells were either treated with 2 μM NU7441 for 1 h prior to infection and gene expression analysis 6 and 16 h after infection (pre-treatment) or infected for 6 h prior to treatment with NU7441 and gene expression analysis 16 h after infection (post-treatment). (I) Gene expression analysis in cells pre-treated and post-treated as described in H. Graph presents the mean (±SEM) of three independent experiments. Statistical significance was calculated by Student's t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. See also Fig. S5.

Fig 4: DNA-PKcs inhibits STING agonists-induced inflammatory responses in murine models. RAW264.7 cells were treated or not with NU7441 1 h before stimulation with 10 µM diABZI or 1 µM E7766 for 6 h prior to gene expression analyses. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by one-way ANOVA with multiple comparisons. ***: P < 0.001; ns, nonsignificant. Related to Fig. 4.

Fig 5: 2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B. Whole-cell extracts (WCEs) from T98G cells were used as input for IPs using mock IgG and DNA-PKcs–specific antibodies prior to incubation with 2′3′-cGAMP and detection of bound 2′3′-cGAMP. (B) WB analysis of DNA-PKcs IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) Graph represents mean (±SEM; n = 3 independent experiment) 2′3′-cGAMP levels as measured in mock and DNA-PK–specific IP performed as in A. Statistical significance was calculated by two-tailed Student t test. (D) Silver staining was conducted on recombinant DNA-PKcs used in Fig. 2 for immunoprecipitation experiments. Representative gel of three independent experiments. (E) WB analysis of TSA, conducted on WCE from THP-1 cells incubated with or without 2′3′-cGAMP or in presence or absence of NU7441. Immunoblot was performed using DNA-PKcs–, STING-, and HSP90-specific antibodies. Representative WB of three independent experiments. (F) Heatmap representation of the relative band intensities quantified from three independent experiments performed as in E. (G) Molecular modelling and docking study of 2′3′-cGAMP into DNA-PKcs. Human DNA-PKcs in ribbon representation with 2′3′-cGAMP docked in its catalytic site. (H) The docking conformation of 2′3′-cGAMP (in red spacefill representation) into the catalytic site of DNA-PKcs in the proximity of the catalytic residues (in ball and stick representation). (I) The docked conformation adopted by 2′3′-cGAMP onto the catalytic site of DNA-PKcs upon the MDSs. (J) The 2D molecular interactions diagram of 2′3′-cGAMP with the catalytic residues of DNA-PKcs. (K) Molecular modelling of ATM and ATR. DNA-PKcs superposed to the models of ATM and ATR (in red, blue, and yellow ribbon representations, respectively). (L) Close-up of the superposed active sites of ATM, ATR, and DNA-PKcs. Each of the three kinases has significant conformational differences and docking of 2′3′-cGAMP to all of them failed to return a thermodynamically viable pose (complex conformation). (M) Experimental scheme for N. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation or not with increasing doses of NU7441 (0, 0.2, 2, and 20 µM) followed by 2′3′-cGAMP incubation, release of bound 2′3′-cGAMP, and detection by ELISA (N) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in DNA-PK–specific IP performed as in M. n = 3 independent experiments. Statistical significance was calculated by two-tailed Student t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Related to Fig. 1. Source data are available for this figure: SourceData FS1. IP, immunoprecipitation.