2 3 cgamp  (InvivoGen)

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: 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 . IP, immunoprecipitation.

Article Snippet: Briefly, 30 units of human recombinant DNA-PK were incubated for 60 min at RT with 1 μM ATP, 1× activator, and 0.2 μg/μl substrate in presence of increasing doses of 2′3′-cGAMP (InvivoGen), 3′3′-cGAMP (InvivoGen), c-di-GMP (InvivoGen), c-di-AMP (InvivoGen), ADU-S100 (MedChemExpress), or E7766 (MedChemExpress).

Techniques: Activation Assay, In Vitro, Recombinant, Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression, Control, Knock-Out, Isolation

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs selectively counteracts CDNs. (A) ATP hydrolysis by DNA-PKcs was measured in vitro in presence of increasing doses (0.8–2,500 µM) of c-di-GMP. Graph presents the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by one-way ANOVA. ns, not significant. (B) T98G 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. (C) As in B, except that gene expression analyses were conducted. Graphs present the mean (±SEM) performed in biological triplicates. Statistical significance was calculated by two-tailed Student's t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05. (D) Experimental scheme for human primary monocyte isolation and treatment . 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. (E) Flow cytometry analysis of macrophages prepared as in . (F) Gene expression analyses were performed on human primary cells treated as described in . Graphs present the mean (±SEM) expression of IFIT1 in three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) 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 analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (H) 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 analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (I) 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 analysis of gene expression. WB analyses were conducted using indicated antibodies and are representative of three independent experiments. (J) Control and DNA-PKcs knockout THP-1 cells were treated with 1 µM E7766 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. (K) Control and DNA-PKcs knockout THP-1 cells were treated with 10 µM diABZI 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. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05. Related to . Source data are available for this figure: .

Article Snippet: Briefly, 30 units of human recombinant DNA-PK were incubated for 60 min at RT with 1 μM ATP, 1× activator, and 0.2 μg/μl substrate in presence of increasing doses of 2′3′-cGAMP (InvivoGen), 3′3′-cGAMP (InvivoGen), c-di-GMP (InvivoGen), c-di-AMP (InvivoGen), ADU-S100 (MedChemExpress), or E7766 (MedChemExpress).

Techniques: In Vitro, Gene Expression, Two Tailed Test, Isolation, Flow Cytometry, Expressing, Control, Knock-Out

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs inhibits STING agonist–induced inflammatory responses in murine models. (A) 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. (B) Experimental scheme: Spleens were harvested from eight mice, primary splenocytes isolated, and treated with NU7441 for 6 h with 3′3′-cGAMP or diABZI prior to gene expression analysis and cytokine measurement. (C) Gene expression analysis was conducted on primary cells treated as described in B with 3′3′-cGAMP. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) Heatmap presents cytokines that were measured in the supernatant of cells from C. (E) Mean centroid analysis of cytokines presented in D. Statistical significance was calculated by two-tailed Student's t test. (F) Gene expression analysis was conducted on primary cells treated as described in B with diABZI. Graphs present the mean (±SEM) of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (G) Heatmap presents cytokines that were measured in the supernatant of cells from F. (H) Mean centroid analysis of cytokines presented in G. Statistical significance was calculated by two-tailed Student's t test. (I) In vivo experimental scheme. 8 μg NU7441 or vehicle (4% DMSO in PBS) was administered i.p. to WT, Sting −/− , and Ifnar −/− mice 1 h before challenge with diABZI (1 µg, i.p.). Parameters were analyzed 6 h after the last administration. (J) Gene expression analyses were conducted in peritoneum of mice treated as in I ( n = 3–8 mice per group). Graphs present the mean (±SEM). Statistical significance was calculated by two-tailed Student's t test. (K) Gene expression analyses were conducted in peritoneal macrophages of mice treated as in I ( n = 4–8 mice per group). Graphs present the mean (±SEM). Statistical significance was calculated by two-tailed Student's t test. (L) Gene expression analyses were conducted on the spleen of mice treated as in I ( n = 3–8 mice per group). Graphs present the mean (±SEM). Statistical significance was calculated by two-tailed Student's t test. (M) Concentration of Cxcl10 in the peritoneal fluid of mice treated as in I was determined by ELISA ( n = 3–8 mice per group). Graph presents the mean (±SEM). Statistical significance was calculated by two tailed Student's t test. (N) The concentration of Cxcl10 in the spleen of mice treated as in I ( n = 3–8 mice per group) was determined by ELISA. Graph presents the mean (±SEM). Statistical significance was calculated by Student's t test. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see .

Article Snippet: Briefly, 30 units of human recombinant DNA-PK were incubated for 60 min at RT with 1 μM ATP, 1× activator, and 0.2 μg/μl substrate in presence of increasing doses of 2′3′-cGAMP (InvivoGen), 3′3′-cGAMP (InvivoGen), c-di-GMP (InvivoGen), c-di-AMP (InvivoGen), ADU-S100 (MedChemExpress), or E7766 (MedChemExpress).

Techniques: Gene Expression, Isolation, Two Tailed Test, In Vivo, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: 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 .

Article Snippet: Briefly, 30 units of human recombinant DNA-PK were incubated for 60 min at RT with 1 μM ATP, 1× activator, and 0.2 μg/μl substrate in presence of increasing doses of 2′3′-cGAMP (InvivoGen), 3′3′-cGAMP (InvivoGen), c-di-GMP (InvivoGen), c-di-AMP (InvivoGen), ADU-S100 (MedChemExpress), or E7766 (MedChemExpress).

Techniques: Virus, Infection, Staining, Gene Expression

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: DNA-PKcs decreases the ability of STING agonists to trigger an antiviral response. (A) T98G cells were treated with the 10 µM diABZI, 1 µM E7766, or 10 mg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441. Cells were subsequently infected or not with VSV-GFP for 16 h prior to DAPI nuclear staining and image acquisition. Images are representative of three independent experiments. (B) As in A, except that cells were infected with the MPXV clade 2b strain S2626 for 48 h. Images are representative of three independent experiments. (C) T98G cells engineered to express control nontargeting or DNA-PKcs–targeting gRNA were treated with 10 µg/ml fluorinated 3′3′-cGAMP in combination or not with 2 µM of NU7441. Cells were subsequently infected or not with VSV-GFP at MOI 0.3 for 16 h, prior to DAPI nuclear staining and image acquisition. Graph shows the mean (±SEM, n = 3 independent experiments) percentage of infected (GFP + ) cells as measured by fluorescent microscopy. Statistical significance was assessed using two-tailed Student's t test. (D) Gating strategy for macrophages used in . (E) Histograms show the percentage of infected (GFP + ) cells as measured by flow cytometry, following treatment with STING agonists, in the presence or absence of NU7441, at two different MOIs. (F) Primary macrophages from healthy donors 1, 2, and 3 were pretreated with NU7441 prior to STING agonist treatment. Cells treated with a STING agonist, and they were set as 100% infection, and the effect of adding a NU7441 was assessed relative to this condition. Scale bars, 500 μm. **: P < 0.01; *: P < 0.05. Data are from at least three independent experiments. Related to .

Article Snippet: Briefly, 30 units of human recombinant DNA-PK were incubated for 60 min at RT with 1 μM ATP, 1× activator, and 0.2 μg/μl substrate in presence of increasing doses of 2′3′-cGAMP (InvivoGen), 3′3′-cGAMP (InvivoGen), c-di-GMP (InvivoGen), c-di-AMP (InvivoGen), ADU-S100 (MedChemExpress), or E7766 (MedChemExpress).

Techniques: Infection, Staining, Control, Microscopy, Two Tailed Test, Flow Cytometry