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 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: The following drugs were used: NU7441 (Bio-Techne/Tocris, #3712, CID: 11327430); DMSO (Sigma-Aldrich, D2650, CID: 679); 2′3′-cGAMP (InvivoGen, tlrl-nacga23-02, CID: 137120248); 3′3′-cGAMP (InvivoGen, tlrl-nacga, CAS number: 849214-04-6); c-di-AMP (InvivoGen, tlrl-nacda, CAS number: 2734909-87-4/54447-84-6 [free acid]); c-di-GMP (InvivoGen, tlrl-nacdg, CAS number: 2222132-40-1/61093-23-0 [free acid]); diABZI (InvivoGen, tlrl-diabzi-2, CID: 137701219, CAS number: 2138299-34-8); E7766 (MedChemExpress, HY-111999A); ADU-S100 ammonium salt (MedChemExpress, HY-12885B); fluorinated 3′3′-cGAMP (InvivoGen, tlrl-nacgaf-05; CAS number: not available).

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: The following drugs were used: NU7441 (Bio-Techne/Tocris, #3712, CID: 11327430); DMSO (Sigma-Aldrich, D2650, CID: 679); 2′3′-cGAMP (InvivoGen, tlrl-nacga23-02, CID: 137120248); 3′3′-cGAMP (InvivoGen, tlrl-nacga, CAS number: 849214-04-6); c-di-AMP (InvivoGen, tlrl-nacda, CAS number: 2734909-87-4/54447-84-6 [free acid]); c-di-GMP (InvivoGen, tlrl-nacdg, CAS number: 2222132-40-1/61093-23-0 [free acid]); diABZI (InvivoGen, tlrl-diabzi-2, CID: 137701219, CAS number: 2138299-34-8); E7766 (MedChemExpress, HY-111999A); ADU-S100 ammonium salt (MedChemExpress, HY-12885B); fluorinated 3′3′-cGAMP (InvivoGen, tlrl-nacgaf-05; CAS number: not available).

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