simplot version 3 5 1  (Simplot Science)

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: 2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B and C. FLAG-tagged DNA-PKcs (F-DNA-PKcs or FLAG-DNA-PKcs) expressed in 293T cells was FLAG was subjected to immunoprecipitation (IP), prior to incubation with 2′3'-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (B) WB analysis of input and FLAG-IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) 2′3′-cGAMP was measured by ELISA on experiment performed as in A. Graph presents the mean ± SEM of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) Experimental scheme for E. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation with 2′3′-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (E) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in mock IgG and DNA-PK–specific IP performed as in D. Statistical significance was calculated by two-tailed Student's t test. n = 3 independent experiments. (F) Experimental scheme for G. FLAG-tagged DNA-PKcs (FLAG-DNA-PKcs) expressed in 293T cells was FLAG purified prior to incubation with biotin or biotinylated 2′3′-cGAMP (C3-2′3′-cGAMP), followed by streptavidin pull-down and WB analysis. (G) WB analysis of input and streptavidin pull-down experiment performed as in F was conducted using a FLAG-specific antibody. Representative WB of three independent experiments. (H) DNA-PKcs (red) and 2′3′-cGAMP (green) subcellular localization was assessed 6 h after iFluor488-2′3′-cGAMP transfection in T98G cells. Immunofluorescence was performed using a DNA-PKcs–specific antibody and DAPI nuclear staining. Representative images of 15–20 images. Scale bars, 5 µm. (I) Quantification of cytosolic DNA-PKcs and iFluor488-2′3′-cGAMP foci colocalization following transfection of T98G cells with mock or fluorescent 2′3′-cGAMP using the CellProfiler software. n = 424 and 558. Statistical significance was calculated by two-tailed Student's t test. (J) Experimental scheme for K. THP-1 cells were processed for TSA in the presence or absence of 2′3′-cGAMP. (K) WB analysis of TSA, as described in J, was conducted using indicated antibodies. Representative WB of three independent experiments. (L) Experimental scheme for M. Purified FLAG-DNA-PKcs was used as input material for TSA in the presence or absence of 2′3′-cGAMP. (M) WB analysis of TSA, performed as in L, was conducted using anti-FLAG antibody. Representative WB of three independent experiments. (N) Representation of the molecular modelling of 2′3′-cGAMP in interaction with DNA-PKcs. (O) ATP hydrolysis by DNA-PK was measured in vitro in presence of NU7441 or increasing doses (300–2,700 µM) of 2′3′-cGAMP. Graph presents the mean of three independent experiments. One-way ANOVA. (P) As in D, except that DNA-PKcs IP was incubated with or without 2′3′-cGAMP in presence or absence of NU7441 (used as a competitor) prior to measurement of bound 2′3′-cGAMP. Graph represents mean (±SEM) 2′3′-cGAMP levels; n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (Q) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were expressed in 293T cells prior to TSA analysis in the presence or absence of 2′3′-cGAMP. WB was conducted with the indicated antibodies. Representative WB of three independent experiments. (R) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were FLAG purified as in A prior to incubation with biotin or biotinylated 2′3′-cGAMP and binding analysis by WB as in G using FLAG antibody. Representative WB of three independent experiments. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . 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: Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant, Purification, Transfection, Immunofluorescence, Staining, Software, In Vitro, Binding 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: 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 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 . Source data are available for this figure: . 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: Incubation, Two Tailed Test, Silver Staining, Recombinant, Immunoprecipitation, Western Blot, 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 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 (T98G cGAS ) 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) T98G cGAS 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 . Source data are available for this figure: . WCE, whole-cell extracts.

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: Transfection, Gene Expression, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Control, Knock-Out, Expressing

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 dampens 2′3′-cGAMP-mediated STING signaling. (A) Graph represents mean (±SEM, n = 3 independent experiment) CXCL10 and CCL5 levels as measured in supernatant of T98G cells treated or not with 2 µM NU7441 for 1 h prior to transfection or not of 10 µg/ml 2′3′-cGAMP for 6 h. Statistical significance was calculated by two-tailed Student's t test. (B) T98G cells were pretreated with the NU7441 (2 µM), NU7026 (10 µM), and AZD7648 (5 µM) DNA-PKcs inhibitors for 1 h prior to transfection of 2′3′-cGAMP (10 µg/ml) for 6 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. (C) T98G cells were pretreated with the NU7441 (2 µM) for 1 h prior to transfection with dsDNA (2 µg) for 6 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. (D) T98G cells were transfected for DNA-PKcs– or KU70-targeting siRNAs or a control nontargeting siRNA for 48 h prior to analysis of knockdown efficiency by WB using the indicated antibodies. Representative WB; n = 3 independent experiments. (E) T98G cells were engineered to express control nontargeting or PRKDC-targeting gRNA. Representative WB; n = 3 independent experiments. (F) THP-1 CTRL, THP1 cGAS−/− , and THP1 STING−/− were pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection or not of 2′3′-cGAMP (10 µg/ml) for 6 h. WB analysis was performed using the indicated antibodies. Representative WB of three to five independent experiments. (G) Densitometric quantification of band intensities of the p-IRF3/IRF3 ratio from the WB in F. Results shown as % of induction of 2′3′-cGAMP response ( n = 3–5 independent experiments). (H) Densitometric quantification of band intensities of the pSTING/STING ratio from the WB in F. Results shown as % of induction of 2′3′-cGAMP response ( n = 3–5 independent experiments). (I) Graph represents mean (±SEM, n = 3 independent experiment) IFNβ levels as measured in supernatant of THP1 CTRL , THP1 cGAS−/− , and THP1 STING−/− pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection or not of 2′3′-cGAMP (10 µg/ml) for 6 h. Statistical significance was calculated by two-tailed Student's t test. (J) T98G cells were engineered to express control nontargeting or PRKDC-targeting gRNA. Representative WB of three independent experiments. (K) T98G cells engineered to express control nontargeting or STING-targeting gRNAs were treated or not with 2 µM of NU7441 prior to transfection or not of 10 µg/ml 2′3′-cGAMP and gene expression analysis. Graphs present the mean (±SEM, n = 5 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (L) As in K, except that WB analysis was performed using the indicated antibodies. Representative WB of three independent experiments. (M) As in L, except that T98G cells expressing an IFNAR-targeting gRNA were used. Representative WB of three independent experiments. (N) THP-1 cells were pretreated or not with the NU7441 (2 µM) inhibitor for 1 h prior to transfection with dsDNA (2 µg) for up to 24 h. Gene expression analysis was conducted at 3, 6, 16, and 24 h. Graphs present the mean (±SEM, n = 3 independent experiments). Statistical significance was calculated by two-tailed Student's t test. (O) Cell culture supernatants were collected at 24 h in experiment performed as in N, and cytokine/chemokine levels were analyzed using a proteome profiler array. Heatmap representation of relative spot intensities is shown (mean of three independent experiments). (P) STING-deficient THP-1 cells engineered to express human STING haplotypes (STING-H232, STING-AQ, and STING-HAQ) were pretreated or not with the NU7441 (2 µM) for 1 h prior to transfection of 2′3′-cGAMP (10 µg/ml) for 6 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. (Q) As in P, except that IFNβ and CXCL10 levels were quantified by ELISA in supernatants. 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. 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: Transfection, Two Tailed Test, Gene Expression, Control, Knockdown, Expressing, Cell Culture, 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 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