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: Endogenous immunoprecipitation was performed using DNA-PKcs–targeting antibody (A300-517A; Bethyl) or Rabbit IgG (Santa Cruz) as a negative control.

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: Endogenous immunoprecipitation was performed using DNA-PKcs–targeting antibody (A300-517A; Bethyl) or Rabbit IgG (Santa Cruz) as a negative control.

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 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: Endogenous immunoprecipitation was performed using DNA-PKcs–targeting antibody (A300-517A; Bethyl) or Rabbit IgG (Santa Cruz) as a negative control.

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