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: To generate the T98G IFNAR−/− knockout and control cell lines, lentiviral particles were produced by co-transfection of 2 × 10 6 293T cells with 5 μg of LentiCRISPRv2GFP plasmid (#82416; Addgene) expressing the gRNA targeting the gene of interest or nontargeting control gRNA, 5 μg of psPAX2, and 1 μg of pMD2.G, using the standard calcium phosphate transfection protocol.

Techniques: Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant, Purification, Transfection, Immunofluorescence, Staining, Software, In Vitro, Binding Assay