c di amp sodium  (MedChemExpress)

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: bioRxiv

Article Title: Type I interferon signaling promotes early innate control of Borrelia burgdorferi infection

doi: 10.64898/2026.02.13.705697

Figure Lengend Snippet: (A) In vitro macrophage assays. RAW 264.7 macrophages were pretreated with c-di-AMP before infection with GFP-expressing B. burgdorferi at low and high MOI. Phagocytosis was assessed by flow cytometry to determine the percentage of GFP-positive cells and GFP MFI. ( B ) Gentamicin protection assay. To assess intracellular spirochete viability, RAW 264.7 macrophages were pretreated with c-di-AMP and subsequently infected with B. burgdorferi for 2.5 hours, followed by treatment with gentamicin (200 µg/mL) to eliminate extracellular bacteria. Following washing and lysis, cell lysates were diluted in BSK-II medium and plated in 96-well plates. Intracellular bacterial viability was assessed by limiting dilution, and data are presented as percentages of B. burgdorferi- positive wells. ( C ) In vivo bacterial clearance. Mice were pretreated intradermally with c-di-AMP before infection with B. burgdorferi (1Χ10 4 spirochetes/ mouse). Spirochetal burden at the site of infection was quantified by qPCR at 5 DPI ( left ), and dissemination was assessed by the frequency of culture-positive tissues at 10 DPI ( right ). Data are presented as mean ± SD of two independent experiments (n = 3–5 mice/ group). Statistical significance was determined using a two-tailed unpaired Student’s t- test for (A) and (B) (*, P < 0.05; **, P < 0.01) and a two-tailed Fisher’s exact test for (C) (***, P < 0.001).

Article Snippet: To elucidate the effect of c-di-AMP in vivo , mice were pre-treated intradermally with c-di-AMP (50 μg/mouse; InvivoGen, tlrl-nacda) 24-hours before infection with B. burgdorferi (1 × 10 4 spirochetes/mouse).

Techniques: In Vitro, Infection, Expressing, Flow Cytometry, Bacteria, Lysis, In Vivo, Two Tailed Test