Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Predicted binding affinity scores of BT against pattern recognition receptors using the DeepAVC model. (B) SPR sensorgrams of BT binding to recombinant human cGAS at the indicated concentrations (0.196-50 µM). (C) Dose–response curve fitted from SPR data in (B). (D) RT-qPCR of IFNB1 mRNA in wild-type (WT), cGAS knockout and STING knockout HT1080 cells treated with BT (50, 75 or 100 µM) or DMSO for 12 h. (E) Secreted type I IFN measured by ELISA in the same cell lines and treatments as in (D). (F) Flow cytometry of VSV-GFP infection in WT, cGAS knockout and STING knockout HT1080 cells treated with BT (50, 75, 100 µM) and infected (MOI = 0.1) for 12 h. (G) Western blots of WT, cGAS knockout and STING knockout HT1080 cells treated as in (D). (H-I) In vivo VSV and HSV-1 challenges in cGAS knockout mice. Left panel is survival curves of mice, right panel is viruses RNA levels in liver measured by RT-qPCR. (J) Confocal micrographs of RAW 264.7 cells treated with DMSO or 50 µM BT for 12 h, stained for DAPI (blue) and cytosolic dsDNA (green). Scale bars, 5 µm. (K) Confocal images of RAW 264.7 cells treated as in (J), stained with DAPI (blue) and MitoTracker (magenta) to visualize mitochondrial morphology. Scale bars, 5 µm. (L) Comparison of mitochondrial structure by confocal versus STED super-resolution microscopy in RAW 264.7 cells treated with DMSO or BT and stained with PK Mito. Scale bars, 2 µm. (M) Transmission electron micrographs of mitochondria in RAW 264.7 cells treated with DMSO or BT. (N) In vitro cGAS enzymatic assays: LC-MS quantification of cGAMP production by recombinant cGAS incubated with dsDNA in the presence of BT (25 or 100 µM). (O) LC-MS analysis of cGAMP production by cGAS incubated with BT (25 or 100 µM) in the absence of exogenous DNA. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Recombinant human cGAS protein were purchased from MedChemExpress (catalog no. HY-P72337).

Techniques: Binding Assay, Recombinant, Quantitative RT-PCR, Knock-Out, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Infection, Western Blot, In Vivo, Staining, Comparison, Super-Resolution Microscopy, Transmission Assay, In Vitro, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: Frontiers in Immunology

Article Title: cGAS–STING and MyD88 Pathways Synergize in Ly6C hi Monocyte to Promote Streptococcus pneumoniae -Induced Late-Stage Lung IFNγ Production

doi: 10.3389/fimmu.2021.699702

Figure Lengend Snippet: cGAS–STING mediate S. pneumoniae -induced lung IL-12p70 production by monocyte/monocyte-derived cells. (A, B) STING −/− , cGAS −/− , and WT littermates mice were given PBS or infected (i.n.) with S. pneumoniae (D39 strain, ~5 × 10 6 CFU). IL-12p70 (24, 48 hpi) were measured by ELISA (n = 3-4 mice per group). Data are representative of two independent experiments. (C) Flow cytometry analysis of IL-12p70 in lung immune cells from PBS or S. p (~5 × 10 6 CFU) infected C57BL/6J mice at 24 hpi (n = 3 mice per group). Data are representative of three independent experiments. (D) Total cell numbers of IL-12p70 + lung immune cells from (C) were enumerated. (E, F) CCR2 −/− and WT littermates mice were infected (i.n.) with S. pneumoniae as in (A) Total cell numbers of lung Ly6C hi monocyte (E) and neutrophils (F) were enumerated (n = 3–4 mice per group). Data are representative of three independent experiments. (G) CCR2 −/− and WT littermate mice were infected with S. pneunoniae as in panel (A) IL-12p70 in lung homogenates (24 hpi) were measured by ELISA (n = 3 mice per group). Data are representative of two independent experiments. Graphs represent the mean with error bars indicating SEM. p -values determined by one-way ANOVA Tukey’s multiple comparison test. Significance is represented by asterisk, where * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: The following strains were obtained from The Jackson Laboratory: CCR2 −/− , cyclic GMP–AMP synthase (cGAS) −/− , IL-12p70 −/− , IFNRA1 −/− , MyD88 −/− , TLR2 −/− , and IFNγ YFP-reporter.

Techniques: Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Comparison

Journal: Frontiers in Immunology

Article Title: cGAS–STING and MyD88 Pathways Synergize in Ly6C hi Monocyte to Promote Streptococcus pneumoniae -Induced Late-Stage Lung IFNγ Production

doi: 10.3389/fimmu.2021.699702

Figure Lengend Snippet: Monocyte expression of STING mediates S. pneumoniae induced late-stage lung IFNγ production. (A, B) STING −/− , cGAS −/− , and WT littermates were given PBS or infected (i.n.) with S. pneumoniae (D39 strain, ~5 × 10 6 CFU). IFNγ in lung homogenates (24 and 48hpi) were measured by ELISA (n = 3–4 mice per group). Data are representative of two independent experiments. (C) IFNAR1 −/− and WT littermate mice were infected with S. pneunoniae as in (A) . IFNγ in lung homogenates (48 hpi) were measured by ELISA (n = 3 mice per group). Data are representative of two independent experiments. (D, E) STING fl/fl CD11C Cre , STING fl/fl LysM Cre , and STING fl/fl littermates mice were infected (i.n.) with S. pneumoniae as in (A) . IFNγ in lung homogenates (24, 48 hpi) were measured by ELISA (n = 3 mice per group). Data are representative of two independent experiments. (F) STING −/− and WT littermates were infected with S. pneumoniae as in panel (A) At 16 hpi, 1 million bone marrow WT Ly6C hi monocytes were adoptively transferred (i.n.) into STING −/− mice (STING −/− + WT monocyte). IFNγ in lung homogenates was measured at 48 hpi by ELISA (n = 3 mice per group). Data were representative of two independent experiments. (G) A diagram of lung IFNγ production by monocyte-derived IL-12p70. (H) STING −/− and WT littermates were infected with S. pneumoniae as in (A) . At the 16 hpi, recombinant IL-12p70 (1 µg) was administered (i.n.) into STING −/− mice. IFNγ in lung homogenates was measured at 48 hpi by ELISA (n = 3 mice per group). Data are representative of two independent experiments. (I, J) STING −/− and WT littermates mice were infected (i.n.) with S. pneumoniae as in (A) . Ly6C hi monocytes (I) were identified in total lung cells at 48 hpi by flow cytometry. Total cell numbers of lung Ly6C hi monocytes (J) were enumerated (n = 3–4 mice per group). Data were representative of three independent experiments. Graphs represent the mean with error bars indicating SEM. p -values determined by one-way ANOVA Tukey’s multiple comparison test. Significance is represented by asterisk, where * p < 0.05, ** p < 0.001.

Article Snippet: The following strains were obtained from The Jackson Laboratory: CCR2 −/− , cyclic GMP–AMP synthase (cGAS) −/− , IL-12p70 −/− , IFNRA1 −/− , MyD88 −/− , TLR2 −/− , and IFNγ YFP-reporter.

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Derivative Assay, Recombinant, Flow Cytometry, Comparison

Journal: Frontiers in Immunology

Article Title: cGAS–STING and MyD88 Pathways Synergize in Ly6C hi Monocyte to Promote Streptococcus pneumoniae -Induced Late-Stage Lung IFNγ Production

doi: 10.3389/fimmu.2021.699702

Figure Lengend Snippet: Ly6C hi monocyte production of IL-12p70 and IFNγ require the activation of both cGAS–STING and MyD88 pathways. (A) Splenocytes isolated from a C57BL/6J mouse were cultured ex vivo for 4 days. Genomic DNA was extracted and run on an agarose gel (Self DNA 1 and Self DNA2). (B, C) Ly6C hi monocyte isolated from C57BL/6J mice were activated with self-DNA (1.5 µg/ml), HKSP (5 × 10 6 CFU/ml), 2′3′-cGAMP (4 µg/ml) or DNA + HKSP, HKSP + 2′3′-cGAMP for 17 h IL-12p70 (B) and IFNγ (C) were measured in the culture supernatant by ELISA. Data are representative of three independent experiments. (D–F) Ly6C hi monocyte isolated from indicated mice were activated with self-DNA (1.5 µg/ml) plus HKSP (5 × 10 6 CFU/ml) for 17 h as in (B) . IFNγ and IL-12p70 were measured in the culture supernatant by ELISA. Data are representative of three independent experiments. (G) TLR2 −/− and their WT littermates were infected (i.n.) with PBS or S. p (D39 strain, ~5 × 10 6 CFU). IFNγ in lung homogenates was measured by ELISA at 24 and 48 hpi (n = 4–5 mice/group). Data are representative of two independent experiments. (H) CCR2 −/− and WT littermates were infected with S. pneumoniae (D39 strain, ~8 × 10 6 CFU). At the 16 hpi, 1 million bone marrow WT, MyD88 −/− , or STING −/− Ly6C hi monocytes were adoptively transfer (i.n.) into CCR2 −/− mice. IFNγ in lung homogenates were measured at 48 hpi by ELISA (n = 4–5 mice/group). Data are representative of two independent experiments. Graphs represent the mean with error bars indicating SEM. p -values determined by one-way ANOVA Tukey’s multiple comparison test. Significance is represented by asterisk, where * p < 0.05, ** p < 0.001.

Article Snippet: The following strains were obtained from The Jackson Laboratory: CCR2 −/− , cyclic GMP–AMP synthase (cGAS) −/− , IL-12p70 −/− , IFNRA1 −/− , MyD88 −/− , TLR2 −/− , and IFNγ YFP-reporter.

Techniques: Activation Assay, Isolation, Cell Culture, Ex Vivo, Agarose Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, Infection, Comparison