Fig 2: GSNO inhibits cGAS-STING activation.Quantitative polymerase chain reaction (qPCR) analysis of Ifnb mRNA expression in mouse peritoneal macrophages (PMs) pretreated with increasing concentrations of GSNO (0, 100, 250, and 500 μM) (a) or SNAP (0, 250, and 500 μM) (b), following herpes simplex virus-1 (HSV-1) infection. c Enzyme-linked immunosorbent assay (ELISA) analysis of interferon (IFN)-β secretion in mouse PMs pretreated with solvent (Ctrl), GSNO, or SNAP, plus stimulation as indicated. d–f Immunoblot assays of p-TBK1, p-IRF3, and p-STAT1 in PMs pretreated with solvent (Ctrl) or GSNO and then stimulated with HSV-1, IFN-stimulating DNA (ISD), or cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP). g qPCR analysis of Isg15, Isg54, Isg56, and Mx1 mRNA expression in PMs pretreated with solvent (Ctrl) or GSNO and then stimulated with cGAMP. h qPCR analysis of IFNB mRNA expression in THP1 cells pretreated with solvent (Ctrl) or GSNO and then stimulated with HSV-1 or cGAMP. i Immunoblot assays of p-IRF3 and p-STAT1 in THP1 cells pretreated with solvent (Ctrl) or GSNO and then stimulated with ISD. j Luciferase activity assays of IFN-β activation in 293-Dual hSTING-A162 cells pretreated with solvent (Ctrl), GSNO, or SNAP, following stimulation by DMXAA. k qPCR analysis of Ifnb mRNA expression in PMs from Sting+/+ or Sting-/- mice pretreated with solvent (Ctrl) or GSNO and then infected with L. monocytogenes. l Immunoblot assays of p-TBK1, p-IRF3, and p-STAT1 in PMs pretreated with solvent (Ctrl) or GSNO and then infected with L. monocytogenes. Data represent mean ± standard deviation (SD) or one representative image from three independent experiments. The p values were calculated using unpaired two-sided t test and adjustments were made for multiple comparisons.

Fig 3: cGAS-STING cytosolic DNA sensing pathway mediates axon regeneration induced by IFNγ(A) Retinal sections from WT or Ptpn2 cKO mice with PBS or IFNγ treatment. The samples were collected 2 dpc and stained for Tuj1 (green), and cGAS (red). Scale bar, 50 μm.(B) Quantification of the percentage of cGAS+ RGCs in (A). ANOVA followed by Dunnett’s test. n = 3–4 mice.(C) Retinal sections from WT intact, cKO intact or WT+CPT mice stained with dsDNA (green), TOMM20 (red), and Tuj1 (gray). Scale bar, 2 μm.(D) Quantification of relative cytoplasmic DNA intensity from (E). ANOVA followed by Dunnett’s test. n = 11–25 cells.(E) Sections of optic nerves from WT, Cgas KO, Sting KO, or Mavs KO mice at 2WPI. The vitreous body was injected with AAV-sh-Ptpn2 plus IFNγ. Scale bars, 200 μm.(F) Number of regenerating axons at indicated distances from the lesion site. ANOVA followed by Tukey’s test, n = 3–4 mice.(G) Quantification of regenerating axons in WT, Cgas KO, Sting KO, or Mavs KO mice. ANOVA followed by Tukey’s test, n = 3 mice.(H) Sections of optic nerves from WT mice at 2 WPI, with PBS or 25-mM cGAMP treatment. Scale bar, 200 μm.(I) Number of regenerating axons at indicated distances from the lesion site. ANOVA followed by Tukey’s test, n = 3 mice. Mean ± SEM; *p < 0.05, **p < 0.01.See also Figure S4.

Fig 4: The combined treatment attenuated expression of kidney injury, inflammation, and fibrosis markers in Pkd1 mutant mouse kidneys. (A) Heatmap showed the increased mRNA expression for kidney injury markers (Havcr1, Lcn2), components of cytosolic DNA sensor (Cgas, Sting), inflammatory transcription factor NFκB subunits (Nfkb1, Rela), inflammatory cytokines (Tnf, Ccl2), fibrosis marker (Acta2), as well as chloride channel (Cftr) in Pkd1 mutant kidneys; all these changes were attenuated by the treatments. (B) Transcript levels (microarray) of Havcr1, Lcn2, Rela, and Ccl2 correlated with 2 KW/BW% in Pkd1 mutant kidneys. (C) Relative protein levels of cGAS, STING, NFκB activity (pSer536 NF-kB p65), TNF-α, and α-SMA (Acta2) in WT, untreated and treated mutant mouse kidneys by Western blot. GAPDH was used as a loading control. (D) Relative mRNA levels of Ccl2 in WT, untreated and treated mutant mouse kidneys by RT-qPCR.

Fig 5: FA inhibits the cellular cGAS activation. A) Immunoblotting analyses of the lysates from cGAS wild type (WT) and knockout (KO) MEF cells treated with 1 µg mL−1 of ISD transfection for 0.5 h and then 1 mm of OA for 8 h. IRF3: interferon regulatory factor 3; p‐IRF3 (ser396): phospho‐interferon regulatory factor 3 (Ser396). WT: wild type; KO: knockout. ISD: oligo immune stimulatory DNA. OA: oleic acid. n = 3. B) Immunoblotting analyses of the lysates from the MEF cells treated with 1 mm of OA for 1 h and then 1 µg mL−1 of ISD transfection for 8 h. IRF3: interferon regulatory factor 3; p‐IRF3 (ser396): phospho‐interferon regulatory factor 3 (Ser396). WT: wild type; KO: knockout. ISD: oligo immune stimulatory DNA. OA: oleic acid. n = 3. C) Ifnb mRNA and cGAMP levels in the cells treated with transfection and then OA. cGAS WT and KO MEF cells were treated with 1 µg mL−1 of ISD or poly (I:C) (pIC) dsRNA transfection for 0.5 h, and then 1 mm of OA for 8 h. Ifnb mRNA and cGAMP levels in the resultant cells were analyzed by qRT‐PCR and MS, respectively. ISD: 45‐bp oligo immune stimulatory DNA. pIC: poly (I:C) dsRNA. Unless specifically noted, mean ± standard deviation (mean ± SD) was used in this study. Unless specifically noted, p values were calculated by unpaired two‐tailed Student's t‐test, and p values < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). n = 6. D) Enzyme linked immunosorbent assays (ELISAs) of the secretion of IFN‐β protein from the cells treated with 1 µg mL−1 of ISD or pIC dsRNA transfection for 0.5 h, and then 1 mm of OA for 12 h. n = 3. E) Ifnb mRNA and cGAMP levels in the MEF cells treated with 1 mm of OA for 1 h, and then 1 µg mL−1 ISD or pIC dsRNA transfection for 8 h. n = 6. F) IFN‐β protein secreted from the cells treated with 1 mm of OA for 1 h, and then ISD or pIC dsRNA transfection for 12 h. n = 3. G) Ifnb mRNA and cGAMP levels in the MEF cells treated with irradiation (IR) and/or OA. cGAS WT and KO MEF cells were treated with 8 Gy of IR. After 1 h, the cells were treated with 1 mm of OA for 24 h. n = 6. H) IFN‐β protein secreted from the cells treated with IR and/or OA as described in (G). n = 3. I) Ifnb mRNA and cGAMP levels in the MEF cells treated with OA and/or herpes simplex virus‐1 (HSV) infection. cGAS WT and KO MEF cells were treated with 1 mm of OA for 1 h, and then five multiplicity of infection (MOI) of HSV infection for 8 h. n = 6. J) IFN‐β protein secreted from the cells treated with OA and/or HSV infection. The MEF cells were treated with 1 mm OA for 1 h, and then 5 MOI HSV infection for 12 h. n = 3.