Fig 2: Recovery of CRIg+ (complement receptor of the immunoglobulin superfamily) macrophage population attenuates obesity‐associated adrenal dysfunction.After 4 weeks of injection with lentivirus harboring deactivated Cas9 and guide RNA‐Vsig4, the abundance of bacterial DNA (A), proinflammatory genes (B), cGAS/STING activation (C) in the adrenal glands, circulating catecholamine (D), and systolic blood pressure (SBP) (E) of 16 weeks high‐fat diet (HFD) wild‐type (WT) mice were evaluated. Data are presented as mean±SEM. P values are determined by unpaired 2‐sided Student t test (A through E). Cas9 indicates CRISPR associated protein 9; cGAS, Cyclic GMP–AMP synthase; CRIgoe, overexpressing CRIg; EPI, epinephrine; HSP90, heat shock protein 90; NE, norepinephrine; pSTING, phosphorylated STING; STING, the cyclic GMP–AMP receptor stimulator of interferon genes; and Vsig4, V‐set and immunoglobulin domain containing 4.

Fig 3: The N-terminal domain and NES regulate cGAS nuclear localization.a The cell lysates of RAW264.7 macrophages were separated by 15–55% sucrose density centrifugation. Fractions were blotted as indicated. The fraction of thyroglobulin (669 kDa), a protein standard, was indicated. b H1299 cells were stimulated with or without 1 μg/mL ctDNA by transfection for 4 h. Then, the cell lysates were fractionated into five fractions: cytoplasmic, membrane, nuclear soluble, chromatin-bound, and cytoskeletal. The fractions were blotted as indicated. STING: membrane marker; TBK1 and β-actin: cytosolic marker; H3: nuclear marker; vimentin: cytoskeletal marker. c Wild type (WT) and cGAS knockout (KO) H1299 cells were either mock stimulated or transfected with ctDNA. After 4 h, cells were fixed and stained as indicated. cGAS: green; DAPI, blue. Bar = 10 μm. d Representative cGAS localization in unstimulated H1299 WT cells in (c). (i) surrounding nucleoli; (ii) micronucleus; (iii) chromosome; (iv) chromatin bridge; (v) perinuclear region. Arrows indicate each distinct localization in (i) to (v). cGAS: green; DAPI, blue. Bar = 10 μm. e Schematic of the LL/RK mutation in the nuclear export signal of mouse cGAS (mcGAS). Red arrows indicate the mutated sites, and the green frame indicates the introduction of a NLS caused by the mutation. f IFA of HEK293 cells stably expressing FLAG-tagged mcGAS or the indicated LL/RK mutant. FLAG: red; DAPI, blue. Bar = 10 μm. The summary of the subcellular localization of mcGAS and the LL/RK mutant was shown in the left panel. C: cytosolic; N: nuclear; C + N: cytosolic and nuclear. Data represent means ± s.d. of three independent experiments (> 200 cells were counted in each field and five fields were counted per experiment). g HEK293 cells stably expressing the delN with LL/RK mutation of mcGAS (mcGAS-delN-LL/RK) were transfected with or without 1 μg/mL ctDNA for 4 h. Then, the cell lysates were fractionated into five fractions: cytoplasmic, membrane, nuclear soluble, chromatin-bound, and cytoskeletal. The fractions were blotted as indicated. C1QBP: membrane marker; TBK1 and α-tubulin: cytosolic marker; H3: nuclear marker.

Fig 4: Glycolysis facilitates STING signaling in DCs from human NSCLC.(A–C) Correlation between LDHA transcripts and STING signature, including STING (A), IFNA (B), and IFNB (C), in TCGA data set of patients with lung cancer with low or high LDHA transcripts (n = 262). (D) ECAR of the freshly isolated DCs from the paracancerous tissue and NSCLC tissue under Bas or Max conditions. (E) Intracellular ATP of the freshly isolated DCs from the paracancerous tissue and NSCLC tissue. (F) Immunoblot analysis of indicated proteins in whole-cell lysates of the freshly isolated DCs from the paracancerous tissue and NSCLC tissue. (G) Immunoblot analysis of the freshly isolated NSCLC DCs treated with 2-DG (5 mM) for 8 hours. Ctrl, without 2-DG treatment. (H) qRT-PCR analysis of DCs isolated from the paracancerous tissue and NSCLC tissue and NSCLC DCs treated with 2-DG (5 mM) for 8 hours. (I) Immunoblot analysis of indicated proteins in whole-cell lysates of human NSCLC DCs that were pretreated with 2-DG (5 mM) for 6 hours and subsequently incubated in the presence or absence of ATP and SLO for 3 hours. The numbers indicate the relative densities of indicated protein bands normalized to β-actin. Data are representative of 3 independent experiments (D–I). Data are shown as the mean ± SEM. Statistical analysis was performed using a paired 2-sided Wilcoxon signed-rank test (A–C), 2-tailed Student’s t test (D and E), and 1-way ANOVA (H); **P < 0.01.

Fig 5: Nuclear soluble cGAS senses HSV-1 infection and instigates innate immune response.a The cGAS KO(LL/RK-NLS) RAW264.7 cells were treated with 2 μg/mL Dox for 24 h, followed by mock-infection or infection with 1 MOI of HSV-1 McKrae for 16 h. Then, cells were fractionated into five fractions and blotted as indicated. STING: membrane marker; TBK1 and α-tubulin: cytosolic marker; H3: nuclear marker. Red arrow indicates nuclear soluble cGAS. b, c The cGAS KO, KO(mcGAS), KO(LL/RK-NLS) RAW264.7 cells were treated with Dox for 24 h and then infected with 1 MOI of HSV-1 KOS d109 for designated times. Real-time PCR was performed to determine the relative mRNA levels of IFNβ (b) and IP-10 (c). Data represent means ± s.d. of three independent experiments. The P-value was calculated by two-way ANOVA followed by Dunnett’s multiple comparisons test. d The cGAS KO, KO(mcGAS), KO(LL/RK-NLS) RAW264.7 macrophages were treated with Dox for 24 h and then infected with 1 MOI of HSV-1 KOS d109 for indicated times. Cell lysates were collected and blotted as indicated. e cGAS KO, KO(mcGAS), KO(LL/RK-NLS) RAW264.7 macrophages were treated with Dox for 24 h and then infected with 1 MOI of HSV-1 KOS d109 for 8 h. The amount of cGAMP in each cell line was determined by ELISA assays. All experiments were repeated three times. The P value was calculated by one-way ANOVA followed by Tukey’s multiple comparisons test. f The cGAS KO(LL/RK-NLS) RAW264.7 cells were treated with DMSO or 25 μM RU.521 for 16 h. Then, cells were mock-infected or infected with HSV-1 KOS d109 for 16 h. Real-time PCR was performed to determine the relative mRNA levels of IFNβ, IP-10, and RANTES. Data represent means ± s.d. of three independent experiments. The P-value was calculated by two-way ANOVA followed by Tukey’s multiple comparisons test. g The cGAS KO(LL/RK-NLS) and KO(LL/RK-NLS)/D307A cells were treated with Dox for 24 h and then mock-infected or infected with HSV-1 KOS d109 for 16 h. Real-time PCR was performed to determine the relative mRNA levels of IFNβ, IP-10, and RANTES. Data represent means ± s.d. of three independent experiments. The P-value was calculated by two-way ANOVA followed by Sidak’s multiple comparisons test.