Fig 2: PCBP2 attenuates the cGAS condensation.a HEK293T cells were transfected with the indicated expression plasmids. After 24 h of transfection, cell lysates were subjected to Co-IP with anti-Flag beads, followed by immunoblotting. b HEK293T cells were transfected with the indicated expression plasmids. After 24 h of transfection, cell lysates were resolved with SDD-AGE or SDS-PAGE, followed by immunoblotting. c Pcbp2+/+ and Pcbp2-/- L929 cells were transfected with HT-DNA (2 µg/ml) for 6 h, and the cell lysates were resolved with SDD-AGE or SDS-PAGE, followed by immunoblotting. d Recombinant cGAS and PCBP2 wild-type or its KH3-deleted mutant proteins were incubated with or without HT-DNA (200 µg/ml) at 25 °C for 1.5 h and then resolved by SDD-AGE and SDS-PAGE, followed by immunoblotting. e HEK293A cells were transfected with GFP-cGAS, together with mCherry-tagged wild-type PCBP2 or its KH3-deleted mutant or mCherry for 24 h. The cells were fixed, stained with DAPI (blue), and observed by confocal microscopy. Scale bars, 10 µm. The number of cGAS granules per cell was quantified (bottom), at least 100 cells from each group were analyzed. p < 0.0001; p < 0.0001 in sequence. P2: PCBP2. f Pcbp2+/+ and Pcbp2-/- L929 cells stably expressing mouse cGAS-GFP were transfected with or without HT-DNA (2 µg/ml) for 6 h. The cells were then fixed, stained with DAPI (blue), and observed by confocal microscopy. Scale bars, 10 µm. The number of cGAS granules per cell was quantified (bottom), at least 100 cells from each group were analyzed. p < 0.0001. g–i Recombinant GFP-cGAS proteins (5 µM) were incubated with 2 µM of GST-tagged wild-type PCBP2 or its KH3-deleted mutant or GST proteins in the presence of 2.5 µM Cy3-ISD (g), Cy3-VACV70 (h), or HSV120 (i) for 5 min. Confocal images are representative of cGAS condensates of all fields. Scale bars, 10 µm. Data shown in e, f are from one representative experiment of at least three independent experiments (mean ± SD, n = 107 cells in e, and n = 133 cells in f). Two-tailed Student’s t-test was used to analyze statistical significance. ***p < 0.001. Source data are provided as a Source data file.

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: Inhibition of HSV-1 replication blocks cGAS release from the chromatin.a RAW 264.7 cells were mock-infected or infected with 1 MOI of HSV-1 KOS d109 for 16 h. Cell lysates were fractionated and blotted as indicated. b RAW 264.7 cells were pretreated without or with 8 μg/mL of acyclovir for 16 h, followed by infection with 1 MOI of HSV-1 McKrae for 12 h. Cell lysates were fractionated and blotted as indicated. The arrow indicates nuclear soluble cGAS. c RAW 264.7 macrophages were treated with dimethylformamide (DMF) as a vehicle mock control, 50 μM cisplatin for 4 h, or infected with HSV-1 McKrae for 12 h. Cell lysates were fractionated and blotted as indicated. The arrow indicates nuclear soluble cGAS. STING: membrane marker; α-tubulin: cytosolic marker; H3: nuclear marker; ICP8: HSV-1 viral protein; γ-H2A.X: DNA damage marker. The arrow indicates nuclear soluble cGAS. d RAW 264.7 macrophages were treated with DMF (mock control), 50 μM cisplatin or infected with HSV-1 McKrae for indicated times. Then, cell viability was determined by MTT assays. 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.

Fig 5: miR-4767 antagomir abolished the protective roles of HDAC3 deficiency in macrophage in LPS-treated mice(A and B) Histological analyses of the H&E staining and lung injury score of LPS-treated HDAC3-CKO mice injected with antagomir NC or miR-4767 antagomir (n = 6).(C) Lung wet/dry weight (n = 6).(D) The level of miR-4767 in lung tissues (n = 6).(E and F) Relative mRNA levels of Tnf-α and IL-6 in LPS-treated HDAC3-CKO mice injected with antagomir NC or miR-4767 antagomir (n = 6).(G) Western blot analysis of cGAS, P-STING, T-STING, NLRP3, and GSDMD-N in LPS-treated HDAC3-CKO mice injected with antagomir NC or miR-4767 antagomir (n = 6). Values represent the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus the matched groups, n.s. represents no significance.