Fig 2: Cell cycle-dependent localization of cGAS.a Cellular localization of cGAS in Raw264.7 cells. Raw264.7 cells were fixed and stained with anti-cGAS and DAPI. b cGAS is associated with chromosomes in different phases of mitosis. MLF cells were fixed and stained with anti-cGAS and DAPI before confocal microscopy. Inter, interphase; Pro, prophase; Prometa, prometaphase; Meta, metaphase; Ana, anaphase; Telo, telophase. c cGAS is associated with chromosomes upon NEBD in living cells. The GFP-cGAS plasmid was transfected into H2B-RFP-expressing HeLa cells for 20 h before live confocal microscopy. Scale bar, 10 μm. Data shown are representative of at least two biological repeats.

Fig 3: The CDK1-cyclin B complex accounts for cGAS phosphorylation and inactivation in mitotic cells.a CDK1 but not AKT or CDK2 inhibitor abolishes mcGAS S291 phosphorylation. Raw264.7 cells were arrested at mitosis with nocodazole (Noc arrest) and then treated with the indicated kinase inhibitors (10 μM) for 15 min before immunoblotting or FACS analysis. b cGAS is a direct substrate of the CDK1-cyclin B complex. In vitro kinase assays were performed with recombinant CDK1-Cyclin B proteins and FLAG-cGAS immunoaffinity-purified from THP1 cells stably-expressing FLAG-cGAS in the presence or absence of RO-3306 (10 μM). The reactions were then analyzed by immunoblotting analysis with the indicated antibodies. c The CDK1-cylin B1 complex impairs cGAS activation. MITA stably-expressing HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. ***P < 0.001 (Student’s t-test, unpaired, two-tailed). Data shown are mean ± S.D. of one representative experiment performed in triplet. Data are representative of three biological repeats.

Fig 4: Omp25 reduces level of cGAS protein and induces proteasome-dependent degradation of cGAS. (A,B) Omp25 interacted with cGAS. Cells were co-expressed with Flag-Omp25 and HA-cGAS for 24 h, and cell lysates were subjected to a co-IP assay using anti-Flag antibody, anti-HA antibody, and anti-β-actin antibody. (C,D) Omp25 degraded cGAS in murine and porcine macrophages. RAW264.7 or 3D4/21 cells were infected with 100 MOI of Lv-Omp25 or Lv-GFP for 36 h, then treated with or without CHX (100 μg/ml) for another 12 h. Level of cGAS was detected by Western blotting. (E,F) Omp25 promoted degradation of cGAS via proteasome-dependent pathway. RAW264.7 or 3D4/21 were infected with 100 MOI of Lv-Omp25 or Lv-GFP for 36 h and treated with dimethyl sulfoxide, CQ (20 μM), or MG132 (5 μM) in presence of CHX for another 12 h, followed by Western blotting to detect levels of cGAS. (G,H) Omp25 induced cGAS degradation through ubiquitin–proteasome pathway in RAW264.7 and 3D4/21 cells. Cells were transfected with vector expressing HA-Ub by Lipofectamine 3000 for 24 h, then infected with 100 MOI of Lv-Omp25 or Lv-GFP. At 36-h post-infection, cells were treated with MG132 (5 μM) for another 12 h; ubiquitination level of cGAS protein was determined by Western blotting.

Fig 5: cGAS is tightly tethered in the nucleus.(A) Mouse and human cell lines were separated into cytosolic and nuclear fractions, followed by sequential stepwise elutions of nuclear pellets with the indicated concentrations of NaCl. cGAS and the indicated control proteins (shown for HeLa cells) were monitored throughout the elution by western blot. (B) Densitometry measurements quantitating the relative amounts of endogenous cGAS protein in the cytosol, the 0 salt nuclear lysis, and the cumulative nuclear pellet.