Extended Data Figure 9: Gap junctions initiate cytosolic DNA response in astrocytes.
From: Carcinoma–astrocyte gap junctions promote brain metastasis by cGAMP transfer
a, Control or Cx43-depleted H2030-BrM3 cells were co-cultured for 18 h with/without astrocytes, and subjected to immunobloting analysis of phosphorylated TBK1 and IRF3 (n = 3 independent experiments). b, Immunoblot of mouse astrocytes depleted of STING with control (non-silencing) or Sting shRNAs. c, Mouse IFNα and TNF were quantified in the conditioned medium after co-culture by ELISA (n = 2 independent experiments with 3 replicates each). d, LLC-BrM growth in syngeneic C57Bl6 mice hosts wild-type (+/+) or knockout (−/−) for Sting. Bottom, diameter of brain metastases. Scale bar, 50 μm. Brains from all mice (n = 22) were sectioned, immunostained, and measured. All GFP+ brain metastases were quantified (2.8 ± 0.67 metastases per Sting+/+ mouse; 1.6 ± 0.55 in Sting−/− mice). e, Quantification of dsDNA in the indicated cellular fractions from 2 × 107 H2030-BrM3, MDA231-BrM2 or human astrocyte cells. Data are mean ± s.e.m. (n = 3 biological replicates; 2 independent experiments). f, Ratio of cytosolic dsDNA and nuclear dsDNA in indicated cancer cells and non-neoplastic cells. g, Representative image of immunofluorescent staining of dsDNA, GFP and CoxIV (a mitochondrial marker) in MDA231-BrM2 cells. h, cGAMP identification. The peak at 4.47 min contains all three selected reaction monitoring (SRM) transitions specific for cGAMP. AA, automatically integrated peak area; RT, retention time. i, j, EdU-labelled MDA231-BrM2 cells were co-cultured with astrocytes for 6 h. Transfer of EdU-labelled DNA from cancer cells to astrocytes was visualized using confocal microscopy (i), or quantified by flow cytometry (j). k, Immunoblot of H2030-BrM3 cells depleted of cGAS with shRNAs or control shRNA. l, Human astrocytes, were cultured for 18 h with/without H2030-BrM cells expressing control or cGAS shRNA. Human IFNα and TNF were quantified in the conditioned medium by ELISA (n = 2 independent experiments in triplicate). m, Quantification of BLI signal from brain metastases formed by H2030-BrM3 cells depleted of cGAS with two independent shRNAs (n = 2 independent experiments with 6 mice total per group).
