Extended Figure 9. Gap junctions initiate cytosolic DNA response in astrocytes.

a, Control or Cx43-deplated H2030-BrM3 cells were co-cultured for 18 h with or without astrocytes, and subjected to immunobloting analysis of phosphorylated TBK1 and IRF3 (n=3 independent experiments). b, Immunoblot of mouse astrocytes depleted on STING with short hairpins or control (non-silencing) sh. c, Mouse IFNa and TNFa were quantitated in the conditioned medium after co-culture (b) by ELISA. (2 independent experiments with 3 replicates each). d, LLC BrM growth in syngeneic C57Bl6 mice hosts wild-type (+/+) or knock-out (−/−) for STING. Diameter of brain metastases (d) Scale bar, 50 μm. Brains from all mice (n = 22) were sectioned, immunostained, and measured. All GFP(+) brain metastases were quantitated (2.8 +/− 0.67 metastases per +/+ 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. Values 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, Cox IV (mitochondria marker) in MDA-BrM2 cells. h, cGAMP identification. The peak at 4.47 min contains all 3 SRM transitions specific for cGAMP. RT: retention time, AA: automatically integrated peak area. i, j, EdU labeled MDA231-BrM2 cells were co-cultured with astrocytes for 6 h. Transfer of EdU-labeled 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 short hairpins or control (non-silencing) sh. l, Human astrocytes (Astro), were cultured for 18 h with or without H2030 BrM cells (BrM) expressing control shRNA (Ctrl sh) or shRNA targeting cGAS; Human IFNa and TNFa were quantitated in the conditioned medium by ELISA (l). (2 independent experiments in triplicate). m, Quantification of BLI signal from brain metastases formed by H2030 BrM3 cells depleted of cGAS with two independent sh. (Data are from 2 independent experiments with 6 mice total per group).