Fig. 4.
DNA demethylation can restore cGAS-dependent STING activation in cGASabsent melanoma cell lines. Immunoblot analysis of cGAS, STING, DNMT1, DNMT3A, and DNMT3B expression in three cGASabsent human melanoma cell lines (526-MEL, A375, and WM39) with or without 5AZADC treatment. α-Tubulin and β-Actin were used as loading controls. Ratio of total cGAS relative to β-actin for each cell line was quantified using ImageJ software (A). Immunoblot analysis and ratio of p-IRF3 relative to IRF3 in indicated melanoma cell lines after 4 h stimulation with dsDNA (B). Induction of IFN-β (Top) and CXCL10 (Bottom) in indicated melanoma cell lines after stimulation with dsDNA measured using ELISA (C). Schematic of the cGAS-dependent STING signaling activation (D). Immunoblot analysis of cGAS, STING, and DNMT1 in wild-type or stably transduced WM39 cells with a lentiviral shRNA specific for sh-STING or sh-control with or without 5AZADC treatment (E). Immunoblot analysis of p-IRF3 and total IRF3 (F) and induction of IFN-β (Top) and CXCL10 (Bottom) (G) in indicated melanoma cell lines in response to stimulation with dsDNA. Data are mean ± SD of two or three biological replicates. Statistical significance was determined by unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant).