Fig. 3.

Reconstitution of STING expression through DNA demethylation can rescue STING signaling in human melanoma cell lines. Experimental setup for 5AZADC treatment of STING^absent human melanoma cell lines at indicated time points (A). Immunoblot analysis of STING, DNMT1, DNMT3A, and DNMT3B expression in human melanoma cell lines with or without 5AZADC treatment. α-Tubulin and β-Actin were used as loading controls (B). Ratio of total STING, DNMT1, DNMT3A, and DNMT3B relative to β-Actin for each cell line with or without 5AZADC treatment (C). Experimental setup for 5AZADC treatment and 2’3′-cGAMP stimulation of STING^absent human melanoma cell lines at indicated time points (D). Ratio of p-IRF3 relative to total IRF3 (E) and immunoblot analysis of p-IRF3 and IRF3 in indicated melanoma cell lines after 4 h stimulation with 2’3′-cGAMP (F). Induction of CXCL10 (Top) and IFN-β (Bottom) in cell culture supernatants measured using ELISA and reported as mean ± SD for two or three biological replicates (G). WM1361A melanoma cells with or without 5AZADC pretreatment were cocultured with HLA-matched human melanoma TIL 40 in the presence or absence of 2’3′-cGAMP. In some experimental conditions, tumor cells were preincubated with an MHC class I blocking Ab (W6/32) to determine whether IFN-γ release was mediated by CD8⁺ TIL T-cell receptor engagement with peptide/MHC class I. Coculture supernatants were collected after 24 h, and IFN-γ and IFN-β concentrations were measured using ELISA and reported as mean ± SD for two or three biological replicates (H). Statistical significance was determined by unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant).