Fig. 4. Demethylation improves melanoma response to STING agonist therapy in STING^gt/gt mice.

Schematic of the STING agonist and 5AZADC treatment schedule. Groups of STING^gt/gt mice were injected subcutaneously with 1.5 × 10⁵ B16-F10 or Yumm1.7 or 1 × 10⁵ B16-ISG or B16-ISG-STING^KO cells on day 0 and were intratumorally treated with 50 μg of ADU-S100 and/or 0.1 mg/kg of 5AZADC or PBS (a). Tumor growth curves of B16-F10 (b), Yumm1.7 (c), B16-ISG (d) and B16-ISG-STING^KO (e) in STING^gt/gt mice treated with PBS, 5AZADC, ADU-S100, or 5AZADC + ADU-S100 according to the schedule presented in (a). Data are shown as the mean ± SEM and are representative of two independent experiments (n = 5 mice per group in (b), n = 4, 4, 4, and 5 mice in (c), n = 4, 6, 5, and 7 mice in (d), n = 6, 6, 5, and 6 mice in (e) for Control, 5AZADC, ADU-S100, and 5AZADC + ADU-S100 groups, respectively). Immunoblot analysis of STING, DNMT3A and DNMT3B expression in tumor lesions of STING^gt/gt mice bearing B16-F10 or Yumm1.7 tumors with or without 5AZADC treatment. β-Actin was used as a loading control (f). Images are representative of two independent experiments. Ratio of total STING relative to β-Actin was quantified using ImageJ version 1.53a software (g). Quantitative RT-PCR analysis of Ifnb1 and H2-k1 mRNA expression in B16-F10 tumors in STING^gt/gt mice treated with PBS, 5AZADC, ADU-S100, or 5AZADC + ADU-S100 (n = 3 biological replicates). Data are shown as mean ± SD and are representative of two independent experiments (h). Statistical significance was determined by two-way (b–e) or one-way ANOVA (h) (ns, not significant).