Fig. 2.

Expression of activated STAT3 (pSTAT3) and PD-L1 in MC-C and LMM3 tumors. pSTAT3 was evaluated by flow cytometry (A) and Western Blotting (B) in LMM3, MC-C, and LMM3 tumor cells that have been treated with 20 ng/ml of JSI-124 for 24 h. Controls with actin and total STAT3 were added. Results are representative of three similar experiments. (C, D) Percentage of cells PD-L1⁺ (C) and mean fluorescence intensity (MFI) (D) in MC-C and LMM3 tumors. Each value represents the mean ± SEM of three assays. (E, F) Representative dot plots of expression of PD-L1 in MC-C (E) and LMM3 (F) tumor and tumor-infiltrating cells. (G) Acquired capacity of LMM3 lysate to promote the maturation of dendritic cells (DC) by pre-treatment with JSI-124. Expression of cell-surface receptor CD86 was evaluated by flow cytometry. DC were incubated with LMM3 lysate or with lysate from LMM3 cells that had been pre-treated in vitro for 24 h with different concentrations (1, 5, 10, and 20 ng/ml) of JSI-124. DC incubated with LPS served as a positive control. Negative controls were immature DC (DCi). Data represent the mean ± SEM of three independent experiments. (H) Vaccinating capacity against LMM3 of DC stimulated with a lysate from LMM3 cells that had been treated in vitro with 20 ng/ml of JSI-124. Two doses of DC of the different groups were inoculated in the footpad of mice 14 and 7 days before the s.c. challenge with different doses of LMM3 tumor cells. Vaccinating capacity was measured as an increase of tumor dose 50 (DT₅₀) of LMM3 tumor in treated mice compared to control. Data represent the mean ± SEM of two independent experiments. In each experiment, 20–25 mice per group were utilized. Statistical comparison between experimental groups and DCi: ## p < 0.01; ### p < 0.001. Statistical comparison among experimental groups: ** p < 0.01; *** p < 0.001