Figure 6. STAT3 signaling is involved in down-regulation of Foxp3 by entinostat.

A and B, Entinostat treatment induces STAT3 acetylation. HepG2 cells or splenocytes were treated for 6 hours, harvested, and lyzed for Western immunobloting or immunoprecipitation. A, Entinostat induced STAT3 acetylation in HepG2 cells. Upper panel: Cell lysates were analyzed directly and blotted with anti-STAT3 and actin. Bottom panel, Cell lysates were immunoprecipitated with anti-STAT3 antibody and then blotted with anti-STAT3 antibody and with anti-acetylated lysine antibody. B, Entinostat induced STAT3 acetylation in splenocytes. IP and Western blot were performed as described in A. C and D, Down-regulation of Foxp3 is inhibited by blocking STAT3 signaling. Splenocytes were harvested from BALB/c mice and put in culture. Cultures were treated with vehicle or 0.5 mM specific STAT3 peptide inhibitor for one hour, followed by vehicle or 0.5 µM entinostat treatment for 23 hours. Cells were harvested and analyzed by flow cytometry using fluorescence-conjugated antibodies specific to CD4 and Foxp3. Fixable live/dead dye was used to stain cells and live cells were gated. Cell culture was in absence (panel C) or in presence (panel D) of IL-2. In each condition, histograms show quantification of Foxp3 expression in Tregs by FACS analysis. Graph shows means, error bars represent standard deviations. In C, p = 0.0002 for entinostat vs. STAT3 inhibitor+entinostat. In D, p = 0.00015 for entinostat vs. STAT3 inhibitor+entinostat. Results are representative of three separate experiments.