Fig. 6: REGγ declines SOD2 expression in a PP2Acα–FoxO3a-dependent manner.

a Protein levels of PP2Acα, P-FoxO3a, and SOD2 in REGγ+/+ and REGγ−/− heart tissue, and siNeg and siREGγ NRCMs and AC16 cells. Knocking out or silencing REGγ decreases FoxO3a phosphorylation levels in (b) murine heart tissue or in (c) human cardiomyocyte AC16 cells. REGγ knockdown promoted the translocation of FoxO3a from the cytoplasm to the nucleus in AC16 cells by d immunofluorescence analysis (scale bars: 20 μm) and e corresponding quantitation graphs, and f cell fractionation assay. (The experiments were repeated three times. Error bars represent standard deviation, **P < 0.01, Student’s t test.) g The levels of P-FoxO3a after OA treatment or PP2Acα overexpression in AC16 cells. h Overexpressing PP2Acα or i blocking PP2Acα activity by OA treatment significantly diminished the change of FoxO3a phosphorylation levels which were caused by REGγ knockdown in AC16 cells, similar results of (j–m) immunofluorescence analysis (scale bars: 20 μm) and corresponding quantitation graphs of FoxO3a translocation, and n cell fractionation assay was observed. (The experiments were repeated three times. Error bars represent standard deviation, **P < 0.01, one-way ANOVA test.) o Expression of SOD2 after PP2Acα overexpression, knockdown or activity inhibition by PP2Acα plasmid, siPP2Acα transfection or OA treatment, or FoxO3a overexpression or knockdown by FoxO3a or siFoxO3a plasmid transfection. p–r PP2Acα knockdown, overexpression, or activity inhibition dramatically rescued or attenuated SOD2 mRNA expression of Ang II stimuli regardless of REGγ levels. s, t FoxO3a overexpression or knockdown diminished the change of SOD2 mRNA expression of Ang II stimuli which REGγ caused. u, v Similar results of luciferase assays were observed in AC16 cells. (The experiments were repeated three times; error bars represent standard deviation, **P < 0.01, ***P < 0.001, one-way ANOVA test).