Fig. 6

SIRT3 deacetylates FOXO3 to regulate Prkn gene transcription. a The molecular docking image of SIRT3 and FOXO3. b, c CoIP of SIRT3 and FOXO3 with or without SIRT3 transfection in MC3T3-E1 cells. d Schematic illustration of the mechanism that SIRT3 deacetylates FOXO3, thereby improve Prkn-mediated mitophagy. e Acetylation status of mitochondrial and cytoplasmic proteins was determined by western blot using pan-acetylated lysine antibody. Blue indicates Lv-Ctrl, dark blue indicates Lv-Sirt3, gray indicates Lv-Ctrl + HGPA and dark gray indicates Lv-Sirt3 + HGPA. The same color scheme applies to (f, h). f Western blot analysis of acetylated FOXO3 in MC3T3-E1 cells with or without SIRT3 overexpression and HGPA stimulation. g Representative IF images of FOXO3 in MC3T3-E1 cells. Scale bar, 20 μm. h Western blot analysis of nuclear-localized and cytosolic FOXO3 in MC3T3-E1 cells. i Schematic representation of CUT&Tag experiment using anti-FOXO3 antibody, followed by DNA purification and qPCR analysis with primers designed for predicted binding sites (generated by Figdraw). j The binding motif for FOXO3 included in the promoter region of the Prkn gene was predicted by JASPAR. k CUT&Tag-qPCR assay of Prkn enriched by FOXO3 antibody. Data presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001