Effect of SIRT1 activity on the nuclear export and acetylation of FoxO3a during CK2 downregulation-mediated senescence. HCT116 and MCF-7 cells were treated with CK2α siRNA (A, C) or pcDNA3.1-HA-CK2α (B, D) in the absence or presence of pECE-flag-SIRT1 (A, C) or pECE-flag-SIRT1 mutant (H363Y) (B, D) for 48 h. (A, B) The subcellular distribution of FOXO3a (red) was determined by immunofluorescence staining (upper panels). Cells were counterstained with DAPI (blue) to visualize the nuclei. Fluorescence intensity was quantified using ImageJ software (bottom panels). Arbitrary intensity values for FoxO3a (red) or DAPI (blue) are shown relative to the reference line (white) used for analysis. (C, D) Cell lysate was immunoprecipitated (IP) with anti-acetyl Lys antibody, followed by western blotting with anti-FoxO3a antibodies. Cell lysates IP with IgG heavy chain served as a loading control. Representative data from three independent experiments are shown (upper panels). The graphs show the relative amount of acetylated FoxO3a (bottom panels).
