FOXO3a regulates expression of ER target genes and CDK inhibitors, and induces apoptosis in MCF-7. (a) Ectopic expression of Forkhead box class O (FOXO)3a reduces the expression of some estrogen receptor (ER)-regulated genes and enhances the expression of cyclin-dependent kinase (CDK) inhibitors in MCF7-FO cells in the presence of 17β-estradiol (E2). Immunoblotting (IB) analyses for HA-FOXO3a, endogenous FOXO3a, p27Kip1, p21Cip1, p57Kip2, cyclin D1, cyclin E, cathepsin D, progesterone receptor (PgR), ER-α, and ER-β protein expression in MCF7-FO33 and MCF7-FO41 cells (constitutively expressing FOXO3a) and in control (MCF-7 and MCF7-C5) cells were performed with specific antibodies, as indicated. Equal loading was confirmed by the same IB analysis with antibodies against β-actin or β-tubulin. (b) MDA-MB-453 (MDA-453, ER-negative) cells were transfected with either FOXO3a or an empty pCDNA3.1 vector (control), as indicated. Total lysates of the transfected cells were prepared and subjected to SDS-PAGE followed by IB analysis with the indicated antibodies.