ER-α and ER-β bind to the amino-terminal and carboxyl-terminal domains of FOXO3a, respectively. (a) Glutathione-S-transferase (GST) – pull down in vitro assays. Whole cell lysates from 293T cells were incubated with the GST-forkhead box class O (FOXO)3a (GST-FO [amino acids 1 to 300] and GST-FO [amino acids 301 to 673]) fusion proteins as indicated and GST alone (negative control), and analyzed by SDS-PAGE and immunoblotting with an antibody (Ab) against estrogen receptor (ER)-α or ER-β (upper panels) and an anti-GST Ab (lower panel) as protein controls. (b) FOXO3a downregulates FOXM1. Immunoblotting (IB) analyses for endogenous FOXO3a, forkhead box M1 (FOXM1), and β-actin (loading control) protein expression in MCF7-FO10 and MCF7-FO41 cells (constitutively expressing FOXO3a) and in control (MCF-7 wt and MCF7-C12) cells were performed with specific antibodies antibodies as indicated. (c) MCF7-d8_pa cells (pooled clones of MCF-7 FOXO3a-knockdown derivatives) were established with retroviruses expressing small hairpin RNA against human FOXO3a. The expression levels of FOXO3a and p27Kip1 in MCF-7 wild-type (wt) and MCF7-d8_pa cells were determined by IB with specific Abs against FOXO3a or p27Kip1 or β-actin (loading control). (d) Silencing endogenous FOXO3a in MCF-7 cells promoted tumorigenesis in vivo. The tumor growth rates of control group MCF-7 wt and knockdown group MCF7-d8-pa were determined after injection of cells (2 × 106cells/mouse) as indicated into the mammary fat pads of female athymic mice not given supplemental 17β-estradiol (E2; indicated as – E2). Data are expressed as means and standard deviations from two experiments with five mice in each group. *P < 0.01 between MCF7-d8-pa group versus control MCF-7 wt group.