Figure 3. HBP1 expression is regulated by FOXO1 activity.

(A–C) Effect of LY294002 on FOXO1-mediated HBP1 expression. (A) HSC-3 cells were treated with LY294002 (20 μM) up to 12 h, followed by detection of p-Akt, p-FOXO1, HBP1, and α-tubulin expression by Western blotting analysis. One representative experiment out of three independent experiments is shown. Values are mean ± SD (*p < 0.05 and **p < 0.01 as compared with vehicle alone). (B) Total RNA isolated from LY294002-treated HSC-3 cells was subjected to RT-PCR analysis for the measurement of HBP1 mRNA level with 18S as an internal control. (C) HEK-293T cells were transfected with a 2-kb HBP1 promoter-luciferase construct, FOXO1 expressing vector, and β-galactosidase plasmid for 24 h, followed by treatment of increasing concentration of LY294002 (0–20 μM) for additional 24 h. Luciferase intensities were measured and normalized to β-galactosidase activities (*p < 0.05; **p < 0.01). (D) Effect of Akt activity on FOXO1-mediated HBP1 expression. A 2-kb HBP1 promoter-luciferase construct (0.2 μg), a β-galactosidase plasmid (0.1 μg), and FOXO1-Flag cDNA (0.2 μg) were co-transfected with 0.2 μg of pcDNA3 empty vector, Akt1, Akt1 T308A/S473A mutant, or constitutively active Myr-Akt1 vector into HEK-293T cells cultured in a 24-well plate. After 48 h of incubation, (D, upper panel) luciferase intensities were measured and normalized to β-galactosidase activities (**p < 0.01), and (D, lower panel) expression levels of p-Akt, Akt, Flag-FOXO1, p-Flag-FOXO1, and HBP1 were measured by Western blotting with α-tubulin as an internal control. (E) Effect of different FOXO1 domain on HBP1 promoter activity. A 2-kb HBP1 promoter-luciferase construct and a β-galactosidase plasmid were co-transfected with a pcDNA3 empty vector, FOXO1, FOXO1-H215R or constitutively active FOXO1-AAA vector (0.2 μg) into HEK-293T cells. After 48 h of transfection, luciferase intensities were measured and normalized to β-galactosidase activities (*p < 0.05; **p < 0.01).