(A) SV40 hepatocytes were transiently cotransfected with reporter plasmids and incubated with FSK-DEX and/or insulin or LY294002. (B) Eight hours after cotransfection, cells were incubated with insulin for 16 hours. (C) Cells were transduced with either empty, Myr-p110, or Myr-Akt adenovirus. After 24 hours, cells were transiently cotransfected with the indicated plasmids and adenoviruses. (D) Deletion analysis of the Trb3 promoter in SV40 hepatocytes cultured in the presence of FSK-DEX (white bars), FoxO1ADA (black bars), or LY294002 (gray bars). *P < 0.05 vs. vector-transfected cells; **P< 0.01; and ^#P < 0.05 vs. β-gal–transduced cells. (E) ChIP in SV40 hepatocytes. We immunoprecipitated the following with anti-FoxO1 (upper panel) or control antiserum (middle panel): lane 1, untransduced cells; lane 2, cells transduced with FoxO1ADA; lanes 3–4, cells transduced with WT FoxO1. Bottom panel: total input DNA. (F) Mutation analysis. Control (WT) or mutant Trb3 promoter with mutations of the Foxo sites (Mutant) were assayed in basal conditions (white bars) and in the presence of LY294002 (black bars) or FoxO1ADA (gray bars). **P < 0.01. (G) Effect of FoxO1 knockdown on Trb3 promoter activity. SV40 hepatocytes were transiently cotransfected with either FoxO1siRNA or control siRNA with Trb3 promoter reporter construct at final concentration of 30 nM. **P < 0.01.