Foxo1 expression confers insulin inhibition on G6p, but not on Pepck. (a) Time-course analysis of dex/cAMP effect in cells transduced with Foxo1. LLC cells were transduced with adenovirus encoding WT Foxo1 and incubated with dex/cAMP for the indicated periods of time. At the end of the incubation, mRNA was extracted and analyzed by Northern blot analysis using the relevant cDNA probes. (b) Time-course analysis of insulin effect. LLC cells were transduced with Foxo1 adenovirus. After 48 hours, the medium was replaced with serum-free medium, and incubation was continued overnight. Thereafter, dex/cAMP was added for 16 hours, followed by 6 hours of treatment in the presence (lanes 2–5) or in the absence of insulin (lanes 6–9) for various lengths of time, as indicated. Northern blot analysis was performed as described above. (c) Northern blot of G6p and Pepck mRNAs following transduction of LLC cells with adenoviral vector encoding Foxo1. LLC cells were incubated in serum-free medium overnight before addition of dex/cAMP for 4 hours (lanes 2 and 3) or 16 hours (lanes 5 and 6), followed by insulin stimulation for 6 hours (lanes 3 and 6). Thereafter, mRNA was isolated and Northern blot analysis was performed with cDNA probes encoding G6p (upper panel), Pepck (middle panel), and β-actin (lower panel). A representative experiment is shown, and data from three independent adenoviral transductions are summarized in (d). Mean ± SEM of the percentage of insulin inhibition of dex/cAMP–induced expression at 16 hours was calculated from three independent experiments using densitometric scanning of the autoradiograms. The mRNA loading was normalized by subsequent hybridization with a β-actin probe. *P < 0.05 ANOVA.