Figure 5.

Akt activation in Hh ligand producing hepatocytes. Primary hepatocytes were isolated from wildtype mice and cultured in the presence of 12.5μg/ml or 25μg/ml Tunicamycin or vehicle control (DMSO) for 1, 6 or 24 hours. (A) Representative western blot analysis for p-Akt, Akt, and lamin (loading control). (B) Cumulative densitometric analyses of p-Akt western blots results (as normalized to lamin expression) are displayed as the mean ±SD. (C) Cell numbers were measured by CCK-8 assay following incubation in 25μg/ml Tunicamycin. Cells were plated in triplicate for each experiment. The results of two experiments were averaged and mean±SD results are graphed. (*p<0.05, 24 hour vs. 1 hour-treated cells). (D) HepG2 cells, a hepatocytic cell line demonstrating basal expression of Ihh, were infected with an adenovirus containing dominant Akt (dnAkt; multiplicity of infection 25) or GFP (multiplicity of infection 25) for 24 h (to inhibit Akt). Cells were harvested at 48 hours and quantitative real-time PCR performed for Ihh. Graphed is one representative experiment; results were reproduced four times. Results were normalized to GFP and graphed as mean ± S.D. of triplicate plates. (**p = 0.003)