Fig. 4. Role of GSK3β signaling in mediating apoptosis and cell cycle arrest by AKT dephosphorylation in hepatocytes treated with leptin. (A, B) Hepatocytes were pretreated with SC79 (4 µg/mL) (A) or IL-1Ra (100 ng/mL) (B) for 1 h followed by leptin (250 ng/mL) for additional 8 h. The levels of phosphor-GSK3β were measured by western blot analysis. (C-F) Hepatocytes were pretreated with SB216736 (5 µM), a pharmacological inhibitor of GSK3β, for 1 h followed by treatment with leptin (250 ng/mL) for 24 h (C, E) or 8 h (D, F). (C) Apoptotic cell death was detected by annexin V/7-AAD binding and flow cytometry analysis. Cell in each quadrant were quantified and presented in the right panel. (D) Expression levels of cleaved caspase-3 were measured by western blot analysis. (E) Cell cycle analysis was performed by PI staining and flow cytometry analysis. Percentage of the cells in each cell cycle phase is presented in the right panel. (F) Expression levels of p16 measured by western blot analysis. In both flow cytometry and western blot analyses, representative images from three independent experiments are shown. Values are presented as mean ± SEM, n=3. *p<0.05 compared with control cells, #p<0.05 in comparison to the cells treated with leptin.
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