Fig. 3. Mediating role of AKT dephosphorylation in apoptosis and cell cycle arrest by leptin in hepatocytes. (A) Hepatocytes were isolated and treated with leptin (250 ng/mL) for the given time periods. Phosphorylated AKT levels were measured by western blot analysis. (B, C) Hepatocytes were pretreated with IL-1Ra (B), or MCC950 or Ac-YVAD (C) for 1 h, followed by treatment with leptin (250 ng/mL) for 8 h. The levels of phosphorylated AKT were measured by western blot analysis. (D) Conditioned media obtained from hepatocyte culture treated with leptin in the absence or presence of Ac-YVAD-cmk were prepared as in methods. A fresh set of hepatocytes were treated with CM for 8 h. Phosphorylated AKT levels were determined by western blot analysis. (E-H) Hepatocytes were pretreated with SC79 (4 µg/mL) for 1 h followed by leptin stimulation for 24 h (E, G) or 8 h (F, H). (E) Cells were stained with annexin V/7-AAD and subjected to flow cytometry analysis for the detection of apoptotic cell death. Cells in lower right quadrant indicate early apoptosis and upper right quadrant indicate late apoptosis. Cells in each quadrant were quantified and presented in the right panel. (F) Expression levels of cleaved caspase-3 were determined by western blot analysis. (G) Cell cycle analysis was carried out by flow cytometry. Representative images from three independent experiments are presented. The percentage of the cells distributed in each cell cycle phase from three experiments is presented in the lower panel. (H) Expression levels of p16 was measured by western blot analysis. In the western blot analyses, β-actin was used as internal control. Band intensities were analyzed by densitometric analysis and are presented in the lower panel of the images. Values are presented as mean ± SEM, n=3. * denotes p<0.05 compared with control cells. # denotes p<0.05 in comparison to the cells treated with leptin.
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