Fig. 5. Role of p38MAPK activation in apoptosis and cell cycle arrest by leptin in rat hepatocytes. (A) Hepatocytes were treated with leptin (250 ng/mL) for the indicated time and phosphor-p38MAPK levels were analyzed by western blot analysis. (B-E) Hepatocytes were pretreated with SB203580 (10 µM), a pharmacological inhibitor of p38MAPK, for 1 h followed by incubation with leptin (250 ng/mL) for additional 24 h (B, D) or 8 h (C, E). (B) Cells were stained with annexin V/7-AAD and apoptotic cell death was determined by flow cytometry analysis. Cells in each quadrant were quantified and presented in the right panel. (C) Expression levels of cleaved caspase-3 were measured by western blot analysis. (D) Cell cycle analysis was performed by PI staining and flow cytometry analysis. Cells in each phase of cell cycle is quantified and presented in the right panel. (E) Expression levels of p16 were measured by western blot analysis. (F-H) Hepatocytes were pretreated with SC79 (4 µg/mL) (F), SB216763 (5 µM) (G), or IL-1Ra (100 ng/mL) (H) for 1 h followed by incubation with leptin (250 ng/mL) for additional 8 h. Phosphor-p38MAPK levels were determined by western blot analysis. Representative images from at least three independent sets of experiment is shown for both flow cytometry and western blot analysis. In western blot analysis, the band intensities of the genes of interest were quantified by densitometric analysis and presented in the lower panel. β-actin or total form of corresponding protein was used as internal control. Values are presented as mean ± SEM, n=3. *p<0.05 in comparison to control cells, #p<0.05 in comparison to the cells treated with leptin.
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