Fig. 2. Implication of IL-1β signaling in apoptotic cell death by leptin in rat hepatocytes. (A, B) Rat hepatocytes were pretreated with IL-1Ra (100 ng/mL) for 1 h (A), or cells were transfected with siRNA targeting IL-1R1 or scrambled control siRNA (100 nM) (B), followed by treatment with leptin (250 ng/mL) for additional 24 h. Apoptotic cell death was determined by annexin V binding assay. Representative images from three independent experiments are presented. Cells in lower right quadrant indicate early apoptotic cells and upper right quadrant indicate late apoptotic cells. The percentage of cells in each quadrant from three independent experiments were quantified and presented in the right panel of this assay. (C, D) Cells were treated with IL-1Ra (100 ng/mL) for 1 h (C), or transfected with siRNA targeting IL-1R1 or scrambled control siRNA (100 nM) (D), followed by treatment with leptin (250 ng/mL) for additional 8 h. Expression levels of total and cleaved cytokeratin-18 were measured by western blot analysis. (E-G) Cells were treated with leptin (250 ng/mL) for 8 h in the absence or presence of IL-1Ra (100 ng/mL). Protein expression levels of (cleaved) PARP (E), Bax (F), and Bcl-2 (G) were measured by western blot analysis. (H) Conditioned media obtained from hepatocytes culture treated with leptin in the absence or presence of Ac-YVAD-cmk were used for the treatment of a fresh set of hepatocytes (CM: fresh WME (1:2)). Expression levels of (cleaved) caspase-3 were analyzed by western blot analysis. (I) Rat hepatocytes were treated with leptin (250 ng/mL) for 8 h in the absence or presence of IL-1Ra (100 ng/mL). Cells were incubated with MitoOrange dye for 30 min and mitochondrial membrane potential was determined by flow cytometry analysis. In the western blot analyses, β-actin or total form of respective protein was used as an internal control. Statistical analysis for the band intensity determined by densitometric analysis are presented in the lower panel. Values are presented as fold change in comparison to control group and expressed as mean ± SEM, n=3. *p<0.05 compared to control cells. #p<0.05 in comparison to the cells treated with leptin.
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