FIGURE 5.

The analysis of cell apoptosis on PANC-1 and BxPC-3 cells with siL21-1 and siL21-2 transfection. (A,B) PANC-1 and BxPC-3 cells were transfected with RPL21 siRNA (siL21-1 and siL21-2) and Mock-siRNA (40 nM) for 72 h, respectively, and then analyzed by Annexin V-FITC/PI staining with Flow Cytometry (FCM) analysis. The cells were analyzed with the BD FACSCalibur and FlowJo software at 10,000 events. The lower right area shows early apoptotic cells, and the upper area shows late apoptotic cells. (C) Summary graphs of the flow cytometry (FCM) results. (D) Effects of RPL21 siRNA on Caspase-8 activities. PANC-1 and BxPC-3 cells were transfected with RPL21 siRNA (siL21-1 and siL21-2) and Mock-siRNA (40 nM) for 72 h, respectively, then the cells were seeded into black wall/clear bottom 96-well plates (50,000 cells/well) and were added with caspase-specific fluorogenic substrate. The activities of Caspase-8 were detected at corresponding excitation/emission wavelength (Ex/Em = 490/525 nm) with microplate reader. (E) Effects of RPL21 siRNA on Mitochondrial membrane potential. PANC-1 and BxPC-3 cells were transfected with RPL21 siRNA (siL21-1 and siL21-2) and Mock-siRNA (40 nm, 72 h), respectively, then the cells were harvested and seeded into 96-well plates (60,000 cells/well) followed by the addition of JC-10 dye-loading solution. The ratio of fluorescence intensities on Em at 525/590 was used for Mitochondrial membrane potential analysis. The control, negative control (NC) and siL21-1/siL21-2 represented the untransfected, Mock-siRNA transfected and RPL21 siRNA transfected groups, respectively. Each bar represents the mean ± SD of three separate experiments with triplicate wells per condition, *P < 0.05.