Figure 3

GRP78-silencing enhances, whereas translational inhibitor cycloheximide reduces cell death mediated by PAC-1. (a and b) MCF7 and HeLa cells were transfected with siGRP78. Silencing of GRP78 was confirmed in blot. Cell death was analyzed by PI staining using FACS after silencing GRP78. The comparison and percentage of PI-positive cells are represented graphically (n=3, mean±S.D.). (c) ER stress was inhibited by using cycloheximide (CHX; 2 μg/ml) along with PAC-1, and caspase activity was evaluated using FACS by employing caspase-3 and -7 specific FRET probe, CFP-DEVD-YFP, stably transfected in HeLa and MCF7 cells. Cleavage of DEVD sequence by activated caspases will cause FRET loss which is reflected as enhancement of CFP/YFP ratio. As shown in the gates (FRET lost-population), cycloheximide reduced percentage of cells with FRET loss, suggesting that ER stress inhibition reduces caspase activation induced by PAC-1. (d) Cell death was analyzed by PI staining using FACS in MCF7 and HCT116 after PAC-1 treatment in the presence and absence of cycloheximide. The percentage of PI-positive cells is indicated in respective histograms, suggesting that cycloheximide inhibited cell death induced by PAC-1. (e) Cell survival was analyzed by clonogenicity assay in PAC-1-treated MCF7 and HCT116 cells, with and without cycloheximide. As noted in images, translational inhibitor cycloheximide rescued cells from PAC-1-induced cell death when used along with PAC-1. Absorbance was measured for the solubilized stain and relative percentage of clonogenicity is represented in graph (n=3, mean±S.D.)