Effects of GRP78 silencing on AST-induced apoptosis (A) and colony formation (B). After 24 h of transfection with GRP78 siRNA or scrambled control siRNA [Scr], the protein levels of GRP78, cleaved caspase 3 and PARP were determined in HCT116 cells with or without AST treatment (80 μg/ml for 24 h) by Western immunoblotting. Data shown are representative immunoblots from four independent experiments with similar findings, normalized by β-actin. Blots were scanned and optical densities were determined using the Quantity One software. Alternatively, native or GRP78 gene-silenced HCT116 cells were cultured in six-well plates at the density of 200 cells/well at 37°C in 5% CO2 atmosphere for 3 weeks with or without AST treatment (80 μg/ml) in the last 14 days to determine colony formation.