Cells were seeded in 48-well plates (1 × 104 per well) and allowed to grow overnight before the addition of IT at different concentrations. After 5 or 24 hr incubation, cells were washed twice with cold phosphate-buffered saline (PBS) containing 0.1% FCS, and then incubated with [3H]-leucine (2 μCi ml-1) in leucine-free medium at 37°C for 45 min. Cells were then washed with 5% trichloroacetic acid (TCA) for 5 and 10 min, respectively, and dissolved in 0.1M KOH for 10-15 min. The resultant solution was transferred to the liquid scintillator. Sample counts were

determined in a liquid scintillation counter. Assays were performed in duplicates and repeated at least three times. selleck compound Counts per minute (cpm) for treated cells were compared to cpm for untreated cells and reported as a percentage of leucine incorporation with the control value set to 100%[16]. The experiment was completed in the isotope laboratory of Nanjing Medical University. Flow cytometric analysis of cell apoptosis Apoptosis were determined by flow

cytometric analysis. Briefly, cells in triplicates, were incubated with or without various concentrations of IT for 24 hr. Cells were then harvested, washed in cold PBS, and fixed with 1 ml 75% ice-cold ethanol at -20°C until processing. An aliquot (1 ml) of fixed cell suspension containing 1 × 106 cells was washed twice in cold PBS and then treated with fluorochrome DNA staining solution (1 ml) containing 40 μg of propidium iodide and 0.1 mg of RNase A in the dark at room temperature for 0.5 hr. Flow cytometric analysis were performed three times [17]. Caspase activity assay Ro 61-8048 purchase Caspase activity was determined in 96-well plates using cell lysates from 1 × 106 cells for each measurement. Caspase-3 and caspase-8 activities were determined using colorimetric assay kits according to the manufacturer’s protocol

(BioVision). GES-1, MKN-45 and SGC7901 cells were treated with anti-c-Met/PE38KDEL (100 ng/ml) for 24 hr prior to the assay. Cell extracts were incubated with 5 μl of 4 mM tetrapeptide substrates (DEVD, caspase-3; IETD, and caspase-8) at 37°C for 1-2 hr. The reaction was measured at 405 nm in a Microplate Reader. Background readings from cell lysates and buffers were subtracted from the readings of both IT-induced and control samples before calculating the Exoribonuclease relative change increase in caspase activity in the IT-induced samples compared to that of the control. IT treated samples were normalized to the caspase activity of the untreated sample, which was set to 1.0. Fold of increases in caspase activities were presented. Statistical analysis Statistical analysis was performed with SPSS 13.0 software. Data were presented as mean ± standard deviation. Student’s t-test was used to compare two samples, and the single-factor analysis of variance (One-way ANOVA) was used to compare multiple samples. A p-value less than 0.