Briefly, DNA was stained with 50 μg/ml propidium iodide (Sigma). Samples were kept for 1 hr in the dark at room temperature and the DNA index was then measured by cytofluorimetric analysis using an FACS Calibur flow cytometer (Becton Dickinson, San Diego, CA). Data were analyzed using CellQuest software. Annexin V/PI for cell apoptotic analysis Cell viability was detected by trypan blue and apoptosis was evaluated by the annexin V/propidium iodide (BD Biosciences) double staining assay following the manufacturer’s instructions. K562 cells were harvested at the end of treatment, rinsed twice with PBS, and

stained with Annexin V-FITC apoptosis detection kit I (BD Biosciences). Analysis was performed on the FACS Calibur using CellQuest software. Western blotting Three groups of K562 cells were cultured at 37°C, 5% www.selleckchem.com/products/VX-765.html CO2 for 24 hrs. SCG-S represented the group of K562 cells cultured without FBS. CCG-S represented the group of K562 and MSCs without FBS. CCG-S+LY294002 represented the group pretreated with 10 μM LY294002 for 1 hr. After incubation, K562 cells were dissolved in lysis buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerin, 200 mM dithiothreitol, plus protease inhibitors) and quantified for proteins by a BCA protein assay kit (Pierce Company, USA). Equal amounts of protein extract

were loaded onto a 12% SDS-PAGE gel and transferred to PVDF membrane (Gibaino Company, Beijing, China). The blot was blocked in 5% fat-free milk at 4°C overnight and then incubated with Selleck LY2157299 mouse monoclonal anti-Akt, p-Akt-Ser-473, anti-Bad, p-Bad-Ser-136 antibodies, (Cell Signal Transduction). Mouse monoclonal anti-beta-actin antibody (Cell Signal Transduction) was used as control. The immunocomplexes were visualized by using a chemiluminescent kit

(Cell Signal Transduction). Statistical analysis Data were presented as mean ± SD, using the SPSS system package for statistical analysis. Student-t-test was used for comparison of two groups of data. One-Way ANOVA was used for more than two groups of data. Multiple comparisons between two groups were analyzed by a SNK-q test. A P value < 0.05 was considered significant. Results MSCs inhibit proliferation Lenvatinib molecular weight of K562 cells under different nutritional conditions As shown in figure 1, the growth of K562 cells was clearly decreased in the absence of serum in culture media. However, even with the addition of 10% FBS, viable cell numbers in the coculture, transwell, and CM experimental groups were significantly decreased compared to the SCG subgroups (p < 0.001). The CCG groups were especially affected. This suggested that cell growth was inhibited when K562 cells were cocultured with MSCs Moreover, the suppression persisted even if the cells were separated in a transwell system or were cocultured in MSC supernatant, which indicated the suppression effect was mediated by some soluble substances, most likely cytokines.