After the treatment, cells were rinsed twice in cold
PBS, resuspended in binding buffer, and then analyzed for apoptosis level by a PE-labeled Annexin-V/7-AAD assay. These cells were directly analyzed in a FACScan (BD FACS Calibur Co., USA) with a sample size of at least 10,000 cells gated on the basis of forward and side scatter. Storing and processing of data were accomplished using FACScan software. Statistical analysis Results are expressed as mean ± standard deviation. Statistical analysis was conducted using SPSS 15.0 software. Differences between groups were examined for statistical significance using a one-way analysis of variance and Student’s t -test; P values less SB203580 supplier than 0.05 were considered statistically significant. Results GSK-3β accumulated in the nucleus of primary ALL cells Using immunofluorescence staining, we identified the localization of GSK-3β in ALL BMMC in 8 children with ALL. As shown in Figure 1, we found nuclear accumulation of GSK-3β in 6 primary pediatric ALL BMMC samples, whereas it was not detected in the nucleus of control BMMC. Figure 1 Immunofluorescence MS275 staining of GSK-3β in ALL cells. Bone marrow samples
were obtained from children with ALL and from control patients. GSK-3β was probed with Dylight 549-labeled anti-rabbit secondary antibody (red fluorescence) and nuclei were counterstained with Hoechst 33342 (blue fluorescence). Nuclear accumulation
of GSK-3β in ALL cells 3-deazaneplanocin A datasheet was detected, whereas only cytoplasmic expression of GSK-3β was observed in control cells. Inhibition of GSK-3β suppressed the binding of NF-κB to the DNA in ALL cells GSK-3β has been shown to play a critical role in NF-κB-mediated survival of cancer cells. The aberrant accumulation of GSK-3β in nuclei of ALL cells prompted us to examine the effect of GSK-3β inhibition on NF-κB activity. Using primary ALL cells, we tested ex vivo the effect of 2 chemically distinct small-molecule inhibitors of GSK-3β: SB216763 (ATP-competitive, arylindolemaleimide) , and LiCl (non-ATP-competitive) . Forty-eight hours after GSK-3β inhibitors treatment, we estimated the level of GSK-3β inhibition by detection of the check details cytosolic/nuclear level of GSK-3β by western blot. We found that both the distinct GSK-3β inhibitors can decrease the level of GSK-3β in nuclear extracts of ALL cells (Figure 2). With the same treatments, nuclear levels of NF-κB p65 in ALL cells were not significantly changed (Figure 2). To further investigate the role of GSK-3β in the regulation of NF-κB activity, we detected NF-κB DNA binding activity by EMSA. The data show that GSK-3β inhibition in ALL cells decreased the binding of NF-κB p65 to its target gene promoter (Figure 3). Taken together, these results suggest that GSK-3β affects NF-κB activity at the transcriptional level in pediatric ALL cells.