As previous reports have demonstrated that disruption of di

Moreover, calculation of the IC50 values indicated that they were nearly similar in both FD Neo and FD Pim44 cell lines. To confirm that the results obtained with the MTT assay reflected cell survival, we stained cells with Trypan blue and counted dye excluding live cells at multiple time points after withdrawal of IL 3. As shown in Figure 1D, FD Pim44 cells Ivacaftor 価格 treated with DMSO were still alive after 72 h, while FD Neo cells stopped growth and started to die already after 12 h. However, when cells were treated with 10 uM DHPCC 9, the protective effects of constitutively expressed 44 kDa Pim 1 were completely lost and the FD Pim44 cells behaved like DMSO treated FD Neo control cells.<br><br> In this assay, DHPCC 9 also reduced the viability of FD Neo cells, which is in line with our previous observations from FDCP1 cells expressing a dominant negative mutant of Pim 1, DHPCC 9 inhibits cellular phosphorylation of Pim substrates such as Bad We had LBH589 費用 recently shown that DHPCC 9 inhibits kinase activities of all three Pim family proteins under in vitro conditions, In order to demonstrate that DHPCC 9 similarly inhibits intracellular activities of Pim kinases, we analysed the phosphorylation status of one of the well established Pim substrates, the pro apoptotic Bad protein, FD Neo and FD Pim44 cells were tran siently transfected with a GST Bad expression vector. Part of the cells were collected 24 h after transfection, while the rest were grown for 8 h in the absence of IL 3, but with increasing concentrations of DHPCC 9.<br><br> In the absence of IL LY2109761 datasheet 3, Ser112 of Bad remained more pronouncedly phosphorylated in FD Pim44 cells than in FD Neo cells, which was well in line with our previous results, However, exposure of FD Pim44 cells to increasing concentrations of DHPCC 9 led to a significant reduction in the level of Bad Ser112 phosphor ylation as compared to Bad expression levels, We also measured Pim protein levels from the cell lysates and noticed that all three Pim family members were expressed there and that DHPCC 9 did not reduce their expression levels either in the presence or absence of IL 3, Thus, our results suggest that DHPCC 9 exerts its cellular effects by inhibiting kinase activities of all Pim family members towards their downstream tar gets.<br><br> Interestingly, while the endogenous expression levels of both Pim 1 and Pim 3 proteins were signifi cantly reduced by IL 3 withdrawal, the levels of Pim 2 remained unchanged, suggesting that its expression in FDCP1 cells may be regulated in a distinct fashion from the two other family members. Pim kinases promote cancer cell migration and invasion During our recent studies we had obtained hints that Pim kinases may be involved in regulation of cell moti lity. Migration of cells is important for many physiological processes including embryogenesis, wound healing and immune responses, but it is also essential for tumor angiogenesis and metastasis. There fore we decided to use DHPCC 9 as a tool to investi gate the possibility that Pim kinases affect migration of adherent cancer cells.