MUC1 cytoplasmic domain dimerization can be disrupted by an

jy9202 pet 11 tra 2014 - 6:14

When we carried out scratch wound assays with PC 3 prostate cancer cells, we noticed that DHPCC 9 decreased the motility of those cells in a dose dependent fashion, but did not reduce endogenous expression levels of Pim kinases, Viability of PC 3 cells also remained unaffected by DHPCC 9, as measured both by the MTT assay and by Trypan blue staining, The inhibitory effects buy Ivacaftor of DHPCC 9 on cell migration were not restricted to PC 3 cells, since similar results were obtained also from UT SCC 12A squamocellular carcinoma cells, We have previously shown that these cells express high levels of Pim 1, but we now demonstrated that they also express Pim 2 and Pim 3, Similarly to PC 3 cells, DHPCC 9 did not decrease either Pim expression levels or viability of UT SCC 12A cells. To further prove that the inhibitory effects of DHPCC 9 on cell motility were mediated via specific inhibition of Pim kinase activity, we carried out wound healing assays in PC 3 cells in the presence of short interfering RNAs targeting either Pim 1, Pim 2 or both. Pim 3 was not targeted, since its expression levels have been reported to be LBH589 supplier significantly lower in PC 3 cells than those for Pim 1 and Pim 2, A larger tip was now used to scratch the wounds to facilitate fol low up of the wound healing processes. While wounds in control cells transfected with non targeting siRNA healed within 48 h, those expressing the Pim specific siRNAs recovered significantly more slowly, These results were not due to differences in cell viability, as confirmed by the MTT assay, Most striking reduction in migration was observed when both Pim 1 and Pim 2 were silenced. In fact, the effects of simultaneously silenced Pim 1 and Pim 2 were initially comparable to those observed in control transfectants treated with DHPCC 9. However, the effects of DHPCC 9 were more sustainable, since they were visible even after the 48 h time point, Here it should also LY2109761 代理店 be noted that the siRNA transfection efficiency was approximately 25%, so only partial silencing of Pim kinases was received, as demonstrated by Western blotting, To follow up the wound healing process in more detail and to compare the effects of DHPCC 9 in the presence or absence of serum, we used the IncuCyte imaging system with a scratch wound application. In the presence of 10% serum, we obtained very similar results as in man ual experiments, while removal of serum reduced cell migration rates, Due to the lower migration rates of serum deprived cells, it was possible to follow the movements of individual cells in movies con structed from the slides produced by the IncuCyte, There DHPCC 9 treated cells seemed to have totally lost their ability to move, whereas only a few of the control cells seemed equally immotile. To determine whether Pim kinases are able to affect invasive properties of PC 3 cells, we carried out Boyden chamber assays with PC 3 cells that had been trans fected with non targeting or Pim specific siRNAs and treated with either DMSO or DHPCC 9. Three days later, cells were fixed and stained to facilitate counting of invaded cells. As summarized in Figure 5D, silencing of Pim 1 and Pim 2 reduced the rate of invasion of PC 3 cells.