We first con firmed that HC11 MECs could undergo differentiation in the presence of lactogenic inhibitor Ruxolitinib hormones, as monitored by the presence of lipid droplets detected by Nile Red, the formation of domes, Inhibitors,Modulators,Libraries and the induction of ? casein expression. In the course of such experiments, cell differen tiation occurred progressively after removal of epidermal growth factor for 24 hours and the subsequent addition of lactogenic hormones over seven days. Quan titative RT PCR and western blot analysis demonstrated a decrease of the steady state levels of MYB mRNA and protein, respectively, as these cells underwent differen tiation. These data suggest that the reduction in MYB levels is part of the normal pathway of differentiation of mammary epithelial cells, and vali date the use of mammary carcinoma cell lines and MECs as models to study the role of MYB in this process.

MYB knockdown promotes differentiation of mammary carcinoma cells We have previously used a doxycycline inducible lentiviral siRNA Inhibitors,Modulators,Libraries system targeting MYB to show that MYB is required for the proliferation of ER positive cell lines. To examine the effect of MYB knockdown Inhibitors,Modulators,Libraries on differentiation, MCF 7 cells stably transduced with this vector, or appropriate controls, Inhibitors,Modulators,Libraries were treated with Dox for 72 hours. The cells were then stained for lipid dro plet accumulation using Nile Red. Approximately 35% of MYB knockdown cells stained positively for lipid dro plet accumulation in contrast to approximately 20% of control cells. This is a modest, yet statistically significant, increase.

It should be noted that the intensity of Nile Red staining was greater in the Inhibitors,Modulators,Libraries MYB knockdown cells, and the number of droplets cell and droplet size in the MYB knockdown cells were greater than those seen in the control cells as visualized by fluorescence micro scopy. In addition, ? casein mRNA levels rose by approximately 3 fold fol lowing MYB knockdown. These experiments were repeated using ZR 75 1 cells, and a similar result was observed. These data suggest that MYB knockdown induces breast tumor cells to initiate the process of differentia tion in the absence of DIAs, albeit with limited efficiency. Synergy between DIA treatment and MYB knockdown As MYB knockdown in mammary carcinoma cells resulted in only a limited amount of differentiation, we next asked whether MYB knockdown was able to act in a synergistic manner with DIAs.

MYB knockdown MCF 7 cells, and appropriate controls, were treated with or without Dox for 24 hours, and then treated with NaBu or VES for a further 72 hours. Staining for lipid droplet accumulation treated with or without Dox showed that the effect of MYB Tivantinib knockdown coupled with the lowest concentration of VES or NaBu caused differentiation comparable with that seen only with the highest concentration of DIAs used alone.