The transfection incubation time for siRNA lipofectamine 2,000 complexes was 24 hours. Chromatin immunoprecipitation assay MCF 7 cells were grown to 95% confluence in phenol red free selleck compound DMEM supplemented with 10% charcoal stripped FBS for 72 hours, at which time, 10 nM b estradiol was added for 12 hours. Chromatin immunoprecipitation Western blot Inhibitors,Modulators,Libraries analysis was conducted as described pre viously. Briefly, extracts prepared in SDS loading buffer were resolved in SDS 10% PAGE gels and trans ferred to PVDF membranes. These were incubated over night in the presence of anti c Myb antibody 1. 1 and were developed by using ECL western blotting sub strates. Results Characterization of, and MYB expression during, the differentiation of human mammary carcinoma cell lines We first characterized the differentiation of MCF 7 cells using several reported markers of this process.

A number of compounds are capable inducing differentiation in breast cancer cells, such as sodium butyrate, Inhibitors,Modulators,Libraries vitamin E succinate or 12 O tetradecanoylphorbol 13 acetate. We examined the formation of lipid droplets as an indicator of breast cancer cell differentiation using the fluorescent dye Nile Red in combination with fluorescence microscopy or flow cytometry. Vehi cle only treated MCF 7 cells expressed only minimal numbers of detectable lipid vacuoles when stained with Nile Red. By contrast, there was a dose dependent increase in the accumulation of lipid droplets in the cytoplasm of the cells treated with NaBu for 72 hours, and in cells treated with Inhibitors,Modulators,Libraries VES and TPA.

As mammary epithelial cells undergo differentiation there is an increase in the transcription of the gene encoding the milk protein ? casein, as such, this can be used as a molecular marker for differentiation. Indeed, quantitative RT PCR showed that ? casein mes sage was significantly increased after treatment of MCF 7 cells with increasing concentrations of DIAs Inhibitors,Modulators,Libraries for 72 hours. Previous studies have shown that differentiation of a number of different cell types, notably hematopoietic and colonic epithelial cells, is asso ciated with a reduction in MYB expression. Therefore, we asked whether the level of MYB would also be reduced when mammary carcinoma cells underwent differentiation. The MYB message was, as expected, readily detectable in control MCF 7 cells by quantitative RT PCR, but treatment with increasing concentrations Inhibitors,Modulators,Libraries of NaBu for 72 hours induced a dose dependent decrease in the level of MYB message.

Western blot analysis of total cell lysates showed a corresponding profile for Crenolanib the MYB protein. MYB expression decreases with differentiation of non tumorigenic mammary epithelial cells HC11 is an immortalized line of non transformed mam mary epithelial cells, which originated from mid preg nant mouse mammary gland tissue, and has retained important characteristics of normal mammary epithelial cells.