These findings suggest that Sin3A may be a new therapeutic target, and identification of an agent that could disrupt Sin3A may be effective in controlling survival of ERa positive tumors. Methods Cell Culture and Hormone Treatments MCF7, MDA MB 231, and Hs578T cells were main tained at 37 C and 10% CO2 in Dulbeccos modified Eagles medium with phenol red and L glutamine, Paclitaxel human endothelial cells supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 ug/ml streptomy cin. T47D cells were maintained at 37 C and 5% CO2 in RPMI 1640 medium with phenol red and L glutamine, sup plemented with 10% FBS, penicillin, and streptomycin as above. For hormone treatments, all cell lines were incu bated at 37 C and 5% CO2 for at least three days in the media described above but without phenol red and containing six times charcoal dextran stripped FBS.

17 b estradiol was added to a final concentration of 10 nM Inhibitors,Modulators,Libraries in all experiments for the length of time indicated in the fig Inhibitors,Modulators,Libraries ures. Ethanol vehicle control was 0. 1% in all samples. Transfection of siRNA One day prior to transfection, cells were plated in 10 Inhibitors,Modulators,Libraries cm plates at a density of 2 106 cells in antibiotic free media. 800 pmol of siRNA was diluted in Lipofectamine reagent and Opti MEM and added to appropriate plates for five hours. Three days later, cells were transfected with siRNA again as above in order to achieve maximum silencing. siRNA duplexes for Sin3A, HDAC1, HDAC2, and a scrambled negative control were predesigned and purchased from Sigma. RNA Isolation and Quantitative RT PCR RNA isolation and quantitative reverse transcriptase real time PCR were carried out as previously detailed.

Primer sequences are available upon request. Ribosomal protein P0 mRNA was used as the internal control. Relative mRNA levels were calculated using the Ct method. For initial screening of candidate Sin3A regulated genes, two complimentary trial RT2 Profiler Human Breast Cancer and Estrogen Receptor Signaling PCR arrays were used. Cell Growth Inhibitors,Modulators,Libraries Assays Cells were transfected with scrambled or Sin3A siRNA as detailed above, changing the media to phenol red free media the day before the second transfection. The day after the second transfection, cells were harvested and pla ted in 6 well plates at a density of 4 105 live cells, as determined by trypan blue exclusion and counting on a hemacytometer. Cells were then treated with either 10 nM E2 or EtOH.

At 24 hour intervals, cells were harvested and resuspended in media. The number of live cells at each time point was determined by hemacytometer counting and trypan Inhibitors,Modulators,Libraries blue exclusion, taking the average of two counts for each sample in each experiment. Flow Cytometry for Cell Cycle and Apoptosis Analysis Knockdown of Sin3A and hormone treatments were performed as described sellckchem above. 72 and 96 hours post treatment, media and cells were harvested and diluted to 1 105 cells in 1 ml of media.