Quantitative real time PCR Both ER positive MCF 7 and ER negative MDA MB 231 and MDA MB 157 cells were cultured and treated as described above. Total RNA from cells or mice tumor tissues was extracted using the RNeasy kit according to the manufac turers instructions. Genes of interest were amplified using 1 ug of total RNA reverse transcribed to cDNA using selleck SB203580 the Superscript II kit with oligo dT primer. In the real time PCR step, PCR reactions were performed in triplicate and primers specific for ER, progesterone receptor, DNA methyltransferase, histone deacetylase and glyceralde hyde 3 phosphate dehydrogenase provided by Inventoried Gene Assay Products were used for Platinum Quantitative PCR Supermix UDG in a Roche LC480 thermocycler. Thermal cycling was initiated at 94 C for 4 min followed by 35 cycles of PCR.

GAPDH was used as an endogenous Inhibitors,Modulators,Libraries control, and vehicle control was used as a calibrator. The Inhibitors,Modulators,Libraries rela tive changes of gene expression were calculated using the following formula fold change in gene expression, Western blot analysis For western blot analysis, protein extracts were pre pared by RIPA Lysis Buffer according to the manufacturers protocol. Proteins were electrophoresed on a 10% SDS polyacrylamide gel and transferred onto nitrocellu lose membranes. Membranes were probed with anti bodies to ER, HDAC1 and DNMT1 respectively, then each membrane was stripped with and reprobed with beta actin antibody as loading control. Molecular weight mar kers were run on each gel to confirm the molecular size of the immunoreactive proteins.

Immunoreactive bands were visualized using the enhanced chemiluminescence detection system following the protocol Inhibitors,Modulators,Libraries of the manufacturer. Immunohistochemical determination of tumor cell proliferation and ER expression Tumor sections were deparaffinized and rehydrated in a series of graded alcohols. Following re hydration, an antigen retrieval process was performed by placing the slides in 10 mmol/L sodium citrate buffer at 95 C for 20 min followed by 20 min cooling at room temperature. The sections were washed in PBS and nonspecific binding sites were blocked with 1% bo vine serum albumin with 2% goat serum in PBS before incubating with either anti Inhibitors,Modulators,Libraries proliferating cell nuclear antigen or anti ER antibody for 2 h at room temperature.

After washing with PBS, the sections were incubated with biotinylated secondary antibody for 45 min followed by horseradish peroxidase conjugated streptavidin, washed in PBS, incu bated with diaminobenzidine substrate, and counterstained with hematoxylin. Photographs Inhibitors,Modulators,Libraries Brefeldin A of representative pictures were taken and the numbers of PCNA positive or ER positive cells were detected and counted using a light microscope. The results are presented as the number of positive cells 100 divided by the total number of cells.