Briefly, primary OSE cells were spun onto slides using a cytospin centrifuge and fixed in cold methanol at 20 C, and subjected to immunofluo rescence staining as described previously. A mouse monoclonal anti cytokeratin antibody was used for cytokeratin staining. Secondary antibody used was FITC conjugated goat anti mouse IgG. The nucleus was counterstained with 4,6 diamidino 2 phenylindole. Y27632 The purity of the epithelial component in each OSE cell culture for the six cases were 100%, 100%, 90%, 90%, 100% and 80%, respectively. For P4 exposures, OSE cells were grown in Dulbeccos Modified Eagles Medium supplemented with 15% fetal bovine serum and streptomycin penicil lin. 48 hours before the hormone treatment, the OSE cells were plated in four T25 flasks, the medium replaced with DMEM Inhibitors,Modulators,Libraries supplemented with 15% charcoal stripped FBS and the cells allowed to reach 70 75% confluence.
The medium was Inhibitors,Modulators,Libraries then replaced with fresh media containing 10 6 M P4 or vehicle. The final concen tration Inhibitors,Modulators,Libraries of ethanol in the media was 0. 03% both in the P4 and control experiments. The hormone treated cells were exposed to P4 for a total of 5 days, with three fresh addi tions of media containing P4 every 48 hours for stable bioavailability as described. At the end of the fifth day, the medium was removed. The cells were washed with PBS, collected following trypsin treatment and immediately frozen for RNA extraction. Cells from duplicate flasks were combined. To test bioavailability of P4 in tissue culture, we used breast cancer cell line MCF 7 to confirm upregulation of human DLG5 gene by P4 and found 5.
2 fold and 2. 2 fold gene expression increases by real time RT PCR at 8 and 24 hours, respectively. RNA extraction The total RNAs were extracted following a commercial protocol that used phenol and guanidine thiocyanate. The total RNAs were resuspended in DEPC treated distilled water and fur ther purified using RNeasy mini columns in preparation for microarray analyses. After column Inhibitors,Modulators,Libraries purification, the total RNAs were quantitated by absorbance at 260 nm using a Beckman DU 64 spectro photometer. Approximately 10g of total RNA were pro vided for microarray analysis of global gene expression patterns. After the RNeasy purification step and demon strating an OD 260 280 ratio of 1. 8 or higher, The Univer sity of Pittsburgh Microarray facility confirmed RNA integrity via Agilent Bioanalyzer 2100.
Microarray hybridization and data processing The high density microarrays used in this study contained 22,283 unique human transcripts derived from the Inhibitors,Modulators,Libraries RefSeq database and were commercially available from Affymetrix, selleck Santa Clara, CA. The hybridizations to microarrays were performed by The University of Pittsburgh Microarray Facility. All RNAs passed quality control tests before they were processed further for microarray hybridizations.