For immunoblot analysis, mouse anti BRCA1 antibody was purchased from Calbiochem. Erlotinib was selleck chemicals Cisplatin purchased from LC Laboratories. Cell culture Informed consent was obtained for the collection of pri mary hMECs from mastectomy specimens of BRCA1 mutation carriers, and cells were isolated as described previously. MECs Inhibitors,Modulators,Libraries were cultured in Mammary Epithelial Cell Growth Medium or HuMEC medium supple mented with bovine pituitary extract. MCF 10A human epithelial cells, Manassas, VA, USA hMEC expressing human telomerase reverse transcriptase cells and immortalized human mammary epithelial cells were cultured in a mixture of Dulbeccos modified Eagles medium Hams F 12 medium supplemented with 5% horse serum, 20 ng ml EGF, 0. 5 mg ml hydrocortisone, 100 ng ml cholera toxin and 10 ug ml insulin.

MCF 7 cells, the HCC1937 BRCA1 mutant breast cancer cell line and HCC1937 cells stably transfected with green fluorescent protein BRCA1 were kept in RPMI 1640 medium with 10% fetal bovine serum. For three dimensional cultures, the cells were embedded in 40 ul Inhibitors,Modulators,Libraries of Geltrex and cultured in eight chamber cul ture slides. Cell viability and luciferase assays For cell viability assays, MECs were seeded at a density of 250 cells well in 96 well plates, and cell viability was determined using the CellTiter Glo Luminescent Cell Viability Assay according to the manufacturers instructions, and absorption was read using a Wallac 3 plate reader.

For luciferase assays, hMEC Inhibitors,Modulators,Libraries or MCF 7 cells were seeded into 24 well plates on day 1, transfected with BRCA1 small interfering RNA 1 or small interfering RNA 2 or control small interfering RNA on day 2 and with control or the full length EGFR luciferase con struct on day 3, followed by a luciferase assay performed on day 4. For each experiment, 2 ug of reporter con struct were transfected in combination with either 1 ng of hMEC or 10 ng of Renilla thymidine kinase, and luciferase activity was determined using a Wallac 3 plate reader. Transient transfec tion of siRNA was performed using siRNA and Hyper Fect transfection protocol according to the manufacturers instructions. Stably infected cells lines were produced using lenti viruses. The sh sequences were cloned into the pLKO. 1 vector, and lentiviruses were produced in the 293FT cell line. The cells were infected and selected with puromycin as previously described.

Flow cytometry To measure the kinetics of binding of EGF, cells were grown for 24 hours in 6 cm dishes and serum deprived for 4 to 6 hours Inhibitors,Modulators,Libraries at 37 C, followed by a 1 hour incuba tion on ice with indicated amounts of Rh EGF. For uptake and binding, cells were incubated on ice with 10 ng of Rh EGF, then the excess Rh EGF was removed with an ice cold Inhibitors,Modulators,Libraries phosphate buffered saline wash and the cells were incubated at 37 C for the indicated time intervals. The reaction was stopped on ice, and inhibitor licensed the noninternalized receptor was stripped with a light acid buffer.