Materials and methods Pharmacological agents BGT226, BKM120 and RAD001 were obtained through material transfer agreements with Novartis. Fulvestrant, LY294002, rapamycin and 17b estradiol were from commercial sources. 17b Estradiol was dissolved in ethanol, chemical information inhibi tors were dissolved in dimethylsulfoxide. Cell culture The HCC712 cell line was kindly provided by Dr Adi Gazdar. Other cell Inhibitors,Modulators,Libraries lines were obtained from American Type Culture Collection. Experi ments with parental cell lines were performed with low passage number cells used within 2 to 3 months fol lowing revival from the supplier. Cell lines were propa gated in RPM1 1640 containing 10% fetal bovine serum with antibiotic and supplements in a humidified 37 C incubator containing 5% carbon diox ide.
LTED MCF7 and T47D cell line variants were pro duced by culturing the parental lines for 9 months in phenol red free RPMI 1640 containing 5% charcoal stripped FBS, Invitrogen, Carlsbad, CA, USA containing antibiotic and supple ments. Inhibitors,Modulators,Libraries Estrogen retreated LTED sublines were created by treating LTED cells growing in CSS medium with 10 nmol l 17b estradiol for at least 4 months prior to experiments. For studies using short term estrogen deprivation parental cell lines, cells were maintained in CSS medium for 1 to 3 weeks prior to experimental treatments. Protein extraction For pharmacological treatments, cells were deprived of serum for 3 to 4 hours, pretreated with the indicated agents for 20 minutes, and then treated with or without 20% FBS for 15 minutes. Lysates were prepared by extracting cells in lysis buffer as previously described.
Cell growth assay and calculation of 50% inhibitory lethal concentrations To determine the effects of estradiol and fulvestrant on the growth of LTED cells, the cells growing in CSS medium Inhibitors,Modulators,Libraries were plated in 96 well Optilux dishes and were treated without or with fulvestrant or the indicated concentrations of 17b estradiol on the day after plating. The medium was replenished every 3 to 4 days and cell growth was assessed after 7 days by mea suring Alamar Blue reduction with a fluorescent microplate reader. For calculation of the half maximal inhibitory Inhibitors,Modulators,Libraries concentration and the 50% lethal concentration, cells were cultured in phe nol red free RPM1 Inhibitors,Modulators,Libraries 1640 containing 5% CSS for at least 1 week prior to plating in 96 well Optilux dishes for drug treatment. Alternatively, cells growing in phenol red RPMI 1640 medium containing 10% FBS were plated in 96 well Optilux dishes and then switched to CSS medium for at least 1 week prior sellekchem to drug treat ment. Five dilutions of each drug were made using a 1,5 serial dilution. Treatments were performed in triplicate and the experiments in each cell line were performed at least twice.