We obtained AU565, HCC1954 and T47D breast cancer cell lines and MCF10A mammary epithelial cells from the American Type Culture Collection (ATCC). We cultured AU565 and HCC1954 cells in RPMI 1640 (ATCC) supplemented with 10% fetal bovine serum (FBS), T47D cells in RPMI 1640 supplemented with 10% FBS and insulin (0.2 U/ml), and MCF10A cells in DMEM/F12 (Invitrogen) supplemented with 5% horse serum, EGF (20 ng/ml), insulin (10 μg/ml), hydrocortisone (0.5 μg/ml), and cholera toxin (100 ng/ml). We added penicillin (50 U/ml) and streptomycin (50 μg/ml) to all growth media.
We purchased dactolisib (BEZ235), GSK1059615 and PP242 from Selleck Chemicals. All compounds were at least 97% pure as evaluated by HPLC and MS analysis. All compounds were dissolved in DMSO as 10 mM stock solutions. For dose-response experiments, we plated cells in 2 replicates at 7,000 cells per well in 96-well plates (Corning) in full growth media for 24 hr and then treated them with 9 doses in serial dilutions (10−10 to 10−5 M) of each compound for 6, 24 and 72 hr.
We purchased dactolisib (BEZ235), GSK1059615 and PP242 from Selleck Chemicals. All compounds were at least 97% pure as evaluated by HPLC and MS analysis. All compounds were dissolved in DMSO as 10 mM stock solutions. For dose-response experiments, we plated cells in 2 replicates at 7,000 cells per well in 96-well plates (Corning) in full growth media for 24 hr and then treated them with 9 doses in serial dilutions (10−10 to 10−5 M) of each compound for 6, 24 and 72 hr.
