Everolimus eve

The U2OS, MDA-MB-231, BT-549 and MCF-12A cell lines were purchased from the ATCC (American Type Culture Collection) and the cell lines were authenticated by Short Tandem Repeat (STR) profiling by ATCC. U2OS cells were maintained in McCoy’s 5A medium supplemented with 10% fetal bovine serum. MDA-MB-231 and BT-549 cells were cultured in Dulbecco modified Eagle medium (DMEM) or RPMI-1640 medium supplemented with 10% fetal bovine serum, respectively. MCF-12A cells were cultured in mammary epithelial growth medium (1:1 DMEM:DMEM/F12 medium, 20% horse serum, hydrocortisone [0.5 mg/mL], insulin [10 μg/mL], recombinant epidermal growth factor [20 ng/mL], cholera toxin [100 ng/mL], and 1:100 penicillin-streptomycin [Invitrogen]). mTOR inhibitors everolimus (EVE) and KU-0063794 (KU) and PARP inhibitors olaparib (AZD2281) and talazoparib (BMN673) were purchased from Selleckchem. Myc-DDK-SUV39H1 plasmid was purchased from OriGene. Antibodies against SUV39H1, p-4EBP1 (Ser65), and Myc-Tag were purchased from Cell Signaling Technology. Vinculin antibody was purchased from Santa Cruz Biotechnology. An Annexin V FITC apoptosis detection kit was purchased from BD Biosciences and an apoptosis assay was performed following the manufacturer’s procedure. The transfection reagent FuGENE 6 and oligofectamine were purchased from Promega and Life Technologies, respectively.