Cell culture-based model system for FOXO3 activity. Cell culture-based model system for FOXO3 activity

MCF7 and MDA-MB-231 breast cancer cell lines were cultured in DMEM-F12 containing 10% FBS (Lonza) 100U/ml penicillin and 100 µg/ml streptomycin (Lonza). Transfecting third generation packaging vectors using Poly-ethylenimine into HEK293T cells generated lentiviral particles (17). MCF7 and MDA-MB-231 cells were stably transduced with lentivirus containing pINDUCER20-FOXO3.A3, allowing doxycycline induced expression of constitutively active FOXO3 (FOXO3.A3). Cells were treated with 20% FBS or 10 µM PI3K inhibitor LY294002 (Selleckchem) for 16 hours to activate and inactivate the endogenous PI3K pathway, respectively. FOXO3.A3 expression was induced by 16 hours treatment with 10 ng/ml doxycycline. Overall design: 3 biological replicates per condition; each experiment contained at least a control and a stimulated condition