Cultivation and Transduction of NHPrE1 Cells

NHPrE1 cells [18 (link)] were maintained in DMEM (Gibco) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA), 1% penicillin/streptomycin (Gibco), 10 ng/ml epidermal growth factor (Sigma– Aldrich, St. Louis, MO), 1% insulin-transferrin-selenium (ITS) (Gibco), and 0.4% bovine pituitary extract (Atlanta Biologicals). NHPrE1 cells were transfected with EGFP-expressing plasmid; GFP-positive NHPrE1 cells were selected by cell sorting. To establish AR-expressing NHPrE1/GFP cells, CMV promoter-driven LNCX or LNCX-AR retroviral vector-based plasmids were transfected into Phoenix packaging cells (ATCC, Manassas, VA). Twenty-four hours later, culture media were collected and used to infect NHPrE1/GFP cells. The infection procedure was repeated twice. The transduced cells, stably expressing AR, were selected by culturing them in the presence of G418 (Sigma, St. Louis, MO, USA). G418 resistant cell populations were used in this study. American Type Culture Collection (ATCC) has authenticated NHPrE1 cells and no contamination from other type of cells was found. PC3/AR [21 (link)] and LNCaP (ATCC) cells were cultured in RPMI1640 supplemented with10% serum.