Cell culture and transfection assays

Two liver epithelial cell lines were used to test receptor activity. HepG2 cells (ATCC, Manassas, VA) were maintained in DMEM supplemented with 10% FCS and non-essential amino acids. ZFL cells (ATCC) were maintained in 50% L-15 Leibovitz, 15% H-12 Ham and 35% DMEM supplemented with 10% FCS (Gibco, Paisley, Scotland, UK) and 50 ng ml-1 EGF (Sigma). Cells were cultured at 37°C (HepG2) or 28°C (ZFL) in 5% CO₂. The transfection experiments were initiated by replacing the tissue culture medium with fresh phenol-free medium supplemented with charcoal stripped FCS and the cells were seeded on 24-well plates. Transfections were performed at 90–95% confluence using 1,5 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and 0,6 μg DNA per well. Transfected DNA contained 60 ng pRL (Promega), 270 ng androgen response element (ARE) luciferase reporter vector, 270 ng stickleback ARβ2 expression vector or 270 ng human AR expression vector (pCMVhAR) while 270 ng empty TNT cloning vector was used to control background luciferase levels following transfections. Two different ARE containing promoter constructs were tested, one containing 3 separate ARE (HRE; slp-ARU) and a second one construct (slp-HRE2) containing 4 copies of the ARE with high affinity for R1881, a synthetic androgen, and low affinity for dexamethasone, a synthetic glucocorticoid [19].

After 16 h the transfection medium was replaced with fresh phenol-free medium, supplemented with charcoal stripped FCS, containing different steroid hormones. The cells were exposed to the hormones for 40 h and luciferase levels were detected using the Dual Luciferase Assay kit (Promega) in a TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA). For each cell line, transfection assay was performed for each concentration with n = 4 and the luciferase value of each assay were normalized to its corresponding Renilla luciferase activity. Each experiment was repeated a minimum of 3 times. The fold-induction is presented as luciferase values normalized against the control. The control levels were arbitrarily set to 1.0 for each cell line.