Figure 2: Membrane androgen binding sites on LNCaP and DU-145 cells assayed by flow cytometry.

(A) Cells were labelled with either BSA-FITC (peak A) or Testosterone-BSA-FITC (peak B) and the percentage of cells that specifically bind testosterone-BSA-FITC is given by Gate R1. (B) OXER1 expression at the mRNA and protein level. OXER1 mRNA expression was assayed by Real Time PCR and was normalized to the expression of Cyclophilin A. Photos present LNCaP cells and DU-145 cells stained with an anti-OXER1 antibody. Representative graphs and photos of three independent experiments are presented. (C) Binding of [H³]testosterone (20 nM) on DU-145 isolated plasma cell membranes in the absence (Total Binding) or in the presence of 5-oxoETE (10⁻⁶ M). Non labelled testosterone (10⁻⁵ M) was utilized for non-specific binding (NSB). Insert shows specific binding. Data presented are the mean ± SEM of three independent experiments (**p < 0.001, NS: non-significant). (D) Membrane steroid binding sites on DU-145 cells transfected with a specific siRNA for OXER1 (right panel), or a scrambled siRNA (left panel) and assayed by flow cytometry. Cells were labelled with either BSA-FITC for non-specific binding (NSB) or testosterone-BSA-FITC, estradiol-BSA-FITC or progesterone-BSA-FITC for androgen, estrogen and progesterone binding sites respectively. The percentage of the specific steroid-BSA-FITC on cells is also shown (Gate R5). Figures are representative of three independent experiments.