Stable Transfection of AR Variants in Prostate Cancer Cells

LNCaP and C4–2 cells were stably transfected as described previously (20 (link)). Briefly, C4–2 or LNCaP cells at 70–80% confluency were transfected with 1 μg GFP-AR^WT or GFP-AR^F876L in 6-well plates using PolyJet™ In Vitro DNA Transfection Reagent (SignaGen) according to the manufacturer’s protocol. Twenty-four hours after transfection, half of the cells from each well were reseeded in 10 cm dishes containing 10 ml regular RPMI 1640 medium. After another 24 hours, medium was replaced with RPMI1640 containing 1000 μg/ml G418 (for C4–2) or 500 μg/ml (for LNCaP) for selection of stably transfected cells. The selection medium was changed every week until the colonies formed were about 1 mm in diameter. Single colonies were transferred individually to separate wells in a 96-well plate. As the colonies were expanded, each was reseeded into a 48-well, followed by a 12-well, then a 10 cm dish. The C4–2 GFP-AR^WT, C4–2 GFP-AR^F876L and LNCaP GFP-AR^F876L stable sublines were monitored using fluorescence microscopy (Nikon Eclipse TS100, Prior Scientific Lumen 200 Fluorescence Illumination System, Tokyo, Japan) and confirmed by western blotting. Unfortunately, we were not able to generate LNCaP-GFP-AR^WT, because LNCaP-GFP-AR^WT cells did not propagate.