One day before transfection, 140,000 cells (DuCaP/VCaP) or 70,000 cells (22Rv1/MIA-PaCa-2) were seeded per well of a 24-well plate, in a total volume of 500 µl medium. After trypsinization, cells were collected and seeded in charcoal-stripped serum-containing medium to wash away traces of androgens previously reported to be present in FCS [50 (link)]. Transfection mixtures were prepared by combining oligonucleotides (AON/SON or GapmeRs) in a desired concentration with X-tremeGENE™ 9 transfection reagent (Roche), both dissolved in Opti-MEM I Reduced serum-free medium (Invitrogen). A mix of transfection reagent alone, i.e., without oligonucleotide, was used as non-transfected control. Mixes were incubated at room temperature for 15 min before addition to the cells in a dropwise manner. For overexpression studies, 140,000 VCaP cells were seeded per well in 24-well plates. Twenty-four hours later, cells were transfected with 250 ng of pCMV-AR-V7 expression vector or empty vector control. For minigene experiments, 70,000 MIA-PaCa-2 cells were seeded per well in 24-well plates and after 24 h, cells were co-transfected with 500 ng of minigene or empty vector and 0.5 µM of the desired oligonucleotide. All experiments were performed at least three times.
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