Targeted siRNA Knockdown and Lentiviral Overexpression in Prostate Cancer

For small interfering RNA (siRNA) transfection, cells were seeded at a density of 0.5 × 10⁵ cells per well in 12-well plates or 2 × 10⁵ cells per well in 6-well plates and transfected with 20 nM nM siRNA targeting the AR exon 7 sequence (UCAAGGAACUCGAUCGUAU), AR-V7 sequence (GUAGUUGUGAGUAUCAUGA) [44 (link)] or siControl (Invitrogen, Catalog# 12935300) using Lipofectamine-iMAX (Invitrogen). The effect of siRNA-mediated gene silencing was examined using qRT-PCR and western blot 2–3 days after transfection. Cells were transiently transfected with plasmids expressing AKR1C3, AR-V7, and HA-ubiquitin using Lipofectamine 2000 (Invitrogen). Lentiviral plasmids encoding shRNA targeting AKR1C3 (TRCN0000026561) were purchased from Sigma-Aldrich. The pLenti-GFP lentiviral vector was used as the control. Lentiviral particles were produced in HEK293T cells after co-transfection of the lentivirus vectors psPAX2 and pMD2.G. The lentivirus-containing medium was collected and the cells were infected.