2.3. siRNA transfection and proliferation assays

siRNA against AR exon3b was obtained from Dharmacon (Lafayette, CO, USA). The target sequence for AR exon3b is 5′‐GUAGUUGUGAGUAUCAUGA‐3′. siRNA knockdown was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. VCaP‐ENZR (5 × 10⁵ cells) and C4‐2B‐ENZR (3 × 10⁵ cells) cells were, respectively, plated and incubated for 24 h. siRNA/transfection reagent complexes were added into each wells. Forty‐eight hours post‐transfection, protein was harvested and analyzed by western blot analysis.

For experiments combining drug treatment with siRNA transfection, VCaP‐ENZR (8 × 10⁴ cells/well) and C4‐2B‐ENZR (2 × 10⁴ cells/well) were plated in 96‐well plates and transfected with siRNA 24 h prior to treatment with enzalutamide for 5 days (VCaP‐ENZR) or 3 days (C4‐2B‐ENZR). BrdU incorporation was measured using BrdU ELISA kit (Roche Diagnostics, Laval, QC, Canada).