Establishment of Enz-Resistant PCa Cell Lines

PCa cell lines CW22Rv1, C4-2, and LNCaP were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 media with 10% FBS and antibiotics (100 units/mL penicillin, 100 µg/mL streptomycin). Cells were maintained in humidified 5% CO₂ environment at 37 °C. For generation of the Enz-resistant (Enz-R) cell line, C4-2 cells were kept in media with 10 µM Enz for at least 3 months before experiments, as in our previous study^{25 (link)}. Hypoxia was achieved by maintaining the cells at 1% O₂, 5% CO₂, and 94% N₂ in a hypoxic chamber (Coy Laboratory Products, Grass Lake, MI, USA) with oxygen sensor controls, with temperature at 37 °C with humidity controls, and CO₂ and N₂ gas regulators.