*4.5. Luciferase Reporter Assay*

LNCaP and C4-2B cells were transiently transfected with pGL3-Basic, pGL3-KLF5, or pGL3-PSA, or pGL3-MMTV and pRL-TK (Renilla luciferase, Promega, Madison, WI) as an internal control. After 48 h of transfection and enzalutamide (Beyotime Biotechnology, Shanghai, China, catalog number: SC0074) treatment, cells were lysed in 5 × lysis buffer (Promega) for 30 min and luciferase activity was measured using a luminometer (Tristar LB941, Berthold Technologies, BadWild, Germany). Firefly luciferase activity was normalized to Renilla luciferase activities in each reaction. Experiments were performed in triplicate.

#### *4.6. Colony Formation Assay*

One thousand C4-2B cells/well or 2000 LNCaP cells/well were seeded in 6-well plates and cultured in the presence of RPMI-1640 medium containing 10% FBS with DMSO or 10 μM enzalutamide (Beyotime Biotechnology,). The plates were incubated for 2 (C4-2B) or 3 (LNCaP) weeks, after which cultures were fixed with 4% paraformaldehyde for 30 min and stained by 0.05% crystal violet (BBI life sciences, Shanghai, China) for 1 h at room temperature and then photographed. In a single experiment, assays were conducted in triplicate and then as three independent experiments.
