Refer to the method in our previous studies [10 (link), 11 (link)]. In brief, HEK293 and LNCaP cells lacking of functional AR were transfected with wild-type AR expression plasmid and reporter genes such as pSG5-AR, pSG5-PR, pSG5-GR, MMTV-Luc, and pRL-TK-luc for AR for 24 h. Then, the cells were cocultured with various concentrations of kaempferol in the presence or absence of 1 nM dihydrotestosterone (DHT) (Sigma, USA) and/or 5 μM hydroxyflutamide (HF) (Sigma, USA) for 24 h. The cells were harvested and assayed for luciferase activity. Data were expressed as relative luciferase activity normalized to the internal Renilla luciferase control.
The mammalian 2-hybrid assay was performed to determine the AR N-C interaction and AR-AR coregulator interaction. After transfection with pGal4-RE-Luc reporter plasmid, pGal4-ARDBD-LBD (AR DNA-binding domain and ligand-binding domain), pCMX-VP16-AR, or pCMX-VP16-ARA70 for 24 h, HEK293 and LNCaP cells were harvested for dual luciferase assay (Promega, WI).
The mammalian 2-hybrid assay was performed to determine the AR N-C interaction and AR-AR coregulator interaction. After transfection with pGal4-RE-Luc reporter plasmid, pGal4-ARDBD-LBD (AR DNA-binding domain and ligand-binding domain), pCMX-VP16-AR, or pCMX-VP16-ARA70 for 24 h, HEK293 and LNCaP cells were harvested for dual luciferase assay (Promega, WI).