*2.2. Cell Culture*

The androgen-sensitive, AR-positive human PCa cell line LNCaP, and the androgen-insensitive, AR-negative human PCa cell line PC-3 were obtained from the American Type Culture Collection (Manassas, VA, USA). LNCaP and PC-3 cells were authenticated by the short tandem repeat method and cultured in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Sigma-Aldrich Co.). Androgen-low-sensitive E9 and F10 cells were established from androgen-sensitive LNCaP cells using a limiting dilution method under regular culture conditions [24,25]. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted conditions [26]. The androgen sensitivity of the parental LNCaP cells and the E9, F10, and AIDL cells were confirmed by the change in *KLK3* (PSA) mRNA expression after treatment with the synthetic androgen R1881 [20].
