Fig. 5: AR suppression upregulates OPRK1, which underpins androgen-independent growth of androgen-independent derivatives of VCaP cells.

a, b Expressions of AR in VCaP treated with siRNA for GFP (siControl) and AR (siAR#1 and siAR#2) assessed by quantitative RT-PCR (a) and western blot (b). **P < 0.01, Normalized by GAPDH (a) and β-actin (ACTB, b). c Expression of OPRK1 in VCaP treated with siRNA for GFP (siControl) and AR (siAR#1 and siAR#2) assessed by quantitative RT-PCR. **P < 0.01, Normalized by GAPDH. d Expression of AR (left) and OPRK1 (right) in VCaP cultured in media supplemented with 10% FBS, charcoal-stripped FBS (CSFBS), and CSFBS plus 1 nM dihydrotestosterone (DHT) assessed by quantitative RT-PCR. **P < 0.01, Normalized by GAPDH. e Summarized results of quantitative RT-PCR for full-length AR (AR-FL), AR variant 7 (AR-V7), KLK3, and OPRK1 normalized by GAPDH in indicated cells. **P < 0.01. f Western blot of indicated proteins in VCaP, PC3, AIVCaP1, AIVCaP2, and AIVaP3. β-actin (ACTB) acted as loading control. g Summarized results of proliferation rates of VCaP and AIVCaP2 (AIVCaP) treated with siRNA for GFP (siControl) and AR (siAR#1 and siAR#2). h Summarized results of proliferation rates of VCaP and AIVCaP2 treated with siRNA for GFP (siControl) and OPRK1 (siOPRK1#1 and siOPRK1#2). i Summarized cell proliferation rates of VCaP and AIVCaP2 (AIVCaP) with regard to the concentration of supplemented nor-BNI, OPRK1 inhibitor. *P < 0.05. j Cell proliferation in VCaP (left) and AIVCaP2 (AIVCaP) treated with siRNA for GFP (siControl), AR (siAR) OPRK1 (siOPRK1), or siAR plus siOPRK1. *P < 0.05, **P < 0.01. k Summarized results of migration (scratch) assay with greater distance indicating higher migration ability. VCaP, AIVCaP, and AILNCaP cells were treated with indicated siRNA and subject to the assay. **P < 0.01.