Figure 6

CXCR7 modulates AR transcriptional activity. (A) Luciferase assay testing the effects of CXCR7 overexpression on the AR-target promoter probasin in LNCaP cells. LNCaP cells were co-transfected with the pGL4.10-Luc2-probasin and pRLSV40 Renilla vectors along with increasing amounts (30 ng experimental + 270 ng pcDNA3, 100 ng experimental + 200 ng pcDNA3, 300 ng experimental + 0 ng pcDNA3) of CXCR7 or ACTN4 cDNA mammalian expression vectors. The maximal amount (300 ng) of the pcDNA3 mammalian expression vector served as the positive control. Cells were subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) and tested for dual luciferase activity. Student’s t-test was used to calculate significant differences (*p ≤ 0.05, n = 3) between control and experimental cells within the androgen-treatment group. (B) Luciferase assay testing the effects of CXCR7 siRNA knockdown and treatment with CXCR7 ligand on the AR-target promoter probasin. LNCaP cells were co-transfected with the pGL4.10-Luc2-probasin and pRLSV40-renilla vectors, along with control or experimental siRNAs (50 nM). Next, cells were pre-treated with indicated ligands (BSA, CXCL11, or CXCL12) for 30 min. Cells were then subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) for 18 hrs and tested for dual luciferase activity. Student’s t-test was used to calculate significant differences (*p ≤ 0.05, n = 3) between control cells and experimental cells within the androgen-treatment group. (C) RNA isolated from LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (10 nM), or CXCL12 (10 nM) for 30 min and subsequently treated with vehicle or androgen (1 nM R1881) for 18 hrs were subjected to qPCR analysis for AR, FASN, NKX3.1, PSA, and TMPRSS2 gene expressions. Student’s t-test was used to calculate significant differences (*p ≤ 0.05, n = 3) between control and chemokine ligand-treated cells.