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. 2007 Nov 5;36(1):51–66. doi: 10.1093/nar/gkm942

Figure 2.

Figure 2.

NRIP is a novel AR-targeted gene. (A) Androgen can induce NRIP and PSA gene expression as measured by RT-PCR in prostate cancer cells (LNCaP). LNCaP cells were grown in RPMI 1640 medium supplemented with 10% FBS or with charcoal/dextran-stripped serum (CDS) and treated with 1 and 10 nM DHT for 24 h. Total RNA was extracted and 5 µg of RNA was amplified by semi-quantitative RT-PCR using NRIP, PSA, AR and β-actin primers. One representative data set from three independent experiments is shown. The expression levels of NRIP, PSA and AR RNA quantified by UVP imaging system were normalized to β-actin, and then set at 1.00 for CDS treatment. (B) AR with androgen can stimulate NRIP gene expression in 293T cells. AR-negative 293T cells were transiently transfected with pcDNA3.0-AR and cultured in CDS medium. After 24 h of 10 nM DHT treatment, total RNA was extracted and RT-PCR was performed using NRIP, AR and β-actin primers. One representative data set from two independent experiments is shown. (C) The promoter activity of NRIP induced by DHT-activated AR. 293T cells were co-transfected with NRIP-P413-Luc and pcDNA3.0-AR or vector (pcDNA3.0) with pRL-CMV and cultured in CDS medium. After 24 h of 10 nM DHT treatment, luciferase activities were measured and normalized. The data are mean ± SD from three independent experiments. The fold change was measured by the luciferase activity of each experimental condition compared to that of the absence of the AR and DHT treatments. (D) AR influences the ARE region of the NRIP promoter. Three site-directed mutants were made by nucleotide substitutions at either ARE or GRE or both sites in NRIP-P413. Wild-type and mutant promoters were transfected with pcDNA3.0-AR and pRL-CMV into 293T cells with or without ligand treatment. The luciferase activities were measured as described above. Panel D(a) depicts the change of relative luciferase activity, which was measured by the luciferase activity of the NRIP-P413 promoter in the absence of AR and DHT treatments. Panel D(b) refers to luciferase activities with DHT relative to that of EtOH for each construct.