Cell Lines, Xenografts and Human Prostatic Tissues. Four human immortalized prostatic epithelial cell lines (HPr-1, HPr-1AR, PNT1 A, PNT2) and seven prostatic cancer lines (CA-HPV-10, CWR22Rv1, LNCaP, C4-2B, DU 145, PC-3, MDA PCa 2b) were used in this study. Total RNA and protein were extracted from cells when they reached 80-90% confluence. Human primary prostate cancer xenograft
CWR22 was grown in male nude mice supplemented with either one slow-release testosterone pellet, while an androgen-independent subline, CWR22R, was grown in castrated nude mice. Benign hyperplastic tissues were obtained from BPH patients by TURP whereas neoplastic tissues were obtained by radical prostatectomy or ultrasound-guided needle biopsy from prostate cancer patients.
RT-PCR. About 5(.ig DNase I-treated total RNA samples extracted from cultured cells, xenografts and human prostatic tissues were reverse transcribed to cDNAs using Oligo(dT) primer and 1 |xl of cDNA samples was used for PCR reaction.
Immunohistochemistry and Western Blot Analysis.A rabbit antiserum against ERRy, generated by a synthetic peptide of mERRy conjugated to KLH (15), was used for immunohistochemistry and Western blot analysis.
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