Fig. 3.

PSF bindings to the target transcripts are enhanced in CRPC. (A) Pathway analysis of PSF-binding genes in 22Rv1 cells is shown. (B) Representative mapping of PSF-binding RNAs in AR locus. (C) Expression levels of PSF, NONO, AR, and AR-V7 in prostate cancer cells. Lysates from LNCaP, LTAD, VCaP, and 22Rv1 cells were used for immunoblots with indicated antibodies. (D) Regulation of AR and AR-V7 expression by PSF and NONO in CRPC model cells. (Left) The 22Rv1 cells were treated with siPSF or siNONO for 72 h. AR and AR-V7 mRNA levels were measured by qRT-PCR (n = 3). (Right) Lysates from 22Rv1 cells transfected with siPSF or siNONO are used for immunoblots to detect AR and AR-V7 protein levels. (E) mRNA stability of AR and AR-V7 was decreased by knockdown of PSF. The 22Rv1 cells are treated with siControl, siPSF #1, or siPSF #2 for 48 h. To inhibit transcription, actinomycin-D (1 nM) was added. After incubation for indicated times, mRNA expression levels were measured by qRT-PCR (n = 3). (F) Immunohistochemistry of AR-V7 in prostate cancer tissues (n = 102) was performed. (G) AR-V7 expression (high, n = 18; low, n = 86) is a strong prognostic factor for prostate cancer patients. Cases with labeling index (LI) > 10% were determined to be AR-V7 high expression. (H) Positive correlation of PSF expression with AR and AR-V7 levels in prostate cancer tissues. LI in each group is shown. (I) NONO as well as PSF was up-regulated in metastatic prostate cancer tissues. Heatmap results using microarray database (GSE35988) is shown. (J) Immunohistochemistry of NONO in prostate cancer tissues (n = 102). (K) NONO expression (high, n = 51; low, n = 51) is a prognostic factor of poor outcome of prostate cancer patients. Values represent the mean ± SD. *P < 0.05, **P < 0.01.