br Western blot analysis br For Western blot analysis total
2.3. Western blot analysis
For Western blot analysis, total protein was extracted using radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, 20 mM EDTA, 1% NP40, 0.1% SDS, 1 mM Na3VO4, 1 mM NaF, and 1 mM phenylmethylsulfo-nylfluoride) (Invitrogen Carlsbad, CA) supplemented with the protease inhibitor cocktail Complete Mini (Roche, Mannheim, Germany). Total protein concentration was measured using bicinchoninic 313263-08-0 protein assay (Pierce, Rockford, IL). Protein samples (20−30 mg) were loaded on 4% to 12 % SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred onto a nitrocellulose or PVDF membrane (Bio-Rad, Hercules, CA) and subjected to electrophoretic analysis and blotting. Membranes were probed overnight at 4˚C with the relevant primary antibodies anti-AR (PG-21), anti-FSHR, anti-b-catenin, and anti-p-Akt from Cell Signaling Technology (Beverly, MA), anti-PSA (Dako, Glostrup, Denmark) and anti-b-actin (Sigma Aldrich), washed and incubated in secondary antibodies horseradish peroxidase (HRP)-conju-gated antimouse Immunglobulin G (IgG) and antirabbit IgG (GE Healthcare, Stockholm, Sweden) for 1 hour at room temperature. Bands were visualized using enhanced chemiluminescence (Pierce, Rockford, IL) and images were acquired using the Bio-Rad Western workflow (Bio-Rad, Hercules, CA). Densitometric quantification of immu-noblots was performed by the ImageJ Image Analysis Software (NIH, Baltimore, MD) and represented as fold change relative to control, normalized relative to b-actin bands.
2.4. Real-time quantitative PCR analysis
Total RNA was isolated from the cells using RNeasy Mini Kit (Qiagen, West Sussex, UK) following the man-ufacturer’s protocol. Each cDNA was synthesized by reverse transcription from 500 ng of total RNA using the RevertAid First-Strand Synthesis System and Oligo(dT)18 primers (Life Technologies, Thermo Fisher Inc.). Real-time polymerase chain reaction (PCR) was performed with SYBR Green QPCR master mix (Life science, Thermo Fisher Inc) in AriaMx detection system (Agilent, Tech-nologies, Willoughby, Australia) with an initiation step at 95˚C for 10 minutes followed by 40 cycles at 95˚C for 30 seconds, 56˚C for 1 minute, and 72˚C for 30 seconds. The following primers for each gene were used: AR (F 50-CCTGGCTTCCGCAACTTACAC-30 and R 50-GGA-CTTGTGCATGCGGTACTCA-30); PSA (F 50-AGGCCT-TCCCTGTACACCAA-30 and R 50-GTCTTGGCCTGGT-CATTTCC-30); NKX3.1 (F 50-GTACCTGTCGGCCC CTGAACG-30 and R 50-GCTGTTATACACGGAGAC-CAGG-30); FSHR (F 50-GATGTTTTCCACGGAGCCTC-30 and R 50-ATCTCTGACCCCTAGCCTGA-30); cMyc (F 50-GGCGGGCACTTTGCACTGGA-30 and R 50-TCG CGGGAGGCTGCTGGTTT-30); b-actin (F 50-CGTGGGG CGCCCCAG-30 and R 50-TTGGCCTTGGGGTTCAGG GGG-30). The mRNA amount was determined by using the DDCt method. Samples were analyzed in triplicates and the data compared with the expression of mRNA in nontreated control which was set as reference value.
2.5. Proliferation assay
Proliferation of PC-3 cells was determined by a viability assay examined by trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium assay (MTS) (Promega Biotech, Nacka, Sweden), according to the protocol from the manufacturer. Approximately 3 £ 103 cells/well were cultured in 96-well plates for 24 hours in 5% serum com-plemented serum before being treated with FSH (100, 400 IU/l, or 10 nM/l DHT) for 24 and 48 hours. Cells were washed with phosphate buffered saline, and then 80 ml new medium containing 20 ml 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium was added to each well. Cells were incubated at 37˚C for 2 hours and measured for optical density values at 490 nm on Milenia Kinetic Analyzer (Diagnostic Products Corpora-tion, DPC, LA). Wells containing medium only served as a blank control. Three independent experiments were carried out.
2.6. Statistical analysis
Results were expressed as the mean § standard devia-tion (SD). Statistical significance was determined with unpaired Student’s t test. All statistical analyses were
conducted using SPSS version 24 (SPSS Inc., Chicago, IL). Values of P < 0.05 were considered statistically sig-nificant.
3.1. Expression of FSHR in CaP cells
Expression of FSHR protein was demonstrated in all 3 cancer cell lines (Fig. 1, upper panels), but not in the PNT1A cells (Supplementary Figure 1). High to moder-ate expression of FSHR protein was found in the C4-2 and PC-3 cells, whereas in LNCaP cells the originally low protein expression was increased following FSH treatment (Fig. 1, upper panels). At mRNA level, FSHR expression was markedly increased in PC-3 and LNCaP cells treated with 100 IU/l FSH as compared with con-trols (Fig. 1, lower panels). C4-2 cells are androgen inde-pendent that have high basal expression of FSHR at protein and mRNA. Hormonal treatment therefore only changed it marginally.