br Cell treatments br For protein detection cells were grown
2.2. Cell treatments
For protein detection, cells were grown in phenol red-free RPMI medium containing 2% charcoal stripped-serum for 24 hours prior to treatment with FSH (Gonal-F, Merck Serono, Darmstadt, Germany) or 5a-dihydrotestosterone (DHT, Steraloids Inc., Newport, RI) for further 24 hours at concentrations of 50, 100, 200, and 400 IU/l or 10 nM, respectively. For AR induction, PC-3 cells were treated with FSH at concentrations of 400, 800, and 1,600 IU/l. In the mRNA detection, assay cells were treated with 50 and 100 IU/l FSH or 10 nM DHT for 6 or 24 hours. For combination treatment, cells were exposed to 10 mM enzalutamide (Astellas Pharma, London, UK) for 1 hour before the treatment with the aforementioned substances for further 24 hours. Forskolin (Sigma Aldrich, St. Louis, MO) is an agent known to induce cAMP by direct activa-tion of adenylyl cyclase, and was therefore used as an FSH control. In controls, cells were left untreated. For the detection of phosphorylated Akt (p-Akt), cells were pretreated with 10 mM of the PI3K inhibitor, LY294002 (Sigma Aldrich) 1 hour before treatment with FSH at concentrations of 50, 100, and 200 IU/l for 24 hours.
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 CP-456773 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.