Laschak et al. BMC Cancer 2012, 12:130 http://www.biomedcentral.com/1471-2407/12/130 with 10% steroid free, dextran charcoal treated FBS (FBSdcc, BioWest, Nuaille, France) [26]. Transfection and reporter gene assays 22Rv1 cells were grown on 24-well plates (Sarstedt, Nüm- brecht, Germany). Transfections were performed using FuGene HD transfection reagent (Roche Diagnostics, Man- nheim, Germany) according to the manufacturer’s instruc- tions. AR- and WNT-specific reporter gene assays were performed as recently described [22,27]. In brief: (1) AR- signalling: Cells were transfected with 200 ng/well of the AR-dependent reporter-plasmid pGL3E-Probasin. The pRL-tk-LUC vector coding for a Renilla luciferase under control of a constitutively active thymidine kinase promo- ter was co-transfected (80 ng/well) to correct for transfec- tion efficiency. After transfection cells were grown in RPMI 1640 with 5% dextran charcoal treated steroid free, dextran charcoal treated FBS (FBSdcc; Biowest, Nuaillé, France) and treated with 5 nM DHT and different concen- trations of JS-K. (2) WNT-signalling: Luciferase reporter plasmids pTopFlash (250 ng/well) and pFopFlash (250 ng/ well) were mixed either with pbCAT expression vector (250 ng/well) or insert-free vector (250 ng/well). pRL-tk- luc (80 ng/well) was co-transfected to correct for transfec- tion efficiency. After an incubation period of 12 h in RPMI-1640 with 5% FBSdcc, medium was changed to RPMI-1640 containing 2.5% FBSdcc with increasing con- centrations of JS-K. AR- and WNT-reporter activities were analyzed after 24 hours using the Dual Luciferase Reporter Assay System (Promega, Mannheim, Germany). Experiments were repeated at least three times and performed in triplicates, unless stated otherwise. Mammalian two hybrid assay To investigate the dimerization of the AR we used the CheckMate/FlexiVector Mammalian Two-Hybrid System from Promega (Mannheim, Germany). VP16 activation domain (pFN10A-AR) and Gal4 DNA binding domain tagged androgen receptor fusion constructs (pFN11A-AR) were cloned according to the supplier’s suggestions by using pSG5-AR as template. DU-145 and PC-3 cells were grown in 24-well plates and co-transfected with 150 ng/ well of pFN10A-AR, pFN11A-AR and the Gal4 reporter construct pGL4.31. After transfection, cells were cultured and luciferase activities were determined as recently described [28]. Nuclear Translocation assay Nuclear translocation of the AR was analyzed in AR-nega- tive DU-145 and PC-3 cells transfected with the green fluorescent AR-EosFP [22,29]. Therefore, prostate cancer cells were transfected in a 24-well plate with pAR-t1EosFP (0.25 μg/well) for 4 hours in RPMI and allowed to grow Page 3 of 10 for another 24 hours in RPMI 1640 supplemented with 5% FBSdcc and antibiotics. Thereafter, cells were grown in RPMI supplemented with 2.5% FBSdcc in the presence/ absence of 5 nM DHT and JS-K for 6 hours. Localization of the AR-fusion protein was subsequently analyzed by fluorescence microscopy. Western Blot analysis Proteins were extracted from cells using RIPA buffer as recently described [30]. Proteins (15 μg lysate) were sepa- rated by Sodium dodecyl sulfate polyacrylamide gel elec- trophoresis (SDS-Page). Subsequently proteins were transferred onto a nitrocellulose membrane (BioTraceNT, Pall Life Sciences, Dreieich, Germany) by tank blotting (transfer buffer: 20 mM Tris/HCl, pH 8.7, 150 mM glycine and 20% (v/v) methanol). Membranes were blocked in phosphate buffered saline with 0.1% Tween20 (v/v) (PBS- T) and 5% BSA (w/v) for 1 hour at room temperature. The membrane was incubated with the primary antibody in PBS-T with 1% BSA over night at 4°C. All primary anti- bodies were used in a dilution of 1:1000, with the excep- tion of the beta-actin antibody, which was diluted 1:20000. The membrane was washed with PBS-T three times before incubating with the peroxidase-coupled secondary anti- body in a dilution of 1:2000 in PBS-T with 1% BSA. Sig- nals were visualized by the SuperSignal West Pico Chemiluminescent Substrate from Pierce (Rockford, USA). Determination of NO NO was determined indirectly by the photometric mea- surement of nitrite according to Green et al. [31] Immunocytochemistry For immunocytochemistry 22Rv1 cells were cultured on glass coverslips (BD Falcon Culture Slides, BD Bios- ciences) washed (2 × 5 min) with PBS and fixed with 3.7% paraformaldehyd for 15 min at room temperature, washed (3 × 10 min) in PBS and permeabilized in 0.2% Triton X-100 with 1% BSA in PBS for 5 min at room temperature. The cells were washed (3 × 10 min) with PBS containing 1% BSA. Cells were then incubated with the monoclonal anti-tyrosine antibody (1:25 in PBS, 1% BSA for 30 min at 37°C, washed (3 × 10 min and incu- bated with the mouse monoclonal anti-goat antibody labeled with Alexa Fluor 488 (Invitrogen, Karlsruhe, Ger- many, 1:100) for 30 min at 37°C. Cells were then washed (3 × 10 min), the nuclei stained with DAPI (2.5 μg/ml in PBS) for 15 min, washed (3 × 10 min) and embedded with glycine and polyvinyle alcohol 4-88 (Fluka Analyti- cal, Deisenhofen, Germany). Cell viability Cell viability was determined by means of a colorimetric MTT-assay measuring the reduction of the water