Fcsdcc

Cell viability was assessed using the WST-1 cell proliferation reagent (Roche Diagnostics, Mannheim, Germany). Cells (10,000 cells in 100 μL) were seeded into 96-well culture plates. After 24 h, the medium was changed to a 50 µL medium containing 5% FCSdcc (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h. Subsequently, cells were treated with the synthetic androgen R1881 or with a combination of R1881 and antiandrogens diluted in 50 µL Medium containing 5% FCSdcc. After 72 h treatment, 10 µL WST-1 solution was added for an additional 2 h. Subsequently, absorbance was recorded at 450 nm (Reference 620 nm) for each well using Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany). After subtracting background absorbance, results were calculated as x-fold of 1 nM R1881 treated cells.

Cell viability was analysed using the Cell Proliferation Reagent WST-1 (Roche, Mannheim, Germany). Cells (10,000 cells in 100 μL) were seeded into 96-well culture plates. After 24 h, the medium was subsequently changed to a 50 µL medium containing 5% FCSdcc (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h. After another 24 h, cells were treated with different concentrations of R1881 (0.01–10 nM). After 72 h incubation, 10 µL WST-1 solution was added for an additional 2 h. Absorbance was measured at 450 nm (Reference 620 nm) using Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany). After subtracting background absorbance, results were displayed as x-fold of untreated cells.

Cells (400,000 cells per well) were seeded into 6-well culture plates for Western blot analysis. After 24 h, the medium was changed to a medium containing 5% FCSdcc (Thermo Fisher Scientific, Waltham, MA, USA). After another 24 h, cells were treated with different concentrations of R1881 (0.01–10 nM) for 72 h. Subsequently, cells were harvested, and protein concentration determination was performed as previously described [26 (link)]. NuPAGE^TM 4–12% Bis-Tris protein gels separated 20 µg protein lysate. As protein standard, 5 µL Spectra Multicolour Broad Range (Thermo Fisher Scientific, Waltham, MA, USA) protein standard mixed with 1 µL MagicMark^TM XP Western Protein Standard (Thermo Fisher Scientific, Waltham, MA, USA) were used. Proteins were transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (all Thermo Fischer Scientific, Waltham, MA, USA). WesternBright Sirius HRP substrate (Advansta, San Jose, CA, USA) was used to detect signals and digitalised using a Microchemi chemiluminescence system (DNR Bio-Imaging Systems, Ha-Satat, Israel). The antibodies used are listed in Table 2. Experiments were analysed with the Image-Studio Lite 5.2 software (LI-COR, Lincoln, NE, USA).