Bicalutamide

LNCaP and MyC‐CaP cells were purchased from ATTC. PC3 and DU145 cell lines were a kind gift from M. Unseld (Medical University Vienna; Comprehensive Cancer Center). LNCaP, PC3 and DU145 cells were routinely maintained in normal RPMI medium containing 10% fetal bovine serum (FBS), phenol red, and antibiotics. MyC‐CaP cells were routinely maintained in normal Dulbecco's modified Eagle's medium (DMEM). For DHT stimulation experiments, LNCaP, MyC‐CaPs, PC3 and DU145 cells were seeded at 50–60% density in RPMI medium or DMEM, without phenol red, containing 5% charcoal‐stripped FBS (Sigma, Vienna, Austria). Transient transfections of all reporter and expression plasmids were performed with Turbofec (Thermo Fisher Scientific, Vienna, Austria), according to the manufacturer's instructions, on the second day of starvation. After 3 days of starvation, cells were stimulated with the indicated DHT concentrations and analyzed at the indicated time points. DHT and bicalutamide were purchased from Sigma.

Dimethyl sulfoxide (DMSO), (17b)-17-hydroxy-17-methyl-estra-4,9,11-trien-3-one (R1881), bicalutamide (BICAL) and dichlone (DCLN) were purchased from Sigma-Aldrich (St. Louis, MO). The 128 chemical test set used to evaluate the AR2 assay, and their respective sources, are listed in Supplementary Table S1. All control and test compounds were solubilized in DMSO, plated in Echo-qualified source plates (Beckman Coulter; Brea, CA), and stored sealed with adhesive foil in a desiccator at −80°C prior to experiments. All compound plates were thawed and stored in a desiccator away from light at ambient temperature 24 h before and between experimental replicates.

The PC3, 22RV1, and HEK293T cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). HEK293T cells were cultured in DMEM medium, while PC3 and 22RV1 cells were cultured in RPMI 1640 medium. To prepare the media, 10% foetal bovine serum and 1% penicillin and streptomycin solution were added before use. The cell incubators were maintained at 37°C and 5% CO₂. Bicalutamide (Sigma, #90357‐06‐5, ≥98%) was used at 10 μM for the treatment of 22RV1 cells.

Antibodies against AnxA6 as well as secondary anti-mouse, anti-goat, and anti-rabbit horseradish peroxidase-conjugated antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibody against HIF-1α was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Antibodies against HIF-2α and EGFR, were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against β-actin (ACTB) was purchased from Sigma Aldrich (St. Louis, MO, USA). Glucose uptake assay kit (Fluorometric; ab136956) and ATP Assay Kit (Colorimetric/Fluorometric; ab83355) were purchased from Abcam (Cambridge, UK). A set of EGFR-TKIs including lapatinib-ditosylate, erlotinib, gefitinib, and canertinib were purchased from BioVision Inc. (Milpitas, CA, USA). The AR antagonists bicalutamide and enzalutamide were purchased from Sigma Aldrich (St. Louis, MO, USA). Except otherwise indicated, all other reagents were purchased from Sigma Aldrich and/or Thermo Fisher Scientific (Waltham, MA, USA).

The human osteosarcoma cell lines, U-2OS (origin from a female 15y old osteosarcoma patient), SaOS, MNNG/HOS, and MG63 were purchased from the American Type Culture Collection (Rockville, MD) in 2014. The human osteosarcoma KHOS (origin from a female 13y old osteosarcoma patient) cell line was provided by Dr. Efstathios Gonos (Institute of Biological Research & Biotechnology, Athens, Greece) in 2008. KHOS and U-2OS are two commonly used cell lines in osteosarcoma research. The human osteoblast cell lines NHOst and HOB-c were purchased from LonzaWalkersville (Walkersville, MD) and PromoCell GmbH (Heidelberg, Germany) respectively in 2013. The osteosarcoma cell lines were cultured in RPMI-1640 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Life Technologies, Carlsbad, CA). The osteoblast cell lines were cultured in osteoblast growth medium (PomoCell) with 10% fetal bovine serum. All cells were maintained in a humidified atmosphere containing 5% CO₂ at 37 °C. Bicalutamide (Sigma-Aldrich, St. Louis, MO) is a synthetic, non-steroidal AR antagonist and a pure antiandrogen used in the treatment of prostate cancer, hirsutism, and other androgen-dependent conditions. Doxorubicin was purchased from APP pharmaceuticals, LLC, (Lake Zurich, Illinois).