R1881

Cells were plated in T75 flasks to 75% confluence in media containing 10% FBS. Prior to treatment with R1881 (Sigma-Aldrich), the media was replaced with a 10% CCS supplemented media for 48 h. R1881 was added to a final concentration of 1 nM or 10 nM for 24 h. Protein extracts were collected in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% [v/v] Triton X-100, 1% [w/v] sodium deoxycholate,. One percent [w/v] Sodium dodecyl sulfate [SDS]) with protease inhibitors (Complete Mini, Roche, Switzerland). Western blot analysis was performed using the following antibodies: rabbit anti-human AR 1:1000 (Cell Signaling), mouse monoclonal anti-human beta-actin 1:1000 (Sigma). Membranes were revealed with HRP-linked goat anti-rabbit IgG 1:2000 (Cell Signaling) or horse anti-mouse IgG 1:2000 (Cell Signaling) using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA).

R1881 and S-(+)-Camptothecin were purchased from Millipore Sigma (Rockville, MD), enzalutamide was purchased from Selleck Chemicals (Houston, TX), and NLG207 was provided by NewLink Genetics (Ames, IA). All doses of NLG207 (mg) were measured using camptothecin (CPT) equivalents, or the mass of CPT contained within the nanoparticle formulation.

Hormone sensitive human PC cell line, LNCaP, cells were maintained in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum (FBS) (Corning) and 100 IU/mL penicillin plus 100 mg/mL streptomycin (Corning) at 37°C with 5% CO₂. Before any experiments, cells were cultured in RPMI 1640 supplemented with 10% charcoal stripped FBS (Gibco) for 72 hours.
The following drugs were used for drug treatment: docetaxel (DTX; Sigma Aldrich), dihydrotestosterone (DHT) analog methyltrienolone, R1881 (Sigma Aldrich).
The following antibodies were used for immunofluorescence staining: mouse monoclonal anti-CD45 directly conjugated with QDot 800 (ThermoFisher Scientific), rabbit monoclonal anti-ARN21, rat monoclonal anti-tyrosinated-tubulin (Millipore Sigma), and mouse monoclonal anti pan-cytokeratin (BioLegend) directly conjugated with CF594 (Biotium). All Alexa-conjugated secondary antibodies were from Invitrogen.

Human prostate cancer cell lines, the androgen independent cell lines PC3 and DU145, the CRPC cancer cell line 22Rv1, and the androgen sensitive human prostate cancer cell lines LNCaP and VCaP were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cell lines were obtained every year between 2010 and 2014 and have been authenticated and tested for mycoplasma in September 2011, June 2012, and November 2013, by Q11 short tandem repeat (STR; method of Masters et al. 2012: Authentication of human cell lines: standardization of STRprofiling; DDC Medical). PC3 AR variant transfectants PC3v7, PCv12 and PC3v567es were generated in this laboratory using plasmids provided by Drs. S. Plymate (University of Washington) and J. Luo (Johns Hopkins Brady Urologic Institute). TGF-β responsive LNCaPTβRII were generated and characterized in this lab (33 (link),34 (link)). All cell lines, but VCaP cells, were maintained in RPMI 1640 (Invitrogen, Grand Island, NY) and 10% fetal bovine serum (FBS), 100units/ml penicillin and 100μg/ml streptomycin in a 5% CO2 incubator (37°C). The VCaP cells were cultured in DMEM (ATCC, Manassas, VA). Cells were seeded in 10% charcoal-stripped serum (CSS) and stimulated by dihydrotestosterone (DHT) (Sigma-Aldrich, St. Louis, MO) or R1881 (1nM).