Dihydrotestosterone dht

The prostate adenocarcinoma cell line LNCaP, castration-resistant adenocarcinoma cell line C4–2, and AR-suppressed prostate cancer cell line PC3 (25 (link)) were obtained from the ATCC (MD, USA) and cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). The RasB1 cell line (an aggressive cell line expressing a constitutively active Ras in DU145 cells and isolated from a bone metastasis) was provided by Dr. Kathleen Kelly (NCI/NIH, MD, USA) and maintained as described previously (24 (link),26 (link)–28 ). The small-cell neuroendocrine carcinoma (SCNC) cell line NCI-H660 was purchased from the ATCC and cultured in RPMI 1640 medium supplemented with 0.005 mg/ml insulin (Sigma-Aldrich), 0.01 mg/ml transferrin (Sigma-Aldrich), 30 nM sodium selenite (Sigma-Aldrich), 10 nM hydrocortisone (Sigma-Aldrich), 10 nM ß-estradiol (Sigma-Aldrich), 4 mM L-glutamine (Invitrogen), and 5% FBS. Dihydrotestosterone (DHT) (Sigma-Aldrich) and LIF protein (R&D Systems) were used to treat cells at 10 nM and 200 ng/ml, respectively, for 24 h in a 10% charcoal-stripped serum (CSS)-containing medium. The AR antagonist enzalutamide (MDV3100) (Selleck) and the first-in-class steroidal LIF inhibitor EC330 (MedChemExpress) were used to treat cells at concentrations of 10 μM and 35 nM, respectively, for 24 h in 10% FBS-containing medium.

Human TNBC cell lines were cultured in 5% carbon dioxide (CO₂). Luminal AR MDA-MB-453 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in DMEM with 10% FBS. SUM159PT cells, expressing moderate levels of AR [35 (link)], were obtained in 2013 from the University of Colorado Cancer Center (UCCC) Tissue Culture Core (Aurora, CO) and maintained in Ham’s/F-12 with 5% FBS, 1% HEPES, 1 μg/mL hydrocortisone and 5 μg/mL insulin. Only cells of under ten passages were used in this study. All cell lines were routinely tested for mycoplasma contamination, and human cell lines were authenticated in 2017 by short tandem repeat analysis in the UCCC Tissue Culture Core. The androgen dihydrotestosterone (DHT; Sigma-Aldrich Corporation, St. Louis, MO) used for in vitro experiments was diluted in ethanol (EtOH). The AR and CYP17 lyase inhibitor seviteronel (Sevi) was provided by Innocrin Pharmaceuticals, Inc. (Durham, NC). The CDK4/6 inhibitor Abemaciclib (Abem) was purchased from Selleck Chemical LLC. (Houston, TX). All drugs were diluted in dimethyl sulfoxide (DMSO).

Fresh tissue samples were transported in ice-cold saline and immediately dissected into portions for fixation in 10% formalin followed by paraffin embedding. For human specimens, a 4-hour enzymatic digestion into single cells was performed at 37°C in 35 mL Hanks’ balanced salt solution (HBSS) containing 5 mg/mL Collagenase Type I (Life Technologies), 10 μM ROCK Inhibitor Y-27632 (StemRD), 1 nM dihydrotestosterone (DHT) (Sigma-Aldrich), 1 mg DNAse I (Roche), and 1% antibiotic/antimycotic solution (100×; Corning).^{4 (link)} Mouse specimens were digested for 1 hour in HBSS containing either 10 mg/mL cold protease or 1.5 to 2 mg/mL Collagenase Type I, plus 10 μM ROCK Inhibitor Y-27632, 1 nM DHT, 1 mg DNAse I, and 1% antibiotic/antimycotic solution.