Cell Line Cultivation and Treatment

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.