Cell lines were cultured in either RPMI medium containing 10% fetal calf serum and 5 mM glutamine (PNT2C2, BPH1 and DU145) or Ham's F12 medium containing 7% fetal calf serum and 5 mM glutamine (PC-3). Normal primary prostate epithelial cells (CC-2555) were purchased from Lonza and cultured in prostate epithelial cell growth medium bulletkit (Lonza, CC-3166). Prostate tissue was obtained with informed consent from patients (Table 1 ) undergoing radical prostatectomy or trans-urethral resection for prostate cancer (TURP), with approval from the local Research Ethics Committee (07/H1304/121). Epithelial cultures were prepared as previously described (17 (link)) and cultured in complete keratinocyte growth medium supplemented with 2 ng/ml leukaemia inhibitory factor, 2 ng/ml stem cell factor and 100 ng/ml cholera toxin. Patient samples were all anonymized.
All cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2. After plating, cells were allowed to attach overnight before treatment with DMSO vehicle (‘untreated’) or the indicated concentration of ML-60218 (Cayman Chemical, Michigan, USA). RNAi was performed with Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's instructions, using Silencer Select siRNAs (Thermo Fisher).
All cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2. After plating, cells were allowed to attach overnight before treatment with DMSO vehicle (‘untreated’) or the indicated concentration of ML-60218 (Cayman Chemical, Michigan, USA). RNAi was performed with Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's instructions, using Silencer Select siRNAs (Thermo Fisher).