Crl 3314

PCa cells lines LNCaP (CRL-1740), and second-generation LNCaP subline: C4-2 (CRL-3314) and C4-2B (CRL3315) (B for Bone Metastatic) [16 (link)] were purchased from American Type Culture Collection (ATCC). LNCaP cells were cultured in Roswell Park Memorial Institute medium containing D-Glucose (4.5 g/L), HEPES Buffer (2383 g/L), L-Glutamine, Sodium Bicarbonate (1.5 g/L), sodium pyruvate (110 mg/L) (RPMI, A1049101, Lonza, Levallois-Perret, France) and supplemented with 10% fetal bovine serum (FBS) (CVF5VF00-01, Eurobio, Les Ulis, France) and 1% penicillin-streptomycin (PS). C4-2 and C4-2B cells are cultured in RPMI medium (BE12-702F, Lonza, Levallois-Perret, France) supplemented with 5% FBS and 1% PS. Cells were maintained in a 37 °C humidified incubator with 5% CO².
The non-steroidal AR inhibitor, Enza (PHB00235) and the SK3 positive modulator CyPPA (Cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine) were purchased from Sigma-Aldrich (St-Quentin Fallavier, France). 1-Ohexadecyl-2-O-methyl-sn-glycero-3-lactose (Ohmline) was synthetised as previously described [17 (link)]. For one-week treatments, cells were treated 3 times per week with Enza, Enza+Ohmline or CyPPA.

In vitro and in vivo experiments were performed using the PSMA-positive C4-2 cell line, a subline of the LNCaP (lymph node carcinoma of the prostate) cell line (CRL-3314; American Type Culture Collection). C4-2 cells were cultivated in RPMI-1640 (PAN Biotech) supplemented with 10% fetal calf serum and stable glutamine (PAN Biotech). Cells were grown at 37 °C and incubated in humidified air equilibrated with 5% CO₂.