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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.

The PCa lines PC3 (CRL-1435), 22RV1 (CRL-2505) and NCI-H660 (CRL-5813) were purchased from American Type Culture Collection (ATTC-HTB-81). PC3 and 22RV1 were cultured in Roswell Park Memorial Institute medium (RPMI, BE12-702F, Lonza, Levallois-Perret, France) supplemented with 5% (PC3) or 10% (22RV1) fetal bovine serum (FBS) (CVF5VF00-01, Eurobio, Les Ulis, France) and 1% penicillin-streptomycin (PS). NCI-H660 were grown in RPMI 1640 Medium (ATCC modification, A10491-01) supplemented with: insulin (0.005 mg/mL); Transferrin (0.01 mg/mL); Sodium selenite (30 nM); Hydrocortisone (10 nM); β-estradiol (10 nM); L-glutamine (2 mM); 5% FBS and 1% PS. All cell lines were maintained in 37 °C humidified incubator with 5% CO₂. The positive allosteric modulator of the CaSR, R-568 (SML2160), was purchased from Sigma-Aldrich (St-Quentin Fallavier, France). The CaSR inhibitor, calcilytic NPS-2143 hydrochloride (362610), was purchased from Tocris Bioscience (Bristol, United Kingdom).