Dmem ham s f 12 with l glutamine

HMVECs (Catalog #: CC-2543) and HUVECs (Catalog #: C2519A) were purchased from Lonza (Slough, U.K.). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 with l-Glutamine (Sigma–Aldrich; Merck KGaA), supplemented with 10% FBS (Sigma–Aldrich; Merck KGaA) and antibiotics (Sigma–Aldrich; Merck KGaA). Cells were maintained at 37°C and 5% CO₂ in a humidified incubator.

PC-3, LNCaP, VCaP, CAHPV-10, PZHPV-7, hFOB1.19, MDA-PCa-2b and DU-145 cell lines were purchased from the American Type Culture Collection (ATCC; Middlesex, UK). Human HECV endothelial cells were purchased from Interlab Cell Line Collection (ICLC; Genoa, Italy). PC-3, LNCaP, VCaP, HECV and hFOB1.19 cells were used in functional assays. PC-3, VCaP and HECV cells were grown in Dubecco’s Modified Eagle Medium (DMEM)/Ham’s F12 with L-glutamine (Sigma-Aldrich, Dorset, UK) and LNCaP cells were cultured in RPMI 1640 medium (Sigma-Aldrich, Dorset, UK). Human hFOB1.19 osteoblasts were maintained in DMEM/Ham’s F-12 without phenol red (Life Technologies, Paisley, UK) containing 0.3mg/ml G418 (Melford Laboratories, Suffolk, UK). All base mediums were supplemented with 10% foetal calf serum (FCS) (Sigma-Aldrich, Dorset, UK) and an antimicrobial solution (Sigma-Aldrich, Dorset, UK). Cells were cultured at 95% humidity, 5% CO₂ and 37° C except hFOB1.19 which were cultured at 34° C.
HGF was a kind gift from Dr T Nakamura (Osaka University Medical School, Japan). Recombinant human ALCAM, comprising a fusion of ALCAM Trp28 - Ala526 and the human IgG Fc region (ALCAM-Fc chimera) was purchased from R&D systems (Abingdon, UK).

The human breast cancer cell lines including T47D and SK-BR-3 were obtained from ATCC (Middlesex, United Kingdom). The cells were subcultured at 37°C, 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM/Ham’s F-12 with L-Glutamine) (Sigma-Aldrich, Dorset, United Kingdom) supplemented with 1× antibiotics penicillin and streptomycin (Sigma-Aldrich, Dorset, United Kingdom) and 10% fetal bovine serum (FBS) (Sigma-Aldrich, Dorset, United Kingdom).