Antimicrobial solution

HLE-B3 cells, a HLEC line immortalized by viral transformation of SV-40, were purchased from the American Type Culture Collection (Manassas, VA, USA). These cells were maintained in Minimum Essential Medium (MEM; Cat # 11095080; Lot No. 2120598; Gibco, USA) containing 10% fetal bovine serum and 1% antimicrobial solution (Sigma-Aldrich, St. Louis, MO, USA) in 5% CO₂ at 37 °C. In the entire experiment, when only media were used, they were described as normal media (NM), and hyperosmolar stress was applied by adding 50, 100, and 150 mOsm/L of NaCl to this baseline media. The osmolarity of the media itself could have a very important effect on this study, so we measured the osmolarity of the normal media using a 2430 Multi-Osmette auto-sampling turntable osmometer (Precision System Inc., Natick, MA, USA). As a result, the osmolarity of the normal media was measured as 291.5 ± 1.0 mOsm/L (n = 4). All cultures were grown to ~80–90% confluency prior to experiments. To induce hyperosmotic stress, sterile sodium chloride (1 M) was added to the culture media. Cells were incubated in hyperosmolar medium for 24, 48, or 72 h. An osmolarity range of 300–450 mOsm/L was selected based on previous data indicating that osmolarity in areas of tear breakup can reach up to 560 mOsm/L in the precorneal tear layer [26 (link)].

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).