Pd173074

Murine mesothelioma AB12 cells (Sigma-Aldrich St. Louis, MO, USA) and human mesothelioma H28 cells (#CRL-5820, American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in RPMI 1640 medium (Lonza, Verviers, Belgium) supplemented with 1% L-Glutamine, 1% penicillin-streptomycin, 25 mM HEPES and 10% fetal bovine serum (FBS) (Gibco, Paisley, UK). Human non-cancerous mesothelial Met5A cells (#CRL-9444, ATCC) were cultured in Medium 199 containing 1.5 g/L sodium bicarbonate (Gibco) supplemented with 3.3 nM EGF, 400 nM hydrocortisone, 1% penicillin-streptomycin, 20 mM HEPES and 10% FBS. Primary Mesothelioma 27 (PM27) cells, generated as described below (see ‘Primary cancerization mouse model and isolation of primary mesothelioma cells’ section), were cultured in DMEM (Gibco) supplemented with 1% L-Glutamine, 1% penicillin-streptomycin and 10% FBS. Cell cultures were incubated at 37 °C in a 5% CO₂ atmosphere and routinely tested for mycoplasma contamination. GI254023X (Sigma-Aldrich) [42 (link)], ionomycin (Sigma-Aldrich) and PD173074 (Abcam, Cambridge, UK) were prepared in DMSO (vehicle). For assays in presence of GI254023X or vehicle, mesothelioma cells were pretreated 16 h before the experiment. Recombinant mouse N-cadherin/Fc Chimera Protein was reconstituted in PBS (R&D Systems, Minneapolis, USA).

NmFGF1 was produced in Escherichia coli and purified to be endotoxin free as previously described (Wu et al., 2005 (link)). The FGFR1-specific inhibitor PD173074 was purchased from Abcam (Cambridge, MA, USA). S1P1 antagonist VPC23019 was purchased from Cayman Chemical (Ann Arbor, MI). The primary antibodies applied in this study including anti-FGFR1 (No. ab824), anti-p-FGFR1 (No. ab59194), anti-S1P1 (No. ab11424), anti-CD31 (No. ab28364), and anti-β-Actin (No. ab8227) were purchased from Abcam (Cambridge, MA, USA), and anti-FGF1 (No. BM5544) was obtained from Bosterbio (Pleasanton, CA, USA). The secondary antibody used were goat anti-rabbit IgG H&L (HRP) (No. ab6721), and Alexa Fluor ^®488-conjugated donkey anti-rabbit (No. ab150073) which were also purchased from Abcam (Cambridge, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and S1P1 agonist FTY720 were purchased from Sigma-Aldrich (St. Louis, MO, USA). EBM-2 medium was purchased from Lonza (Hopkinton, MA, USA). Matrigel matrix was obtained from Corning Inc (Tewksbury, MA, USA). All other chemicals were of analytical-reagent grade.