Ecl plus chemiluminescence kit

Total (100 μg) or nuclear (50 μg) protein extracts were prepared from cells treated as follows. (i) The cells were exposed to 1 μM dAza for 1 or 2 days (24 h apart), in the presence or the absence of HGF 200 ng/ml.^{4 (link), 50 (link)} For HGF treatment, the cells were starved overnight. For 30 days dAza treatment, 0.1 μM dAza was added to the cells every three days^{50 (link)}; corresponding control cells (c30) were carried out. (ii) Some cells were transfected with 400 ng/ml of WWOX e.v. (S. Semba, Kobe University, Japan)^{4 (link)} during the last 1 day of the dAza treatments. (iii) Some cells exposed to 1 day or 30 days dAza, and the respective controls, were treated with CHX (100 μg/ml) for various times,^{6 (link)} and the Western blots were performed with total or nuclear protein extracts. Primary antibody dilutions were: anti-Met (C-12, 1 : 200) (Santa Cruz Biotechnology), anti-phosphoMet 1234/35 (catalytic site) (1 : 2000) (Transduction Laboratories), anti-αHGF (C20, 1.5 μg/ml) (Santa Cruz Biotechnology), anti-Wwox (N19, 1 : 200) (Santa Cruz Biotechnology) and anti-phosphoWwox (1 : 500) (Abcam, Cambridge, UK). Densitometric analysis was performed after reaction with ECL plus chemiluminescence kit from Thermo-Fisher Scientific (Waltham, MA, USA).

Western blotting assays were performed as previously described (73 (link), 74 (link), 75 (link)). Briefly, cells were lysed with ice-cold radioimmunoprecipitation assay lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1% Triton X-100), and the concentrations of the extracted proteins in supernatant were quantified by bicinchoninic acid protein assay kit (Beyotime Biotech). About 30 μg of each protein sample was loaded on SDS-PAGE gel. After the SDS-PAGE electrophoresis, proteins were transferred on a polyvinylidene difluoride membrane, and blocked with 5% skim milk in Tris-buffered saline with Tween-20 (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween-20). Specific proteins were probed with the relevant primary antibodies followed by secondary antibodies. The immunoblots were visualized with ECL plus Chemiluminescence kit (Thermo Fisher Scientific). The antibodies used are listed in Table S4.

Some flasks containing 1833 cells infected with miR-125b mimic were exposed to hypoxia^{50 (link)}, or to 100 ng/ml HGF under starvation^{9 (link)}, and total and nuclear protein extracts were prepared. To evaluate ETS1, HIF1A and PTGS2, immunoblots were performed with anti-Ets-1 (1:2000), anti-HIF-1α (clone54) (1:350, BD-Transduction Laboratories, Franklin Lakes, NJ, USA), and anti-COX2 (1:100). Also, anti-E-cadherin (1:1000, clone 36 Transduction Laboratories, Bedford, MA, USA), anti-MMP2 (1:500, Novus Biologicals, Abingdon, UK), anti-vimentin (1:500 Santa-Cruz Biotechnology), anti-SPARC (1:200, H-90 Santa Cruz Biotechnology), anti-osteocalcin (5 μg/ml, Abcam, Cambridge, UK), anti-B23 (1:1000, H106 Santa Cruz Biotechnology), or anti-vinculin (1:1000, Cell Signaling, Leiden, The Netherlands) was used for immunoblotting. Densitometric analysis was performed after reaction with ECL plus chemiluminescence kit from Thermo-Fisher Scientific (Waltham, MA, USA) or Clarity Max Western ECL Substrate-Luminol Solution (Biorad, Hercules, CA, USA).