Densitometric image analysis software

Proteins obtained from lysate tissue after 24 and 48 h were size fractioned in 4–12% SDS-polyacrylamide gel, before being transferred to PVDF membrane (GE Healthcare, U.K.) by using Trans-Blot Turbo System (Bio-Rad, U.S.A.). Membranes were blocked for 2 h with 5% bovine serum albumine (BSA) in transfer buffer saline (TBS: 2.5% Tris-HCl, 8% NaCl, 0.1% Tween 20, pH 7.4) at room temperature. Afterward the membranes were incubated in 3% TBS–BSA with primary antibodies overnight at 4°C (Supplementary Table S2).
After two washes in PBS 1×, the membranes were incubated with the secondary conjugated antibodies antimouse Cy3 or antirabbit Cy5 (GE, U.S.A.) diluted 1:2500 in 3% TBS-BSA for 2 h (Supplementary Table S2). Immunolabeling was visualized using the ECL Plex procedure (GE) and the laser scanner Pharos FX (Bio-Rad). Bands were quantitated using densitometric image analysis software (Bio-Rad) or ImageJ software. Molecular mass was determined using a wide range protein marker 8–200 kDa (Bio-Rad). Protein loading was controlled by antiactin (Sigma) detection and was statistically evaluated.

MMP2 and MMP9 activity were measured in 24 h culture media by zimography assay. After centrifugation (300 g for 10 min), the supernatant was separated and the protein concentrations were measured using Bradford protein assay. Total 20 µg of proteins per lane were added to the sample buffer (1 M Tris-HCl, pH 6.8, 2% sodium dodecyl sulphate (SDS), and 10% glycerol] and loaded onto a 10% SDS–polyacrylamide gel containing 1 mg/ml gelatine porcine (Sigma). Gel was stained in 0.1% Coomassie Brilliant Blue R-250 for approximately 1 h and destained until the gelatino-lytic bands were clearly seen. The MMP activities, indicated by clear bands of gelatin digestion on a blue background, were quantitated using densitometric image analysis software (Bio-Rad).