Gelatin Zymography Assay

Gelatin-coated polyacrylamide gel electrophoresis for MMP-2 and MMP-9 activity assay was performed. After any relevant treatment, the cell medium was centrifuged and the supernatants were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 10% gels with 1 mg/mL gelatin (Sigma-Aldrich) at 100 V for 2 hours at 4°C. After electrophoresis, the gels were washed twice in 2.5% Triton X-100 (Sigma-Aldrich) for 30 minutes at room temperature and then incubated for 16 hours in zymography developing buffer containing 50 mM Tris-HCl and 10 mM CaCl₂ (pH 7.5; Sigma-Aldrich). The gels were subsequently stained with Commassie Brilliant Blue R-250 and band densities were determined by the Quantity One 4.6.3 software (Bio-Rad Laboratories, Inc, Berkeley, CA).