Development buffer

Zymography was performed in 10 % gelatin polyacrylamide gel (Novex; Life Technologies). Medium samples (10 μl each) were mixed with zymogram sample buffer (BioRad, Hercules, CA, USA) and subjected to SDS-PAGE. The gels were then equilibrated with renaturation buffer (BioRad) and incubated with development buffer (BioRad) overnight at 37 °C. Bands were visualized by staining gels with Simply Blue Safe Stain (Invitrogen, Themo Fisher, Grand Island, NY, USA). At least two replicates were carried out for each sample analyzed by zymography. The MMP2 and MMP9 bands were digitally imaged and semiquantified by Image J 1.45s (NIH, Bethesda, MD, USA).

Zymography assay was performed to examine the MMP activity. In brief, indicated cells were cultured in serum-free DMEM medium for 48 h before the culture media were collected and centrifuged. Equal amounts of supernatants were added with equal amounts of 2× Zymogram sample buffer (Bio-Rad, Hercules, CA) and subjected to Ready Gel Zymogram gels (Bio-Rad) under nonreducing condition. After electrophoresis, the gel was renatured in renaturation buffer (Bio-Rad) and developed in development buffer (Bio-Rad). Finally, the gels were stained with 0.5% Coomassie brilliant blue R-250 and photographed.

Samples for gelatin zymography were prepared following Western blot procedures, but differed in sample preparation, using the zymogram sample buffer (Bio-Rad Laboratories, Hercules, CA, United States). About 40 μl of the sample was loaded per lane onto a polyacrylamide gel containing 0.1% gelatin (Sigma-Aldrich) for electrophoresis. Then, the gel was washed in renaturation buffer (Bio-Rad Laboratories) for 1 h. After rinsing with distilled water 10 times, the gel was incubated in development buffer (Bio-Rad Laboratories) at 37°C for 48 h. Staining was performed using Coomassie Brilliant Blue R-250 staining solution (Bio-Rad Laboratories) for 1 h and followed by a destaining solution (10% acetic acid and 40% methanol). The activity of the matrix metalloproteinases was demonstrated as clear zones in a blue background. Quantification analysis of the intensity was measured using Image J software (Image J 1.4) (Egashira et al., 2015 (link)). The activity of MMPs was represented as the intensity ratio of active form/proform.