Gelatin zymography

To measure the gelatinase activity of the experimentally manipulated cell culture, 2 × 10⁶ cells were first switched to serum-free DMEM/F12. After 24 h, the conditioned media were collected and concentrated by Amicon Ultra-15 centrifugal filter (Merck Millipore). Subsequently, all samples were run on 8% SDS-PAGE with 0.1% gelatin. Gels were washed twice with 2.5% Tritox X-100 for 30 min at room temperature, and then incubated with development buffer at 37°C overnight. Lastly, gels were stained with Coomassie blue to determine the MMP2 and MMP9 activity.