Metformin Modulates Gelatinase Activity

Gelatinase activity was detected in HEK293A supernatants and recombinant human MMP-9 protein (911-MP; R&D Systems Incorporated, Minneapolis, MN, USA) after metformin incubation for 24 h. Zymography was performed according to the manufacturer’s instructions (Applygen, P1700, Beijing, China). Following electrophoresis, the gels were washed twice with 2.5% Triton X-100 to remove sodium dodecyl sulfate and further washed with 50 mmol·L⁻¹ Tris–HCl pH 8.0. Gels were incubated for the following 20 h in an activation buffer (50 mmol·L⁻¹ Tris–HCl supplemented with 5 mmol·L⁻¹ CaCl₂). The gels were stained with Coomassie brilliant blue R-250 and de-stained with 20% methanol and 10% acetic acid in distilled water until clear bands were visualized.