Plasmin enzymatic activity assay

CASKI or C-33A cells were seeded (3×10⁴/well) onto 96-well plates in RPMI medium supplemented with 10% FBS. When necessary, 100 µg/mL cetuximab was added to the wells after seeding and incubated for 30 min, 37°C, 5% CO₂. Then, EGF was added to the wells of interest at 10, 50, or 100 ng/mL and the plates were incubated for 18 h at 37°C, 5% CO₂. The wells were washed twice with FBS/phenol red-free medium and uPA inhibitor (BC-11 hydrobromide, Abcam, USA) was added at a final concentration of 50 µM for approximately 15 min at room temperature. After BC-11 incubation, lys-plasminogen was added at 0.5 µM, and 250 µM of plasmin-chromogenic substrate S2251 (Diapharma, USA) was immediately pipetted to the wells. Plates were analyzed on a Spectramax 190 plate reader (Molecular Devices, USA) for 5 h, at 37°C, during which 405 nm reads were performed at 4 min intervals. All experiments were performed in duplicate.