Cells were treated with 10 μg ml−1 Mitomycin C (Sigma) for 1 h before the assay. Cells were rinsed once with 1 × PBS (Invitrogen), and treated with TrypLE (Invitrogen) for 5 min at 37 °C. TrypLE was neutralized with DMEM/F12 (Invitrogen)+10% FBS (HyClone). Cells were centrifuged at 1000 r.p.m. for 5 min, and resuspended in their respective basal media. A total of 20,000 cells was seeded onto the upper chamber of each transwell (BD). 1 ml culture media was applied to the lower chamber of each transwell. Migration assay was performed for 24 h in an incubator set at 37 °C in the presence or absence of MMP-9 Inhibitor I (10 nM), the metabolic modulators 2-DG (5 mM), FCCP (0.5 μM), Rot/AA (0.1 μM) and the chemotherapeutic compounds Nelarabine (10 μM), letrozole (10 μM) and capecitabine (10 μM). The cells attached onto the upper side of the transwell filter were removed by a cotton stick. The migrated cells attached onto the bottom side of the transwell filter were fixed with 4% PFA (Sigma), and visualized with 0.5% crystal violet
