After being treated with drug materials for 24 hours under hypoxia condition (1.0% O2), 4 × 104 cells were collected with serum-free medium containing 0.2% BSA and seeded into upper chamber, while the lower chamber was filled with 600 μl of medium containing 2.5% FBS. After that, cells migrated for about 10 hours and washed twice with ice-cold PBS. Before being imaged and calculated, cells on the lower surface of the film were fixed, washed, and stained with 0.1% crystal violet, while cells on the upper surface were cleared. The Transwell plate (3422) was obtained from Corning.

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