HCC cells were cultured in 10 cm dish with serum-free medium for 24 h. Marigel (Corning) was even plated onto the upper chamber of a Transwell insert (Corning, NY, USA). 5 × 104 HCC cells were suspended with 100 μL of serum-free medium and seeded into the top chamber. The lower chamber was incubated with serum-contained medium. 24 h later, the inserts were fixed with 4% paraformaldehyde (PFA) for 30 min. Cells on the top surface of the inserts were removed and migrated cells on the lower surface of the inserts were stained with 0.05% crystal violet. Images of cells on the Transwell membrane were taken with a microscope and cell numbers were counted. Data were shown as mean ± SD of three independent experiments.

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