Transwell assay was used to analyze cell invasion and migration. Transwell chambers (Corning, Inc.) were precoated with (invasion) or without (migration) Matrigel (Becton, Dickinson and Company) at 37°C for 30 min. Following cell digestion using 0.25% trypsin solution, the culture medium was discarded by centrifugation (300 × g for 5 min at 37°C) and the cell pellet was washed with PBS 1–2 times. A549 cells were treated with shikonin (0, 10, 20 or 50 µM) for 24 h at 37°C. Next, cells were incubated in serum-free DMEM at a density of 5×105 cells/ml for 24 h at 37°C, then 100 µl cell suspension was added to the upper chamber of the Transwell plate. A total of 600 µl medium supplemented with 20% FBS was plated into the lower chambers. Following incubation for 24 h at 37°C, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and stained with 0.1% crystal violet for 10 min at room temperature. Finally, the cell numbers were counted under a light microscope (magnification, ×200) using ImageJ software.

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