A549 and H446 cells were digested using 0.25% trypsin solution (Gibco; Thermo Fisher Scientific, Inc.) and cultured in a 6-well plate at a density of 5×105 cells/well overnight at 37°C. Upon cells reaching 90% confluence, a single vertical scratch was made in the cell monolayer with a 10-µl micropipette tip. The cells were washed with PBS thrice to remove the detached cells. Subsequently, A549 and H446 cells were cultured in serum-free DMEM with/without shikonin (0, 10, 20 or 50 µM) at 37°C or transfected A549 cells were cultured in serum-free DMEM with 50 µM shikonin at 37°C with 5% CO2 in an incubator. At 0 h and following 24 h of incubation, the wound area was visualized and photographed using a light microscope (magnification, ×100) and the migration was analyzed using ImageJ software (version 1.8.0; National Institutes of Health).

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