2.11. Wound Healing Assay

A549, H292, and H460 cells were seeded in six-well plates at 1 × 10⁵ per well and incubated in RPMI-1640 for 24 h. After becoming a confluent monolayer, the cell layers were scratched with a pipette tip and washed with PBS to remove cell debris and immediately treated with silibinin for 48 h, except control cells. Using microscopy, photos were taken at different time intervals to evaluate the wound edges, and the relative area of wound closure was measured using ImageJ software (NIH Image, Bethesda, MD, USA).