2.10. Wound healing—cell migration assay

Wound healing assays were utilized to test cell migration activities. KHOS and U2OS cells were seeded into 6‐well plates at a density of 4 × 10⁵ cells per well and incubated at 37 °C overnight. A sterile 30 µL tip was then used to scrape two parallel lines within the adherent cell layer. Next, 10 nm of OTS514 was added and left to incubate for 72 h in a low‐serum medium containing 2% FBS. Pictures of wounds were captured using a microscope (Nikon Instruments Inc.) with NIS‐Elements platform after 0, 24, 48, and 72 h of OTS514 treatment. The distance between the two edges of the scratch at five different sites of each image was measured to represent the average wound width. The following formula was applied to calculate the cell migration distance: (wound width at 0‐hour time point – wound width at the observed time point)/2.