*2.7. Wound Healing Migration Assay*

The effect of deflamin on cancer cell migration was analyzed by the wound healing assay. CRC cell lines were seeded in a 24-well plate at a density of 1 × 105 cells/well and cultured until a monolayer of ~85% confluence was reached. A central scratch-like gap was created with a pipette tip and a medium containing increasing concentrations of deflamin (0, 20, 40, or 80 μg/mL) was added to each well. The migration ability of the cancer cells was assessed by the capacity to close the "wound" after 48 h of deflamin treatment. The cell-free area in the well was calculated with the Fiji/ImageJ software, and the relative values were calculated in percentage of control condition area values.
