*4.6. Scratch-Wound Assay*

Cell migration assay was performed as previously described [44]. Briefly, HT-29 cells (3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL) were seeded into 6-well plates and 24 h later the culture medium was removed and straight scratch was created using a 200 μL sterile pipette tip. Cells were washed twice with PBS to remove cellular debris, replaced with appropriate complete culture media containing rHct-S3 (1, 2, and 4 μM) and incubated for 96 h. All experiments were repeated at least three times in each group. For the image analysis, cell migration into the wound area was photographed at the stages of 0 and 96 h using a microscope, Motic AE 20, and ImageJ software. The cells migration distance was estimated by measuring the width of the wound and expressed as a percentage of each control for the mean of the wound closure area.
