Wound healing insert

Each cell group (CMB, PTX, PTX-CMB, CRISPR/Cas9-CMB and CRISPR/Cas9-PTX-CMB groups) was cultured in a Medium petri dish containing complete culture medium as aforementioned for 24 h at 37°C and 5% CO₂. The cells were trypsinized and centrifuged at 80 × g for 3 min at room temperature. After aspirating the supernatant, 1 ml serum-free medium [DMEM (Gibco; Thermo Fisher Scientific, Inc.) + 5% penicillin-streptomycin (Biosharp Life Sciences)] was used to resuspend the cells. Then, 190 µl serum-free medium was added to 10 µl resuspended cells, followed by thorough mixing. Then, 10 µl cell suspension was removed for counting. The density of HEC-1A cells was maintained at 7×10⁵/ml. Next, 70 µl cell suspension was placed in the wound-healing inserts (ibidi GmbH) and cultured in a 37°C incubator until the cells in the chambers on both sides of the wound-healing inserts covered the field of view. The scratch cell was pulled out vertically and the cells washed gently with PBS. Then, 1 ml serum-free medium was added and culturing allowed to continue. Images were recorded under a light microscope (magnification, ×100) every 4 h and stopped when the cells began to fuse. ImageJ (National Institutes of Health, v1.50i) was used to measure the healing area of each group of cells.

4T1 cells with defined cell-free gaps were prepared through the wound healing inserts (Ibidi, USA), followed by an incubation with CbTX, PL/CNC, VAP/CNC or pVAP/CNC at a CbTX concentration of 50 ng/mL for 48 h (n = 4). The cell migration in different groups were measured and calculated through an Image J 1.53e software (USA).