Cell proliferation, colony formation, and cell migration assays

Cell proliferation was determined using an MTT kit (Abcam, #ab211091) according the protocol provided by the manufacturer. Briefly, cells were seeded into 96-well plates with a density of 1×10³/well. After culturing at 37°C for 4 h (0 day), or 1, 2, 3, 4, and 5 days, cells in each well were incubated with 20 μL MTT reagent. The microplates were further incubated at 37°C for 4 h and cells were dissolved with acid-isopropanol. The absorbance was measured at OD₅₉₀ nm. For colony formation assay, cells (~500) were plated into 6-well plates and maintained in DMEM medium for 14 days, with a medium change every three days. The colonies were washed twice with PBS buffer, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet (Sigma-Aldrich, #C0775). The colonies were photographed, and colony numbers were counted manually. Cell migration assays were performed using the Boyden Chamber assay following a previous protocol ²². Briefly, a cell suspension in serum-free DMEM medium was loaded into the upper insert of the Boyden chamber (Sigma-Aldrich, #ECM550). The lower inserts were filled with DMEM medium containing 10% FBS. The entire chamber was incubated at 37°C for 24 h, and cells on the lower chambers were fixed with 4% paraformaldehyde, followed by staining with 0.1% crystal violet. The invading cells were photographed, and colony numbers were counted manually.