Giemsa

The monolayer cultured cells (250/ml) at logarithmic growth phase were collected, digested with 0.25% trypsin at room temperature for 3-4 min and pipetted into individual cells. The cells were suspended in a culture solution containing 10% FBS, and the cell suspension was diluted and then inoculated in DMEM. The culture was terminated when macroscopic clones appeared in the dish. The supernatant was discarded and the cells were carefully rinsed twice with PBS. At room temperature, acetic acid/methanol at 1:3 was used to fix the cells for 15 min. After removing the fixation solution, the cells were properly stained with Giemsa (Beyotime Institute of Biotechnology, Inc.) at room temperature for 25 min. The cell clones were counted with the naked eye and the colony formation rate was calculated as follows: Clonal formation rate = (number of clone formation/number of cells inoculated) ×100%.

For colony formation assay, cells were transfected for 48 h and plated into three 6 cm cell culture dishes (1,000 cells). Cells were incubated for 2 weeks in medium. Plates were washed with phosphate-buffered saline (PBS) and stained with Giemsa (Beyotime Biotechnology, Shanghai, China). The number of colonies with more than 50 cells was counted. The colonies were manually counted using a microscope.
For MTT assay, 24 h after transfection, cells were plated in 96-well plates at a concentration of ~2,000 cells per well and cultured for 5 days. For quantitation of cell proliferation rate, 20 μL of 5 mg/mL MTT (thiazolyl blue) solution was added to each well and incubated for 4 h at 37°C. The medium was removed from each well and 150 μL of dimethyl sulfoxide was added to the well. The plate was measured at 490 nm.

Two-dimensional cell migration was analyzed using a scratch wound assay. SMMC7721 cells were cultured in 6-well plate. The scratch was performed by using a 200 µl pipette tip to press firmly against the top of the tissue culture plate and swiftly make a vertical wound down through the cell monolayer. SMMC7721 cells were fixed with 70% ethanol and captured using a light microscope at 48 h after the scratch was made. The farthest distance that the cells migrated from the wound edge was measured by calculating the mean of three independent microscope fields in each of the three independent experiments.
Three-dimensional cell migration was determined using a Transwell migration assay as per the manufacturer's instructions (Corning Life Science, Corning, NY, USA). A total of 5,000 cells in serum-free DMEM were added into the top chamber. The bottom chamber contained 1% or 10% FBS in DMEM as a chemoattractant. After 24 h, the cells that had not penetrated the filters were scraped from the upper surface. The membrane was fixed with formalin, and the cells that migrated to the bottom surface of the filter were stained with Giemsa at room temperature for 5–10 min (Beyotime Institute of Biotechnology) and counted using a light microscope (magnification, ×200). An average of four randomly chosen fields were counted in each of the three independent experiments.

HCT-116 and HT-29 cells were seeded in triplicate in 96-well plates containing 100 µl of medium at densities of 3×10³ cells and collected for the MTT assay at time points ranging from 1–5 days. The cells were treated with 20 µl of MTT (5 mg/ml; Sigma-Aldrich) for 4 h, after which the MTT solution was removed. The crystals were dissolved in 200 µl of DMSO (Sigma-Aldrich). The absorbance of the solution was measured at 570 nm using a spectrophotometer.
HCT-116 and HT-29 cells were treated with 0.25% trypsin and dispersed into a single cell suspension in medium supplemented with 10% FBS (Gibco) prior to use. Cells were seeded into culture dishes at a density of 200 cells/dish and cultured in a 5% CO₂ atmosphere at 37°C for 2–3 weeks. When cell clones appeared, they were fixed with 4% paraformaldehyde for 10–30 min. Then, the cells were stained with Giemsa (Beyotime) for 10–30 min and counted under a light microscope.