Crystal violet

The two transfected or control groups were planted into six‐well plates at 1.5 × 10³ cells and preserved in RPMI 1640 containing 10% FBS for 8‐14 days. Afterwards, the cells were rinsed with phosphate‐buffered saline (PBS), fixed with 4% PFA (paraformaldehyde Sigma, USA) for 30 minutes. Finally, plates were stained with crystal violet (Sinopharm Chemical Reagent, Beijing, China). All the assays were performed in triplicate.

Transwell chambers with polycarbonate filters (Corning Cat. No. 3464, 24-well insert, pore size: 8 μm) were used for cell migration assays. The upper section was seeded with 3 × 10⁴ SW620 cells or 1 × 10⁴ LoVo cells in a serum-free medium (150 μL). The lower chamber was filled with medium (500 μL) containing 20% FBS. The plates were subsequently placed in a 37°C incubator for 48 hours. After incubation, non-migrated cells on the upper surface of the chamber were removed using a cotton-tipped swab. The migrated cells on the lower surface of the section were fixed with methanol, stained with 0.5% crystal violet (Sinopharm Chemical Reagent, Shanghai, China), and counted under a microscope. The number of migrated cells was quantified using Image J software (NIH, Bethesda, MD, USA).

After transfecting SW620 and LoVo COAD cells with pcDNA3.1-3×HA-KLF7 or siKLF7 for 48 hours, the cells were seeded at a density of 1,000 cells per well into 96-well plates and cultured for 0, 1, 3, 5, or 7 days. The 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reagent (5 mg/mL; Sigma) was added to each well, and the cells were incubated for four hours. After incubation, the MTT crystals were dissolved in DMSO and the absorbance was measured at 490 nm using the iMark Microplate Absorbance Reader (Bio-Rad, USA).
To assess cell clones' formation, transfected cells were seeded into 6-well plates at a density of 500 cells per well. The culture medium was changed every five days. After 14 days of culture, the cells were fixed with methanol for one hour, stained with 0.5% crystal violet (Sinopharm Chemical Reagent, China) for one hour, and photographed the colonies using a digital camera. We then counted the number of colonies using Image J software (NIH, Bethesda, MD, USA).

SW620, LOVO or HCT116 cells were plated in 6-well plates at 1 × 10³ cells per well and maintained in DMEM containing 10% FBS for 2 weeks with the medium replaced every 4 days. After 2 weeks, the colonies were washed with PBS for 2 times, fixed with methanol and stained with crystal violet (Sinopharm Chemical Reagent, China). The number of colonies was counted under a microscope.

SW620 and LOVO cells were transfected with miR-600 mimic or NC, and HCT116 cells were transfected with miR-600 inhibitor. After 72 h, 3 × 10⁴ cells were collected and placed in 100 μl serum-free DMEM medium in the upper chamber coated with Matrigel (BD, USA). DMEM medium supplemented with 10% FBS was added into the lower chamber. Twenty-four hours later, cells that migrated through Matrigel were fixed with methanol and stained with crystal violet (Sinopharm Chemical Reagent, China) for 15 min. To observe the invasion ability, cells were incubated for 48 h. Image J was used to quantify the number of migration and invasion cells.