Imagequanttl7.0 image analysis software

For CCK8 assay, HCT-116 cells and HCT-116 corrected clone cells were seeded into 96-well cell culture plates (5000 cells/well). After 24, 48, and 72 hrs, CCK8 solution (Beyotime, China) was added for an additional 4 hrs at 37°C. Absorbance at 450 nm was determined for each well using a microplate enzyme-linked immunosorbent assay reader.
For EDU dyeing, HCT-116 cells and HCT-116 corrected clone cells were seeded into 96-well cell culture plates (5000 cells/well). After 24 hrs, 25 µM 5-ethynyl-2ʹ-deoxyuridine (EDU) was added for an additional 2 hrs at 37°C. Then, the cells stain with Cell-LightTM Apollo Stain Kit (RIBOBIO, Guangzhou, China) after washed with PBS and fixed with 4% formaldehyde for 30 mins. Before stained with Hoechst (RIBOBIO, Guangzhou, China) for 15mins, the cells were permeabilized with 0.5% Triton X-100 (Solarbio, Beijing, China) for 15 min. Images were obtained using a fluorescent microscopy (BX-51, Olympus Corporation, Tokyo, Japan).
For clonogenic assay, cells were plated onto 6-well plates (500 cells/well). Plated cells were cultured in 5% CO₂ with 95% humidity at 37°C for 14 days. The plates were then fixed with 4% formaldehyde and stained with Giemsa. The colonies containing more than 50 cells were counted using ImageQuantTL7.0 Image Analysis Software (GE Healthcare, Buckinghamshire, UK).

The clone formation test was performed to measure cellproliferation. MDA-MB-231^NC and MDA-MB-231^KD cells were cultured in 6-well plates at a density of 200 cells/well for 2 weeks. Cell clones were immobilized with methanol and stained with crystal violet at room temperature for 15–20 min. Colonies containing >50 cells were counted using CKX41 Inverted MicroScope (OLYMPUS Corporation, Tokyo, Japan) at ×10 magnification and ImageQuant TL7.0 Image Analysis software was applied to image analysis (GE Healthcare Life Sciences, Little Chalfont, UK).