Colony formation assay

The human breast cancer cell lines were treated with different concentrations of SL4 for 24 h. Then the cells were washed in PBS, seeded in 35 mm dishes and cultured for an additional 2–3 weeks. Finally, the cells were stained with 0.5% crystal violet solution after washing with PBS and fixing with paraformaldehyde, and the colonies (>50 cells) were counted under an inverted microscope. Treatments were carried out in triplicate. The colony formation inhibition rate was calculated as the following formula: Inhibition rate = (1 − various concentrations of colony forming number/control group of colony forming number) * 100%.