2.6. Colony Formation Assay

MDA-MB-231 cells were seeded in 12-well adhesive culture plates at a density of 800 cells/well and allowed to attach overnight. The cells were cultured for 15 days at 37 °C in standard growth media and incubated with different concentrations of usenamine A (5, 10, and 20 μM) to assess colony growth. The colonies were washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and stained with 0.04% crystal violet for 15 min. After washing twice with distilled water, the colonies were observed under a light microscope. The colonies were counted in three independent experiments [22].