Smurf2 silencing inhibits colony formation. A soft agar assay was conducted to examine the colony formation ability of (A) MDA-MB-231 cells and (B) MCF-7 cells. Twenty-four hours after the siRNA transfection, MDA-MB-231 and MCF-7 cells were seeded in 0.35% agarose in DMEM medium supplemented with 10% FBS at a density of 1 × 103 per 35-mm culture dish and allowed to grow for 21 days. The dishes were stained with 0.01% crystal violet, and the colonies were examined with microscope. Results are representative picture of colonies of two independent experiments done in triplicate. (C), (D) The number of colonies of MDA-MB-231 and MCF-7 cells were counted and the result represented as mean ± standard deviation (n = 3). *P < 0.05 compared with control siRNA. NC-Negative control without siRNA.