Silencing of IKKε inhibits focus formation and colony formation. Forty-eight hours after the siRNA transfection, (a) SK-BR-3 cells and (b) MCF-7 cells were seeded in six-well plates, and the medium was changed every 2 days. Cells cultured for 9 days were washed twice with ice-cold medium, fixed with ice-cold methanol, and stained with 0.2% crystal violet. Images of the colonies were obtained with a digital camera. (c), (d) The focus number was counted and the result represented as mean ± standard deviation (n = 3). (e) A soft agar assay was conducted to examine the colony formation ability of MCF-7 cells. Twenty-four hours after the siRNA transfection, MCF-7 cells were seeded in 0.35% agarose in RPMI-1640-supplemented 10% FBS at a density of 1 × 104 per 35-mm culture dish and allowed to grow for 14 days. Results are representative picture of colonies of two independent experiments done in triplicate. *P < 0.05. NC, negative control siRNA.