Soft-agar colony formation assay

A series of MCF-10A cells (2 x 10^4 per well) were mixed with 0.3% agarose, low gelling temperature (Cat#35640, SIGMA) in the full growth medium, plated on top of a solidified layer of 0.6% agarose in full growth medium [25] in a 6-well plate, and fed every 3 days with full growth medium. Photographs were taken by an OLYMPUS CKX53 inverted microscope with a DP20 digital camera (OLYMPUS). Finally, the colonies were stained with MTT (1 mg/ml in PBS solution) and imaged using an EPSON GT-X900 scanner. The images were analyzed with ImageJ (Fiji), extracting the number of colonies that exceeded a certain threshold intensity. When we combined soft agar assay with the siRNA experiment, 1 nM siRNA-treated cells were embedded in the soft agar, fed only one week later and cultured for 2 weeks. MCF-10A cell lines were transfected with 1 nM siPOOLs, maintained for 2 days in partial growth medium, and then 2 x 10^4 cells were embedded in full growth medium-based soft agar. In the case of cancer-derived cell lines, cells were first transfected with 1 nM siPOOLs in RPMI supplemented with 10% FBS. One day after transfection, the cells were embedded in the soft agar based on RPMI supplemented with 10% FBS. The cell number embedded in the soft agar is as follows: A549, 2 x 10^4 cells; H358, 4 x 10^4 cells; A375, 1 x 10^4 cells.