SP and non-SP fractions in LC-42 lung cancer cells. A. One representative experiment of the side population in LC-42 cell line as compared to the blocking control with verapamil. B. Sorted SP and non-SP cells were cultured in Defined Keratinocyte-SFM medium supplemented with EGF and FGF, only SP cells have capability of forming spheres in serum-free medium (a). These spheres began to attach to the bottom of the wells during the 2nd week under the same culture condition, three distinct patterns of colony morphologies, which were holoclone (a), meroclone (b) and paraclones (c), could be easily identified. Non-SP cells were not able to form cell sphere. Representative colony images were acquired at 40× magnification. C. Comparison of the expression pattern of stem cell related genes between SP and non-SP cells by real-time PCR analysis, indicating similar levels of the expression of these genes in SP cells compared to non-SP cells. The error bar reflected the variation within the triplicates. D. Colony-forming ability of sorted SP and non-SP LC-42 cells at different cell seeding numbers using soft agar clonogenic assay. The numbers of colonies formed from each cell population were compared at different cell seeding numbers. The clonogenic ability showed significant difference between SP and non-SP cells at lower cell seeding number of 10 cells per tube, showed no significant difference at higher seeding density. The mean value reflected the average of three individual analyses.