Proliferation and tumorigenesis of the CSC+ (or CSC−) cells from different generations are comparable.

(A) Schematic diagram shows the cell classification based on generation and CSC marker expression. Cells from the original tumor cell lines were arbitrarily classified as generation 1 (G1) cells and sorted into CSC marker positive (G1+) and negative (G1−) cells. G1+ and G1− cells were then propagated (from 1×102 to approximately 1×108) to generate G2 (including G1+G2+, G1+G2−, G1−G2+ and G1−G2−) cells. We repeated the procedure until G3 cells were obtained. (B and C) CSC+ and CSC− cells from different generations were used for proliferative and tumorigenic assays. Histograms show the percentage of Ki-67 positive cells, the percentage of Annexin V+ 7-AAD− cells, and the frequency of tumorigenic cells of CSC+ cells (B) and CSC− cells (C) from different generations in KG-1, MCF7, SHG44, Caco-2 and A375. Frequency of tumorigenic cells was calculated using Extreme Limiting Dilution Analysis software. Other data are expressed as mean ± SEM from 3 independent experiments.