Figure 2. AB4 eliminates colorectal cancer stem cells. (A) Totally 1000 single colorectal cancer cells (HCT116 and HCT116/FU) were cultured in suspension culture system with gradient AB4 treatment (0, 5, 25, 50 μM). Representative images of tumor cell spheres were captured in the 10th day. (B) The numbers of tumor spheres at the tenth day were counted and compared between groups. (C) Confocal assays were performed for the expression of LGR5 (Green) in HCT116 and HCT116/FU cells, which was treated with AB4 for 24 days. Nuclei (blue). (D, E) The percentage of LGR5+ cells of HCT116/FU and HCT116 were analyzed with flowcytometry, which were treated with different concentration of AB4 (0, 5, 25, 50 μM) for 48 h. (F) Relative mRNA expression of cancer stem cell-related genes in AB4 (25 μM)/5-FU (20 μM) treated HCT116/FU cells and only 5-FU treated cells. Data represent three independent experiments, and shown as the means ± SEM. *P < 0.01.