Figure 2. ALDHhigh cells have a character of CR-CSCs/CICs.

(A) ALDEFLUOR assay using adherent-cultured cells. Results of an ALDEFLUOR assay using adherent-cultured CRC21 cells are shown. ALDH^high cells were defined by DEAB, an ALDH inhibitor. The ratio of ALDH^high cells was 2.1%. (B) Sphere-forming assay using ALDHhigh cells and ALDHlow cells. Numbers of spheres from 2500 ALDH^high cells and 2500 ALDH^low cells are shown. Data are shown as means ± SD. An asterisk indicates statistical difference. (C) In vivo tumorigenicity of ALDHhigh cells and ALDHlow cells. Four thousand ALDH^high cells and 4000 ALDH^low cells were injected into NOD/SCID mice. Tumors were monitored every week until 7 weeks after injection. Data are shown as means ± SD. An asterisk indicates statistical difference. (D) Resistance to 5-FU. ALDH^high cells and ALDH^low cells were incubated in culture media containing 5-FU at several concentrations for 4 days. Cell viability was examined by the WST-1 assay. Data are shown as means ± SD. An asterisk indicates statistical difference. (E) Quantitative RT-PCR analysis of stem cell-related gene expression in ALDHhigh cells and ALDHlow cells. Expression levels of ALDH1A1, NANOG, POU5F1 and SOX2 were examined by qRT-PCR. Data are shown as means ± SD. An asterisk indicates statistical difference. (F) Self-renewal and differentiation of ALDHhigh cells and ALDHlow cells. ALDH^high and ALDH^low cells were isolated from adherent-cultured cells. ALDH^high cells were cultured in an adherent condition. ALDH^low cells were cultured in an adherent condition and sphere condition. The ratio of ALDH^high cells in adherent-cultured ALDH^high cells was 2.7%. The ratios of ALDH^high cells in adherent- and sphere-cultured ALDH^low cells were 0.4%.