Research Paper Volume 13, Issue 8 pp 11170—11187

The critical role of peroxiredoxin-2 in colon cancer stem cells

PRDX2 knockdown inhibits the self-renewal of CCSCs. (A) HCT116 and HT29 cell lines were first transfected with a lentiviral vector-mediated specific shRNA (shPRDX2) to silence PRDX2 or with a negative control lentivirus vector. Then, shPRDX2-CD133+CD44+ and control-CD133+CD44+ CCSCs were isolated from the transfected HCT116 and HT29 cells by magnetic bead sorting, which resulted in a considerable enrichment of CD133+CD44+ CCSCs (regular purity > 90%), as identified by flow cytometry with a PE-labeled anti-CD133 antibody and a PE-Cy5-labeled anti-CD44 antibody. (B) Western blot analysis of PRDX2 expression and the protein expression levels of stemness-related genes such as Nanog and Sox2 in CD133+CD44+ CCSCs generated from HCT116/HT29 control or shPRDX2 cells. GAPDH was used as the loading control. (C) Left panel: Representative image of spheres formed from CD133+CD44+ CCSCs derived from HCT116/HT29 control or shPRDX2 cells. Right panel: The number of cell spheres per 5000 cells was counted. The data are presented as the means ± SD of sphere numbers from three independent experiments performed in triplicate. Statistical analysis: Student’s t-test, ****p

Figure 2. PRDX2 knockdown inhibits the self-renewal of CCSCs. (A) HCT116 and HT29 cell lines were first transfected with a lentiviral vector-mediated specific shRNA (shPRDX2) to silence PRDX2 or with a negative control lentivirus vector. Then, shPRDX2-CD133+CD44+ and control-CD133+CD44+ CCSCs were isolated from the transfected HCT116 and HT29 cells by magnetic bead sorting, which resulted in a considerable enrichment of CD133+CD44+ CCSCs (regular purity > 90%), as identified by flow cytometry with a PE-labeled anti-CD133 antibody and a PE-Cy5-labeled anti-CD44 antibody. (B) Western blot analysis of PRDX2 expression and the protein expression levels of stemness-related genes such as Nanog and Sox2 in CD133+CD44+ CCSCs generated from HCT116/HT29 control or shPRDX2 cells. GAPDH was used as the loading control. (C) Left panel: Representative image of spheres formed from CD133+CD44+ CCSCs derived from HCT116/HT29 control or shPRDX2 cells. Right panel: The number of cell spheres per 5000 cells was counted. The data are presented as the means ± SD of sphere numbers from three independent experiments performed in triplicate. Statistical analysis: Student’s t-test, ****p < 0.0001.