Figure 5.

KDM2B and EZH2 were indispensable for CCS-like cell maintenance in vitro via regulating the PI3K/AKT pathway. The CRC cancer stem cell population (CD133⁻/CD144⁻ and CD133⁺/CD144⁺) were isolated from HT-29 cells by MACS. (A) Protein expression levels of KDM2B and EZH2 confirmed in CD133⁻/CD144⁻ and CD133⁺/CD144⁺ cells by Western blot. The cluster bar chart represents the Western blot quantification (A) *p < 0.05. (B) Immunofluorescence staining of KDM2B and EZH2 in CD133⁻/CD144⁻ and CD133⁺/CD144⁺ cells (Scale bar: 100 μm). (C) CD133⁺/CD144⁺ cells were transfected via the lentiviral expression vector of short hairpin RNA (shRNA) against KDM2B and EZH2 (shKDM2B and shEZH2) and negative control, and more than 70% of cells had green fluorescence under the fluorescence microscope. Representative microscopic images were taken. (D) Sphere formation in untreated, control, shKDM2B, and shEZH2 groups. The expansion of tumor spheres was analyzed at 40 × magnification under a microscope (bar = 50 μm; magnification, 400 ×). The knockdown of KDM2B and EZH2 activated the PI3K/AKT pathway in the stem cell population in CRC. (E) The expression of p-PI3K, PI3K, p-AKT, AKT, KDM2B, and EZH2 was analyzed by Western blot Assay after KDM2B and EZH2 knockdown in CD133⁺/CD44⁺ cell population. β-tubulin was used as a loading control. (F) The downregulation of KDM2B and EZH2 weakens self-renewal markers in CRC cells. The assessment of protein expression of stemness markers including CD44, CD133, and ALDH-1 was analyzed by Western blotting. The data was statistically significant at **p < 0.01, ***p < 0.001 as compared to the control. Data are represented as mean ± SEM of three independent experiments.