Figure 1
Identification of BEX2 as a cancer stem cell-related molecule. (A) Heatmap of gene expression microarray analysis. CD274-knockdown and control RBE cells were used and gene expression data were analyzed by a rank product method. (B) Tumorigenicity assay of HuCCT1 control and BEX2-knockdown cells (102 cells per site) were subcutaneously injected into NOD/SCID/γcnull (NOG) mice and tumor development over time. Control: n = 12, shRNA#1 and shRNA#2: n = 6. *P < 0.05. (C) Expression levels of CD274 and BEX2 in several cancer cell lines. CD274 was determined by mean intensity of flow cytometry analysis and BEX2 was detected by western blotting analyzed using ImageJ software. (D) Expression of BEX2 mRNA in the mouse hepatoblast cell line (HBC-3) and differentiated cells. Data were obtained from Gene Expression Omnibus (GDS970). (E) MTT assays using BEX2-knockdown cells. n = 5. (F) ALDEFLUOR assay using HuCCCT1 control and BEX2-knockdown cells. ALDH-positive gates were determined with N,N-diethylaminobenzaldehyde (DEAB, an ALDH inhibitor) treatment. SSC, side scatter. Test, assay without DEAB treatment. (G) Gemcitabine sensitivity in HuCCT1 control and BEX2-knockdown cells determined by MTT assay. n = 3. IC, inhibitory concentration. (H) (left panel) Representative graphs of cell cycle assay using HuCCT1 cells fixed with 70% ethanol and stained with PI and Ki67 followed by flow cytometry. (right panel) Summary of the percentage of G0 population (n = 3, *P < 0.05).
