Figure 2.

BMI1 and EZH2 are highly enriched in CD133⁺ cells. A, Frozen GBM neurospheres were immunolabeled with antibodies against BMI1 (top), or GFAP and MAP2 after plating in medium-induced differentiation (bottom). DAPI stains nuclei in blue. B, RT-PCR analysis performed on cultured neurospheres with primers against GAPDH, BMI1, MUSASHI, SOX2, and LHX2. C, Western blot analysis performed on freshly isolated GBM sample and neurospheres using anti-EZH2 and anti-β-actin antibodies. D, CD133⁻ and CD133⁺ purified GBM cells with CD133-coupled magnetic beads were analyzed by real-time PCR for the expression of PROMININ (CD133), BMI1, and EZH2 transcripts. Data are expressed as fold change over gene expression in negative cells (CD133⁻), which was set at 1. Results are mean ± SD (n = 3; *p < 0.05; **p < 0.01). E, Dissociated neurospheres were stained with anti-BMI1 and anti-CD133 antibodies. The R1 gate delineates the cell population analyzed. Values are the percentages of cells in the corresponding regions. F, Gene copy number was assessed by quantitative real-time PCR in GBM samples. Data are expressed relative to healthy human retina genomic DNA and calibrated to β-actin used as internal standard. Another internal standard was used which consisted of PAX6. Scale bars, 60 μm.