Figure 2

CXCR4 is required by glioblastoma cell lines for maintaining self-renewal characteristics of cancer stem-like cells. (A) Flow cytometry using anti-CD133 and RT-PCR indicated that RG2 had a low level of CD133. (B) Spheroid formation of the control shGFP and shrCXCR4 RG2 after culture in an ultralow dish for 10 d. Quantitative analysis showed a reduced number (C) and size (D) of spheres derived from the shrCXCR4 RG2 clone. Similar results were obtained from 2 independent experiments, and each experiment was performed in triplicate. (E) Histograms of cell cycle analysis showed that the deficiency of CXCR4 caused the reduction in G1 and the increase of G2/M populations. After being cultured in an ultralow dish 7d, the spheres derived from shGFP and shrCXCR4-1 were collected and trypsinized, and cell suspensions were subjected to PI staining. Comparing the cell cycle profiles revealed that the percentage of G2/M populations within cells collected from shrCXCR4-1 spheres (S-shrCXCR4, 19.5 ± 0.7%) was higher than those derived from shGFP (S-shGFP, 15.5 ± 0.6%) (*P < 0.05). By contrast, the percentage of G1 population within cells collected from shrCXCR4-1 (68 ± 0.8%) was lower than those from shGFP (72 ± 1.5%, *P < 0.05). (F) Western blot analysis showed that disrupting CXCR4 reduced the level of stem-cell-associated genes, including Oct4 and Nanog. (G) RT-PCR assay indicated that the expression of several genes, including stem-cell markers, Aldh, Nestin, Msi, and the proliferation promoting gene MELK increased in the control clone after forming spheroids, but decreased in the shrCXCR4-1 RG2. The shown were one of the similar results from 2 independent experiments.