Figure 5

CST6 expression and methylation status in neural stem cells and glioma tumor initiating (tumor stem) cells. (a) Semi-quantitative RT-PCR analysis showing that CST6 is expressed at moderate to high levels in two neural stem cell lines (SCP-23 and SCP-27) cultured under non-differentiating conditions. In contrast, the glioma initiating line H1228 does not express CST6 but can be induced to re-express CST6 following treatment with 5 μM 5-azadC for 10 days (‘+’ lane). Untreated and 5-azadC-treated T98G cells serve as negative and positive controls for CST6 expression, respectively. Expression of Nestin and SOX2 demonstrate the multipotent phenotype of normal stem cells and tumor initiating cells. Amplification of β-actin serves as a loading control. (b) CST6 BGS methylation analysis of neural stem cells and the tumor initiating line H1228. High-level methylation of the CST6 promoter in H1228 is consistent with the lack of CST6 expression in the absence of 5-azadC treatment. (c) RT-PCR analysis showing that CST6 expression is induced in neural stem cells following differentiation, whereas this upregulation is blocked in H1228 cells. The graph shows representative gel quantitations and is presented as the fold change in CST6 expression upon differentiation (relative to the undifferentiated cells). ‘NC’—not calculated as no CST6 expression was detectable. ‘D’ differentiated, ‘U’ undifferentiated.