RT-PCR analysis of allele-specific expression of the Gst gene family in undifferentiated NSCs, and following differentiation to neurons and astrocytes.

<p>Numerical values are shown for all RT-PCR samples with at least a 0.8 preference for the B6 (B) or JF1 (J) allele. Other values are listed as biallelic (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031751#pone.0031751.s004" target="_blank">Figure S4</a> for representative data and technical replicates.) <i>n.d.</i>, not detectable.</p><p>**Asterisks denote concordance with undifferentiated NSCs, <i>p</i>-value<0.001.</p><p>Daggers indicate results for samples evaluated by Illumina sequencing:</p>†<p>Biallelic expression at <i>P_B6 or P_JF1</i><0.85,</p>††<p>Monoallelic expression at <i>P_B6</i> or <i>P_JF1</i>> = 0.85.</p

Outline of the cSNP-seq assay.

<p>The diagram shows a hypothetical single-exon gene with 3 SNPS (asterisks), a pooled SNP depth of 10, and a 90% preference for the B6 allele (the red vertical bar shows a JF1 sequence at the cSNP indicated.).</p

Selected list of autosomal genes with monoallelic expression by Illumina sequencing validated by RT-PCR.

†<p>Number of cell lines with monoallelic expression/number of evaluable cell lines. In column 2, the expressed allele(s) are shown in parentheses.</p><p>The values shown for columns 2–4 are from Illumina sequencing (see text).</p

Heatmaps showing results of cSNP analysis.

<p>A. X linked genes (n = 235) evaluable for at least 2 out of the 4 cell lines. Insets expand results of hierarchical clustering. <i>Right inset</i>: Unique pattern of the <i>Xist</i> gene. <i>Left inset:</i> Genes that escape X-inactivation (see text). B. Autosomal genes (n = 152) that show monoallelic expression in at least 2 of 3 evaluable cell lines. <i>Inset:</i> Genes that show imprinted gene expression. C. Autosomal genes (n = 476) with monoallelic expression in at least 1 of 2 evaluable cell lines.</p

Expression levels <i>vs.</i> monoallelic or biallelic expression.

<p>Results are plotted for 313 genes that show monoallelic expression in some NSC lines and biallelic expression in others. For each gene the average log2(RPKM) in NSC lines with monoallelic expression (y-axis) is plotted vs. the log2(RPKM) in cell lines with biallelic expression.</p

Allele-specific expression of <i>Gstp1</i>.

<p>A. Structure of the gene and results of Illumina sequencing in four NSC lines. The chromosomal location, start site and orientation of transcription, the 7 exons, and a CpG island (green rectangle) at the 5′ terminus are shown. The cSNPs in exons 3, 6 and 7 are indicated by the vertical lines above the respective exons. The table shows that the B6 allele is expressed in cell lines 2A1 and 4A5a, while both alleles are expressed in cell lines 2A5 and 3A1. B. RT-PCR results. <i>First row:</i> Chromatograms show a C/T cSNP between B6 and JF1 transcripts, and the presence of both alleles in genomic DNA of NSC lines 2A1 and 4A5. The arrow in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031751#pone-0031751-g004" target="_blank">Figure 4A</a> indicates the location of the cSNP. <i>Second to fourth rows:</i> Analysis of RNA isolated from five NSC lines in the undifferentiated state, and differentiated to astrocytes and neurons, as indicated. The B6 allele is expressed in cell lines 2A1 and 4A5, while both alleles are expressed in cell lines. 2A5, 3A1 and 4B3.</p

miR-124 targets NRAS and PIM3 expression.

<p><b>A.</b> The base-pairing of hsa-miR-124 with the 3′ UTR of NRAS gene. <b>B.</b> miR-124-mediated repression of luciferase reporter gene downstream of 3′ UTR of NRAS. Luciferase reporter gene under the control of wild type (WT) or mutant (MT) NRAS 3′ UTR was transfected into HEK 293 cells along with control, miR-124 RNA duplexes, or the combination of miR-124 RNA duplexes and a miR-124 inhibitor. *p<0.001 by student's <i>t</i>-test. <b>C.</b> Western blot analysis of NRAS expression in control RNA, miR-124 RNA duplexes, or the combination of miR-124 RNA duplexes and a miR-124 inhibitor-transfected GSC1 cells. <b>D.</b> Expression of NRAS in GSC1 and NSC1 determined by real-time RT-PCR analysis. *p<0.05 by student's <i>t</i>-test. <b>E.</b> The base-pairing of hsa-miR-124 with the 3′ UTR of PIM3 gene. <b>F.</b> miR-124-mediated repression of luciferase reporter gene downstream of 3′ UTR of PIM3. Luciferase reporter gene under the control of wild type (WT) or mutant (MT) PIM3 3′ UTR was transfected into HEK 293 cells along with control, miR-124 RNA duplexes, or the combination of miR-124 RNA duplexes and a miR-124 inhibitor. *p<0.001 by student's <i>t</i>-test. <b>G.</b> Western blot analysis of PIM3 expression in control RNA, miR-124 RNA duplexes, or the combination of miR-124 RNA duplexes and a miR-124 inhibitor-transfected GSC1 cells. <b>H.</b> Expression of PIM3 in GSC1 and NSC1 determined by real-time RT-PCR analysis. **p<0.001 by student's <i>t</i>-test. For all panels, data shown are mean ± standard deviation of three replicates.</p

Expression of miR-10b targets in glioblastoma stem cells.

<p><b>A, B.</b> The base-pairing of hsa-miR-10a and hsa-miR-10b with the 3′ UTR of CSMD1 gene. <b>C.</b> miR-10a-mediated repression of luciferase reporter gene downstream of 3′ UTR of CSMD1. Luciferase reporter gene under the control of wild type (WT) or mutant (MT) CSMD1 3′ UTR was transfected into HEK 293 cells along with control, miR-10a RNA duplexes, or the combination of miR-10a RNA duplexes and a miR-10a inhibitor. * p<0.001 by student's <i>t</i>-test. <b>D.</b> miR-10b-mediated repression of luciferase reporter gene downstream of 3′ UTR of CSMD1. WT or MT CSMD1 3′ UTR luciferase reporter was transfected into HEK 293 cells along with control, miR-10b RNA duplexes, or the combination of miR-10b RNA duplexes and a miR-10b inhibitor. * p<0.005 by student's <i>t</i>-test. <b>E.</b> Expression of CSMD1 in glioblastoma stem cell line 1 (GSC) and neural stem cell line 1 (NSC) determined by real-time RT-PCR analysis. <b>F.</b> Expression of HOXD10 in GSC and NSC determined by real-time RT-PCR analysis. For all panels, data shown are mean ± standard deviation of three replicates. ** p<0.01 by student's <i>t</i>-test for both panels <b>E</b> and <b>F</b>.</p

Pathways targeted by deregulated miRNAs in glioblastoma stem cells.

<p>Common miRNA targets were subjected to DAVID functional annotation with KEGG pathway analysis. <b>A.</b> The up-regulated miRNAs in glioblastoma stem cells were predicted to target the p53 pathway. The p53-centered pathway has been shown to regulate cell cycle, apoptosis, angiogenesis, metastasis, and genome stability. <b>B.</b> The down-regulated miRNAs were predicted to target components of the IGF pathway. Various components of the IGF signaling pathways were targeted by down-regulated miRNAs. The IGF pathway has been shown to enhance cell growth, survival, and migration.</p

The morphology, differentiation and growth curve of glioblastoma stem cells (GSCs) and neural stem cells (NSCs).

<p><b>A.</b> Representative images of neurospheres from normal human neural stem cell lines 1–3 (NSC1–3) and glioblastoma stem cell lines 1–3 (GSC1–3). <b>B.</b> The multipotency of NSCs and GSCs. When induced into differentiation, both NSCs and GSCs gave rise to Tuj1+ neurons (green) and GFAP+ astrocytes (red). Representative images of NSC1 and GSC1 differentiation were shown. Nuclear Dapi staining was shown in blue. <b>C.</b> H&E staining of coronal sections from GSC-transplanted brains. The tumor region was indicated by an arrow, shown in dark purple color.</p