Characterization of differentiated NSCs by immunocytochemistry.

<p>(<b>a</b>) After 7 days in culture, differentiated NSCs were positively stained with β III-tubulin (neurons), GFAP (astrocytes), and Gal-C (oligodendrocytes) and negatively stained with CD11b (microglia); (<b>b</b>) nuclei stained with DAPI, and (<b>c</b>) merged images. The images were taken at the magnification of 40X. Scale bars = 25 µm.</p

DNA methylation analysis.

<p>MethylScreen analysis of two CpG sites, CpG3 and CpG6, in the <i>FXN</i> upstream GAA repeat region of DNA from Y47R cells and YG8R cells. UM = unmethylated, IM = intermediately methylated, DM = densely methylated. The experiment was repeated twice for each cell type and each reaction was carried out in duplicate. Error bars = s.e.m.</p

Susceptibility to oxidative stress in Y47R and YG8R cells.

<p>Treatment of Y47R and YG8R cells with 100 µM H₂O₂ or FAC (100 µg/ml) and BSO (1 mM) significantly reduced the cell viability in all cell types, but to greater extent in YG8R cells compared to Y47R cells. The experiment was repeated twice for each cell type and each reaction was carried out as 3–6 replicates. Error bars represent s.e.m (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

Mouse NSCs and differentiated NSCs in culture.

<p><b>a</b>) NSC appeared as “Neurospheres” after 10–14 days of culture with NSC medium supplemented with rhEGF and rhFGF growth factors (10X magnification). <b>b)</b> The differentiation of NSCs was induced by incubating the cells in the NSC medium with differentiation supplements. The early stages of the differentiation, where mixed cells are seen emerging from the neurospheres (10X magnification). Scale bars = 100 µm.</p

Antioxidant gene expression analysis.

<p><i>Catalase</i>, <i>Sod1</i>, <i>Sod2</i> and <i>Gpx1</i> gene expression levels were determined in all three Y47R and YG8R cell types by qRT-PCR. Values were expressed relative to the levels of both <i>Gapdh</i> and <i>β2M</i> and in each case all gene expression levels were normalized to the mean expression levels of the <i>Catalase</i> gene of Y47R cells set at 100%. Two individual cDNA samples were analyzed for each cell type and each reaction was carried out in triplicate. Error bars represent s.e.m (*<i>p</i><0.05, **<i>p</i><0.01, ***0.001).</p

<i>FAST1</i> expression analysis in Y47R and YG8R cells.

<p><b>(a)</b> Strand specific RT-PCR using a FAST-RT primer showed the presence of an antisense transcript. The <i>FAST1</i> PCR product did not amplify in the absence of reverse transcriptase (RT-). <b>(b)</b> qRT-PCR analysis of <i>FAST1</i> levels in fibroblasts, NSCs and differentiated NSCs showed increased levels of <i>FAST1</i> in fibroblasts but no statistical difference was detected in the other two cell types of YG8R cells compared to Y47R cells. Two individual cDNA samples were analyzed for each cell type and each reaction was carried out in triplicate. Values were expressed relative to both <i>Gapdh</i> and <i>Hprt</i> expression levels. Error bars represent s.e.m (*<i>p</i><0.05). M = 1 kb plus DNA marker.</p

GAA repeat instability analysis.

<p>Ethidium bromide-stained agarose gels showing inverted images of GAA repeat PCR products obtained from the successive passages of Y47R and YG8R cells: <b>a</b>) primary fibroblasts (p4-p11), <b>b</b>) NSCs (p4-p12), and <b>c)</b> differentiated NSCs obtained from NSCs (p4-p11). M = 1 kb plus DNA marker, M* = 100 bp DNA marker.</p

Aconitase activity and <i>Pgc-1α</i> expression levels in Y47R and YG8R cells.

<p>(<b>a)</b> Aconitase activities for Y47R and YG8R mouse cell samples. The experiment was performed twice on two samples in triplicate with values being calculated relative to citrate synthase activity. Values were expressed relative to the Y47R mouse cell value set at 100%. (<b>b)</b><i>Pgc-1α</i> expression levels in Y47R and YG8R cells were quantified by qRT-PCR analysis using both <i>Gapdh</i> and <i>β2M</i> as endogenous controls. The mean values of YG8R data are normalized to the mean <i>Pgc-1α</i> mRNA level of the Y47R cells set at 100%. In each experiment, two individual cDNA samples were analyzed for each cell type and carried out in triplicate. Error bars represent s.e.m (*<i>p</i><0.05, **<i>p</i><0.01).</p