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The goal of this study was to examine global alterations in gene …


Biology Articles » Biochemistry » Lipid Biochemistry » Alterations in lipid metabolism gene expression and abnormal lipid accumulation in fibroblast explants from giant axonal neuropathy patients » Figures

Figures
- Alterations in lipid metabolism gene expression and abnormal lipid accumulation in fibroblast explants from giant axonal neuropathy patients

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Figure 1.GAN mutations in GAN fibroblasts. (A) Sequencing of the 1.8-kb RT-PCR product from MCH070 (wild-type) and WG0791 (GAN) cells. The WG0791 product contains an A→T missense mutation. The affected codon is underlined. (B) Sequencing of the 1.7-kb RT-PCR product from WG0791 (GAN) and the 1.8-kb product from MCH070 (wild-type) cells. GAN exon 2 is skipped in the 1.7-kb WG0791 product, resulting in an out-of-frame premature stop codon (*). (C) Sequencing of the RT-PCR products from MCH070 (wild-type) and WG0321 (GAN) cells. The WG0321 product includes part of intron 9. (D) Schematic representation of the GAN mutations in WG0321 and WG0791 patients. Because of a 9.3-kb deletion in the WG0321 allele, a portion of intron 9 is included in the mRNA (gray box). The intronic mutation in WG0791 is indicated with an arrowhead. The positions of the RT-PCR primers used to amplify GAN cDNA are also shown (arrows).

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Figure 2.Cytological analysis of normal and GAN fibroblasts. (A) Fractions of wild-type and GAN cells containing vimentin aggregates in normal and low-serum media. Upon low-serum treatment, there was a dramatic increase of vimentin aggregates in GAN cells, from 12% to 43% for WG0791 cells and from 19% to 89% for WG0321 cells. Vimentin did not form aggregates in normal cells under any culture conditions. (B and C) Immunostaining of MCH070 (wild-type) cells with a polyclonal anti-vimentin antibody (B) and a monoclonal anti-pan-keratin antibody (C). A small percentage of MCH070 fibroblasts expressed both vimentin and keratins. Both of these intermediate filament proteins could form an extensive filament network. (D and E) Immunostaining of WG0321 (GAN) cells with a polyclonal anti-vimentin antibody (D) and a monoclonal anti-pan-keratin antibody (E). Similar to MCH070, a small percentage of WG0321 fibroblasts expressed vimentin and keratins. Both proteins could be found in the aggregates (white arrows). Note that some vimentin aggregates did not contain keratins (arrowheads in D). Scale bar, 10 μm.

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Figure 3.Quantitative RT-PCR analyses of the GAN and lipid-metabolism-related genes in fibroblast explants grown in low-serum conditions. The expression of each gene in MCH070, WG0791 and WG0321 cells was compared to that in MCH068 cells. Each data point is the mean of three separate runs. GAPDH was used for normalization.

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Figure 4.Cytological staining of normal and GAN fibroblasts grown under low-serum conditions. (A) Percentage of cells containing Red-Oil-O-positive droplets. (B-D) Fibroblasts MCH070 (B), WG0791 (C) and WG0321 (D) were stained with Oil Red O and Hematoxylin dyes. Lipid droplets were stained in red and nuclei were stained in blue. Lipid droplets accumulated in WG0791 and WG0321 cells but not in MCH070 cells. (E-G) Fibroblasts MCH070 (E), WG0791 (F) and WG0321 (G) were immunostained with monoclonal anti-vimentin V9 antibody and Oil Red O. Vimentin filaments were stained in green and lipid droplets were stained in red. Scale bars, 10 μm.

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