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Total RNA was isolated from fibroblasts using Trizol reagent (Invitrogen). First-strand cDNA was synthesized with oligo-dT primers and reverse transcriptase (Invitrogen). The procedures were performed according to the manufacturer's protocol. Gigaxonin cDNAs were amplified from the cDNA pool using forward primer, 5'-TTGATGGCTGAGGGCAGTGCCGTGTCTG-3' and reverse primer, 5'-TTCCTCCTCAAGGGGAATGAACACGAAT-3'. After electrophoresis, PCR products were purified by the GeneClean purification system (Q-Biogen) and were sequenced with the BigDye™ sequencing kit (Applied Biosystems). The shorter gigaxonin cDNA products from explant WG0791 were first cloned into pCR2.1-TOPO vector (Invitrogen) before sequencing. The conditions used for genomic PCR and the sequences of the PCR primers have been reported elsewhere .
Fibroblast explants were obtained from the repository for mutant human cell strains at McGill University. They were maintained at 37°C and 5% CO2 in MEM Eagle medium (Earle's) with 10% fetal bovine serum. For low-serum treatment, cells were incubated in medium containing 0.1% fetal bovine serum for 72 hours. All experiments were performed on cells from passages 13–18. For immunocytochemical analyses, cells seeded on coverslips were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.1% Triton-X100 for 5 minutes. Fixed cells were incubated with primary antibodies at room temperature for one hour, followed by several washes with PBS and incubation with appropriate secondary antibodies for 30 minutes. The coverslips were then washed with PBS and mounted onto slides with Aquamount (Lerner Laboratories) for immunofluorescent microscopy. To co-stain lipid droplets and vimentin filaments, formaldehyde-fixed cells were permeabilized with 0.05% Saponin and incubated with an anti-vimentin antibody overnight. Before mounting onto slides, cells were stained for lipid droplets with 0.5% Oil Red O in propylene glycol (Poly Scientific). To stain the nuclei and the lipid droplets, cells were fixed in 4% paraformaldehyde and incubated with 0.5% Oil Red O in propylene glycol and Gill's Hematoxylin I (Poly Scientific) for 20 minutes. Antibodies used: monoclonal mouse anti-vimentin, clone V9 (Sigma), monoclonal mouse pan anti-keratin (Sigma), and polyclonal anti-vimentin .
Human 133A Genechips from Affymetrix were used to study the expression profiles. Biotin-labeled cRNA probes were prepared according to the manufacturer's protocol. In brief, cells were treated with low-serum medium for 72 hours, and total RNA was extracted. First-strand cDNAs were synthesized with oligo-dT-T7 primers and reverse transcriptase (Invitrogen). After second-strand cDNA synthesis, double-stranded cDNAs were used to produce biotin-labeled cRNA probes by T7 polymerase (Enzo Laboratory). The cRNAs were fragmented before being used for hybridization. Hybridization and scanning were carried out at the GeneChip analysis facility at Columbia University.
GeneChip results were analyzed by Affymetrix Microarray Suite (version 5.0). To reduce background noise, a four-way comparison of MCH068, MCH070, WG0321 and WG0791 cells was performed. Only genes that showed consistent changes in both WG0321 and WG0791 fibroblasts when compared to both MCH068 and MCH070 cells were selected. Genes that exhibited more than three-fold differences were considered as significantly altered.
To determine the relative expression levels of a gene, quantitative PCR was performed on cDNAs prepared from normal and GAN fibroblasts. Single-stranded cDNAs were generated from total RNA with Oligo-dT primers and reverse transcriptase (Invitrogen). Quantitative PCR was performed on a SmartCycler II PCR machine (Cephid) with gene-specific primers, SyBr Green fluorescence dye, and OmniMix HS PCR master mix (TakaRa). The sequences of the PCR primers and the PCR conditions are shown in Table 2. GAPDH was used as the internal control, and the fold difference of a gene-of-interest in MCH070, WG0321 and WG0791 relative to MCH068 was calculated using the ΔΔCt method .
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