Primary mouse embryonic fibroblasts and RNA
Mice lacking part of the helicase domain of the Wrn gene were generated by homologous recombination in mouse embryonic stem cells . In the process, 121 amino acid residues of the Wrn protein were deleted (amino acids 710 to 831). The genetic background of these mice was both 129/Sv and Black Swiss (129/Sv/Black Swiss genetic background). Care of mice was in accordance with the guidelines of the Committee for the protection of animals at the Université Laval. Generation and maintenance of the embryonic cells has been described previously . Briefly, healthy 15.5-day old embryos were minced in 6-well plates and maintained in low glucose DMEM supplemented with 10% heat-inactivated calf serum at 37°C in an atmosphere of 5% CO2. Adherent cells established from embryonic tissues were passaged as soon as they reached confluence. Cytoplasmic RNA was extracted according to standard protocols . After DNAse treatment and phenol:chloroform extraction, precipitated cytoplasmic RNA was dissolved in RNase-free water and purity was verified with an Agilent 2100 bioanalyzer (Agilent, Palo Alto, CA).
Duplicated biological RNA samples (wild type versus H2O2-treated wild type; Wrn
Δ hel/Δ hel
versus H2O2-treated Wrn
Δ hel/Δ hel
cells; wild type versus Wrn
Δ hel/Δ hel
) were labeled with Cyanin-3 or -5 labeled CTP (PerkinElmer, Boston, MA). Labeled cRNAs were purified using the RNeasy Mini kit (Qiagen, Mississauga, ON) and were hybridized onto Whole Mouse Genome Agilent 60-mer Oligo Microarrays (44,000 probes/microarray) using the in situ Hybridization Plus kit (Agilent, Palo Alto, CA) following the manufacturer's instructions. Arrays were scanned using a dual-laser DNA microarray scanner. Data were then extracted from images by the Feature Extraction software 6.1 (Agilent, Palo Alto, CA). Lists of differentially expressed genes were generated using limma in BioConductor http://www.bioconductor.org. The data were background subtracted and normalized using the loess method. Correction for multiple hypothesis testing was performed using the Benjamini-Hochberg method. We have deposited all the raw data in the NCBI public database Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo/ [accession series number GSE19007].
The PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system is a unique resource that classifies genes by their functions, using published scientific experimental evidence and evolutionary relationships to predict function even in the absence of direct experimental evidence. Proteins are classified by expert biologists into families and subfamilies of shared function, which are then categorized by molecular function and biological process ontology terms. This program is implemented in the DAVID (Database for Annotation, Visualization, and Integrated Discovery) web site . Enrichments for specific biological functions using PANTHER were considered significant with a Benjamini value smaller than 0.05. The Benjamini value corresponds to an adjusted p -value using the Benjamini-Hochberg method to correct for multiple hypotheses tested during gene enrichment analyses.
Measurements of reactive oxygen species
Mouse embryonic fibroblasts were incubated with 10 μg/μl of the dye 2'-7' dichlorofluorescein diacetate (Sigma-Aldrich Canada Ltd., Oakville, ON) for one hour at 37°C. This dye is highly fluorescent upon oxidation. After this incubation time, cells were harvested and lysed in RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate] for 10 minutes. Cell debris were spun down and 100 μl of lysate was transferred to 96-well plates for fluorescence measurements with a Fluoroskan Ascent fluorescence spectrophotometer (Thermo Electron Inc., Milford, MA). The excitation and emission wavelengths used were 485 nm and 527 nm, respectively. The final result was expressed as units of fluorescence per μg of proteins. Protein concentrations were measured using the Bradford assay.
Measurements of oxidative DNA damage
DNA was extracted from mouse embryonic fibroblasts with the DNeasy Tissue Kit (Qiagen Inc., Mississauga, ON) according to the manufacturer's protocol. Abasic sites in genomic DNA were quantified with the Oxidative DNA Damage Kit from Kamiya Biomedical Company (Seattle, WA). Briefly, this kit contains an aldehyde reactive reagent (N'-aminooxymethylcarbonylhydrazino-D-biotin), which reacts with the open ring form of apurinic sites in DNA. Oxidative attack by hydroxyl radicals on the deoxyribose moiety will lead to the release of free bases from DNA, generating abasic or apurinic sites. The aldehyde reagent will thus tag apurinic sites with biotin. The number of biotin-tagged apurinic sites was quantified using conjugated avidin-horse radish peroxidase followed by a colorimetric detection of peroxidase.
Measurements of reduced glutathione
Intracellular levels of glutathione (GSH) were quantified with the ApoGSH Glutathione Detection Kit from Bio Vision (Mountain View, CA). The assay utilizes monochlorobimane (MCB), a dye that appears to form adducts exclusively with GSH. The GSH-bound MCB dye fluoresces blue. Fluorescence was measured with a Fluoroskan Ascent fluorescence spectrophotometer (Thermo Electron Inc., Milford, MA). The excitation and emission wavelengths used were 355 nm and 460 nm, respectively.
Measurements of ATP and ADP
Intracellular levels of ATP and ADP were quantified with the ApoSensor ADP/ATP ratio assay kit from Bio Vision (Mountain View, CA). The assay utilizes the enzyme luciferase to catalyze the formation of light from ATP and luciferin. ADP levels are measured by conversion to ATP that is subsequently detected using the same reaction. Luminescence was measured with a Luminoskan Ascent luminometer (Thermo Electron Inc., Milford, MA).
Measurements of inner mitochondrial transmembrane potential
The inner mitochondrial transmembrane potential of mouse embryonic fibroblasts was determined using tetramethylrhodamine ethyl ester (TMRE). Cells were incubated in a culture medium containing 100 μM TMRE for 15 min in the dark at 37°C. Fresh medium was then added after washing cells twice with PBS to remove extracellular TMRE. Cells were harvested and fluorescence intensity of TMRE was detected with a Fluoroskan Ascent fluorescence spectrophotometer (Thermo Electron Inc., Milford, MA). The excitation and emission wavelengths used were 544 nm and 590 nm, respectively.
Data are presented as means ± SEM. The unpaired Student's t -tests were all performed using two-tailed hypothesis and equal variance. Differences between classes were considered significant at P -value lower than 0.05 in all statistical analyses. All statistical analyses were performed with R version 2.6.0 http://www.r-project.org.