PMC:4504148 / 11299-21356 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4504148","sourcedb":"PMC","sourceid":"4504148","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4504148","text":"Material and methods\n\nYeast\n\nYeast strains and growth conditions\nIn this study, we used the BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) wild-type strain (Brachmann et al., 1998). Full-length cDNA of either wtDFNA5 or mutDFNA5 was isolated and amplified as previously described (Gregan et al., 2003). Amplified products were ligated into yeast pYX212 plasmid containing an HA-marker (Clontech, Mountain View, CA, USA) using EcoRI and BamHI restriction sites. All constructs were verified by bidirectional sequencing on an ABI genetic analyser 3130 × l (AppliedBiosystems, FosterCity, CA, USA).\nYeast strains were grown at 30°C in selective medium containing 2% glucose (SD-URA). Fifty milliliter yeast cultures, transformed with either wtDFNA5 or mutDFNA5, were harvested in mid-exponential phase (OD600 nm = 3.5−4.2) and at the post-diauxic shift (OD600 nm = 7.4−8.4) (Figure 1). Standard transformation techniques were applied for these transformations (Gietz et al., 1992).\nFigure 1 Yeast microarray design. (A) Illustration of the different comparisons that were made between the RNA samples. RNA was collected from yeast strains transformed with either wtDFNA5 or mutDFNA5 at two different time-points. The bold and dashed lines represent the color flip of each RNA sample. (B) Growth profile of the Saccharomyces cerevisiae BY4741 background strain transformed with either wtDFNA5 (gray squares) or mutDFNA5 (black squares). The blue and red ellipse illustrate respectively the mid-exponential and post-diauxic time-points when RNA was collected.\n\nRNA extraction yeast\nRNA was collected from yeast at mid-exponential phase and just after the post-diauxic shift using RNA pure kit following the manufactures instructions (GenHunter® Corporation, Nashville, TN, USA). All steps were performed in duplo at 4°C to avoid RNA degradation. This resulted in eight different RNA samples: mid-exponential wtDFNA5, (WT_E1 and WT_E2), mid-exponential mutDFNA5 (Mut_E1 and Mut_E2), post-diauxic wtDFNA5 (WT_S1 and WT_S2), and post-diauxic mutDFNA5 (Mut_S1 and Mut_S2) (Figure 1).\n\nMicroarray design and analysis\nMicroarray experiments were performed at the VIB Nucleomics Core (www.nucleomics.be). Before labeling, RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies). RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 1 μg of total RNA spiked with 10 viral polyA transcript controls (Agilent, Santa Clara, CA, USA) was converted to double stranded cDNA in a reverse transcription reaction. Subsequently, the samples were converted to antisense cRNA, amplified and labeled with Cyanine 3-CTP (Cy3) or Cyanine 5-CTP (Cy5) in an in vitro transcription reaction according to the manufacturer's protocol (Agilent, Santa Clara, CA, USA). A mixture of purified and labeled cRNA (Cy3 label: 5 pmol; Cy5 label: 3.5 pmol) was hybridized on Agilent Yeastv2 arrays followed by washing, according to the manufacturer's procedures. To assess the raw probe signal intensities, arrays were scanned using the Agilent DNA MicroArray Scanner and probe signals were quantified using Agilent's Feature Extractor software (version 10.5.1.1).\nIn total four different comparisons were made. Each comparison was done in duplo by a color flip. The different comparisons are shown in Figure 1A. Gene expressions of strains transformed with wtDFNA5 were compared to strains transformed with mutDFNA5, both in mid-exponential phase (comparison 1a) and at the post-diauxic shift (comparison 1b). Additionally, comparisons were made between the mid-exponential and the post-diauxic shift of either wtDFNA5-(comparison 2b) or mutDFNA5-(comparison 2a) transformed cells.\nAnalysis of the microarray data was performed using the R package LIMMA (http://www.bioconductor.org) (Gentleman et al., 2004). Fold changes were computed using raw Cy3 and Cy5 intensities values provided by Agilent's Feature Extractor software (version 10.5.1.1) and loess normalization and background correction were performed to determine the log2-ratios per array. Differential expression was assessed via the moderated t-statistic, described in (Smyth, 2004). To control the false discovery rate, multiple testing correction was performed. This generated four differentially expressed gene lists. All the (raw) data files have been completely uploaded to the gene expression omnibus and have been stored under accession number GSE70169.\n\nHEK293T cells\n\nCell culture and growth conditions\nThe microarray experiments in human cell lines were performed using Human Embryonic Kidney 293T cells (HEK293T). HEK293T cells were subcultured in 60 mm dishes at a density of 2 × 106 cells in Dulbecco's modified Eagle's medium containing 4500 mg/L glucose supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. Overnight cultures were transfected with either control (empty EGFP vector), wtDFNA5 or mutDFNA5 using lipofectamine. Six hours post transfection, cells were harvested using Triple-x reagent for RNA extraction. All products for human cell line cultures were obtained from Invitrogen (San Diego, CA, USA). Full-length cDNA of either wtDFNA5 or mutDFNA5 was isolated and amplified as previously described (Gregan et al., 2003).\n\nRNA extraction\nAn RNeasy mini kit was used for RNA extraction from transfected HEK293T cells (Qiagen, Hilden, Germany) at different time-points. For the microarray experiment, RNA was extracted 12 h post-transfection. For the gene analysis by real time rtPCR, RNA was extracted at either 3, 6, 12, 15, 18, 20, or 24 h post-transfection. For the microarray experiment, the integrity of the resulting RNA was checked on an automated Experion electrophoresis system (Biorad, Hercules, CA, USA).\n\nMicroarray design and analysis\nThe “Totalprep RNA Amplification” kit was used to amplify the RNA samples (Illumina, Ambion, Austin, TX, USA). Doublestranded-cDNA was generated from the mRNA fractions followed by an in vitro transcription reaction which produced cRNA strands containing biotin-UTP nucleotides. Seven Hundred Fifty nano gram of the resulting cRNA samples were hybridized to an Illumina human HT12v3 beadchip (Illumina, San Diego, CA, USA).\nSix independent biological replicates were used for either wtDFNA5- or mutDFNA5-transfected HEK293T cells and loaded on the chip. Overnight hybridization at 58°C was followed by washing and streptavidin-Cy3 dye labeling (Amersham, Buckinghamshire, England). An Illumina Iscan equipped with Iscan control software was used to measure the intensity values and XY coordinates for every probe on the array. The resulting data files were then analyzed using the R package “Beadarray v1.14.0” (Dunning et al., 2007) followed by a quality control and quantile normalization. LIMMA v3.2.1 was used for the further analysis of the normalized intensity values to determine the differentially expressed genes (Smyth, 2004).\nAll the (raw) data files have been completely uploaded to the gene expression omnibus and have been stored under accession number GSE70169.\n\nReal time rtPCR\nTo confirm the results obtained from the microarray data in HEK293T cells, gene expression was studied in human HEK293T cell lines using Power SYBR Green RNA-to CT 1 Step Kit (Invitrogen, San Diego, CA, USA). Each reaction mixture contained 200 nM final primer concentration (primer pairs are shown in Supplemental Data Table 1) and 30 ng RNA template. All reactions were performed in triplicate on a LightCycler 480 system (Roche, Basel, Switzerland) and resulting data were analyzed by Qbase plus (Biogazelle, Ghent, Belgium). Three housekeeping genes were used each time as a reference, namely GAPDH, RPL13A, and YWHAZ.\n\nWestern blot analysis\nFor western blotting, cells were lysed using RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Pierce, Rockford, IL, USA) containing a PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche), an EDTA-free protease tablet and 10 μl (25 units/μl) benzonase (70746-4 Novagen®, Merck Millipore, Darmstadt, Germany). Transfected HEK293T cells were lysed for 20′ at 4°C and centrifugated at 2000 g at 4°C to obtain protein lysates.\nProteins were electrophoretically separated and blotted onto a nitrocellulose membrane (Whatman, Kent, UK). This membrane was blocked for 1 h in 5% non-fat dry milk and afterwards incubated overnight (4°C) in one of the following primary antibodies: anti-phospho-SAPK/JNK (Thr183/Tyr185, #4668), anti-phospho-p44/42 (ERK1/2, #4370), anti-SAPK/JNK (#9252), anti-p44/42 (ERK1/2, #9102) (Cell Signaling Technologies, MA, USA), or anti-β-Actin (A5316, Sigma Aldrich, MO, USA). After washing, the membranes were incubated with either a secondary goat anti-rabbit (ab6721, Abcam, Cambridge, UK) or sheep anti-mouse (NA931, GE Healthcare, Buckingmersham, UK) antibody. Finally, the corresponding proteins were visualized using Enhanced ChemiLuminescence Western Blotting Substrate (Thermo Scientific, IL, USA).\n\nMAPK inhibition\nThe SP600125 JNK inhibitor was used to inhibit the MAPK pathway. Twelve hours post-transfection, HEK293T cells were incubated with 25 μM for 12 h. Next, cells were collected and viability was measured by flow cytometry (CyflowML, Partec, Germany) using propidium iodide as a cell death marker.\n\nGene ontology analysis\nIn addition to the determination of the differentially expressed genes, GO analysis was performed in order to identify enriched GO annotations. We used an open-source application, the Ontologizer, as a tool to statistically analyse the high-throughput data (Ashburner et al., 2000). A standard method for statistics, the “Term-for-Term analysis,” was used followed by Benjamini-Hochberg correction for multiple testing (Hochberg and Benjamini, 1990; Tavazoie et al., 1999). Differentially expressed genes with a corrected p-value below 0.05 and a log2 (fold change) of 1.5 and 0.5 in yeast and human cell lines respectively, were selected for this analysis.\n","divisions":[{"label":"Title","span":{"begin":0,"end":20}},{"label":"Section","span":{"begin":22,"end":4477}},{"label":"Title","span":{"begin":22,"end":27}},{"label":"Section","span":{"begin":29,"end":1554}},{"label":"Title","span":{"begin":29,"end":64}},{"label":"Figure 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