RAW 264.7 cells were cultured in RPMI 1640 supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum. Primary peritoneal mouse macrophages were isolated by peritoneal lavage with 15 ml PBS, supplemented with 10% fetal calf serum and cultured in RPMI 1640 with supplements as described. Cells were kept at 37°C in a humidified atmosphere with 5% CO2.
6 × 106
RAW 264.7 cells were seeded in 10-cm dishes one day prior to experiments. The following day, medium was changed, cells were treated as indicated and harvested. Nuclei were prepared in 200 μl hypotonic lysis buffer (10 mM Hepes, 0.1 mM EDTA, 2 mM MgCl2
, 10 mM KCl, protease inhibitor cocktail, 1 mM DTT, 5 mM PMSF, pH 7.9) and incubated on ice for 10 min. After centrifugation (500 × g, 30 min, 4°C), the supernatant was discarded and nuclei were resuspended in 150 μl nuclei storage buffer (50 mM Tris-HCl, 0.1 mM EDTA, 5 mM MgCl2
, and 40% glycerol, pH 8.3). Stalled transcriptions were allowed to resume following exogenous addition of nuclear run-on reaction buffer and reaction proceeded for 30 min at 30°C. Nuclear RNA was isolated, extracted and cDNA was hybridized to a mouse genomic array chip containing 32000 genes as previously described [26
1 × 107 nuclei in 150 μl storage buffer were added to 150 μl nuclear run-on reaction buffer (10 mM Tris-HCl, 5 mM MgCl2, 300 mM KCl, 1 mM ATP, 1 mM GTP, 1 mM CTP, 1 mM UTP, pH 8.0) and nuclear run-on was performed for 30 min at 30°C. Nuclear RNA was isolated using the peqGOLD RNAPure kit according to manufacture's instructions (Peqlab, Erlangen, Germany).
Identification of differentially expressed genes between normoxic and hypoxic treated NPC cells was carried out using GeneChip_Mous Genome Arrays (Affymetrix, Santa Clara, CA), which represented 32000 transcripts. In brief, 10 μg of total RNA was used to synthesize cDNA. The cDNA was then used as a template to generate biotinylated cRNA by in vitro transcription. The cRNA was subsequently fragmented. The quality and size distribution of total RNA, cRNA and fragmented cRNA were checked on an Agilent 2100 Bioanalyzer (Agilent, Amstelveen, The Netherlands), using RNA 6000 Pico assay. The fragmented cRNA was hybridized to the GeneChip arrays. The arrays were then washed, stained and finally scanned with a laser scanner. The scanned images were processed with GeneChip_Microarray Suite 5.0 (Affymetrix). Comparison between expression profiles of the normoxic and hypoxic and/or NO were performed using GeneSpring_software version 7 (Silcongenetics, Redwood, California). Gene expression data were first normalized by GC-RMA (robust multichip average) preprocessor. Normalized values below 0.001 were set to 0.001. The normalized expression values were then compared between normoxic and hypoxic, normoxic and nitric oxide, as well as for normoxia and hypoxia plus nitric oxide. Fold change differences were calculated to identify the up- and down-regulated genes. Statistical algorithms implemented in the Affymetrix microarray analysis software were applied to identify genes whose overall expression level in three independent experiments was altered at least 2-fold up or down by hypoxia and/or NO compared to untreated controls. To compare the gene profiles of the different treatments and to determine the number of transcripts regulated by each treatment, we eliminated duplicates of transcripts represented more than once as well as transcripts with unknown gene identification numbers.
Quantitative real time RT-PCR
RT-PCR analyses were performed for a selection of upregulated and downregulated genes to confirm their differential expressions. 2 × 106 RAW 264.7 cells and/or primary peritoneal macrophages were seeded in 10-cm dishes one day prior to experiments. The following day medium was changed and cells were treated as indicated. Total RNA was isolated using the peqGOLD RNAPure kit (Peqlab, Erlangen, Germany). The reverse transcription was completed from 1 μg RNA with a IScript™ cDNA Synthesis RT-PCR kit. The quantitative real time PCR was performed by MyiQ (Bio-Rad). Reaction mixtures containing SYBR Green were composed according to the manufacturer protocol. The cycling program was: 50°C, 2 minutes; 95°C, 15 minutes; followed by 35 cycles at 95°C, 15 seconds; 60°C, 30 seconds; 72°C, 30 seconds. Values of Atf3, Ddit4, Vegf1, Bnip3, Sesn2, Cdkn1a and Frat2 were then normalized to 16S ribosomal protein. The following primer pairs were selected for quantitative real time PCR: Vegf1 forward: 5'-CAGGCTGCTGTAACGATGAA-3', Vegf1 reverse: 5'-GCATTCACATCTG-CTGTGCT-3'; Bnip3 forward: 5'-GGTTTTCCCCAAAGGAATA-3', Bnip3 reverse: 5'-TGACCACC-CAAGGTAATGGT-3'; Atf3 forward: 5'-CAGAGCCTGGTGTTGTGCTA-3,' Atf3 reverse: 5'-GGTGTCGTCCATCCTCTGTT-3'; Ddit4 forward: 5'-T-TCATTCGGA-TAGCAG-3', Ddit4 reverse: 5'-TCAGGTTGGCCAGGTG-3'; Sesn2 forward: 5'-GCATT-ACCTGCTGCTGCATA-3', Sesn2 reverse: 5'-AAGGCCTGGATATGCTCCTT-3'; Frat2 forward: 5'-GAATCGGGAGGGCTTCTAAC-3', Frat2 reverse: 5'-GCTCT-GCAATTGTA-GCACCA-3'; Cdkn1a forward: 5'-GGGATGGCAGTTAGGACTCA-3', Cdkn1a reverse: 5'-GTGGGGC-AAGTGCCTAGATA-3'; 16S ribosomal protein forward: 5'-AGATGATCGAGCCGCGC-3'; 16S ribosomal protein reverse: 5'-GCTACCAGGGCCTTTGAGATGGA-3'
2D DIGE gel analysis
Cells were washed twice in PBS, pelleted by centrifugation, and lyzed in lysis buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 30 mM Tris-HCl, pH 8.5) by sonication. Insoluble material was removed by centrifugation at 12000 g for 5 min at 4°C. Precipitation of proteins was performed using the 2D clean-up kit (GE healthcare) according to manufacture guidelines and protein concentration was determined using the RC/DC protein assay (Bio-Rad). 50 μg of each lysate was labeled with Cy3 and Cy5 (400 pmol) in the dark for 30 min and quenched with 50-fold molar excess of free lysine-to-dye. Samples were reverse-labeled in order to enable all comparisons and eliminate any dye-labeling bias. Additionally samples were mixed and run on the same gels with an equal amount (50 μg) of Cy2-labeled standard. Cy2 was used as a standard on all gels to aid image matching and cross-gel statistical analysis.
Isoelectric focusing (IEF) was performed with IPGphor apparatus (GE Healthcare) according to manufacturer's instruction. Briefly, immobilized 24 cm linear pH gradient (IPG) strips, pH 3-10, were rehydrated (30 mM Tris Base,7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG buffer, 50 mM DTT PH 8,5) overnight. The strips were focused according to the following protocol: linear ramp to 1000 V over 2 h, linear ramp to 4000 V over 1 h, linear ramp to 8000 V over 1 h and 8000 V for 68 kVh. Strips were equilibrated for 15 min in equilibration buffer (1.5 M TrisCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and trace amounts of bromphenol blue) containing 1% (w/v) DTT and thereafter in SDS Equilibration buffer containing 4% (w/v) iodoacetamide for 15 additional min. After equilibration, proteins were separated on 12% polyacrylamide gels using an Ettan Dalt Six device (GE Healthcare) at 22°C. Gels were run at 2.5 W/gel for 30 min and then 15 W/gel until the tracking dye had migrated off the bottom of the gel.
Gels were scanned with a Typhoon 9410 variable mode imager (Amersham Biosciences) using excitation/emission wavelengths specific for Cy2 (488 nm/520 nm), Cy3 (532 nm/580 nm), and Cy5 (633 nm/670 nm). Images were normalized, statistically analyzed, and differentially expressed proteins were identified and quantified using DeCyder (GE Healthcare). MS identification was carried out for proteins that showed more than two-fold variation in abundance. Briefly, the protein spots were cut out of 2-D gels using Gelpix Spot-Excision Robot (Genetix, Hampshire, UK) and then washed three times with Milli-Q water. According to the manufacture of ZipPlate micro-SPE Plate, gel pieces were transferred into ZipPlate micro-SPE Plate wells (Millipore, Billerica, MA, USA). The gel pieces were dried in a vacuum and the proteins were digested overnight in 10 ml trypsin (10 ng/ml, Trypsin Gold, mass spectrometry grade, Promega, Madison, WI, USA) in 25 mM ammonium bicarbonate at 37°C. Peptide fragments were extracted with 0.2% TFA for 30 min, applied onto the C18 resin, and then desalted with 0.2% TFA. The tryptic peptide mixtures were recovered with 5 ml elution solution containing 50% ACN/0.1% TFA by centrifugation for 15 s at 17506 g and spotted onto a MALDI sample target plate. Peptide mass spectra were obtained on a MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA, USA).
Protein identification was processed and analyzed by searching the Swiss-Prot and NCBI protein database using the MASCOT search engine of Matrix Science, integrated in the Global Protein Server (GPS) Workstation. The mass tolerance, the most important parameter, was limited to 50 ppm. The results from the MS and MS/MS spectra were accepted as a good identification when the GPS score confidence was higher than 95%.
The nuclear run-on data was statistically analyzed by Genedata AG (Basel, Switzerland). The p-value cut-off was set to 0.0001 or 0.001 and the expected number of transcripts with p-values below these thresholds are about 15 or 150, respectively, assuming random effects only (33155 transcripts on the microarray, thereof ~15000 with valid values). False discovery rates (FDR) were estimated according to a balanced permutation test with 500 repeats. In addition the ratios of the groups means (N-fold changes) were determined. The significant transcripts were in addition determined on the condition that at least 2 out of 3 experiments have valid values in each group. Representative data are shown. For the quantitative real time verification of microarray data, gene expression was shown as mean values ± standard deviation (SD). Means were checked for statistical significance using 1-way analysis of variance (ANOVA), followed by Tukey tests. Differences having a P value < 0.05 were considered to be statistically significant.