Targets of Wnt-1 in C57MG
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE1899 | GEO | 2005/04/07
SECONDARY ACCESSION(S): PRJNA90987
REPOSITORIES: GEO
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2008-06-11 | E-GEOD-1051 | biostudies-arrayexpress
Project description:Pulmonary fibrosis is a chronic, progressive, and lethal interstitial lung disease. It is characterized by extracellular matrix deposition, fibroblast proliferation, and accumulation. Fibroblasts from normal or UIP histology were cultured and analyzed. Keywords: Fibroblasts from normal histology lung tissue or UIP histology lung tissue Five samples of primary human lung fibroblasts were obtained from explanted lungs of patients with Usual Interstitial Pneumonia (UIP) at the University of Pittsburgh Medical Center (Pittsburgh, PA, USA) and three samples of primary normal human lung fibroblasts were acquired from normal histololgy lung tissues of organ donors. The experimental protocol was approved by the University of Pittsburgh Institutional Reviews Board (IRB). Total RNA was extracted and used as a template to generate double-stranded cDNA and biotin-labeled cRNA, as recommended by the manufacturer of the arrays. Fragmented cRNA was hybridized to Codelink Uniset I slides. After hybridization, arrays were washed and stained with streptavidin-AlexaFluor 647. The arrays were scanned using a Genepix 4000B microarray scanner. Images were analyzed using Codelink expression II analysis suite, and visually inspected for defects and quality control parameters as recommended by the manufacturer.
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Project description:Cells of the DC-like cell line XS52 (Xu et al., 1995, J Invest Dermatol. 1995 Dec;105(6):831-6) were lentivirally transduced with a copGFP reporter (pGreenFire) or the mmu-miR-223 gene locus (both from SBI Biosciences, Palo Alto, CA) and cultured in the presence of puromycin to select for transductants as described (Bollmann et al., 2014, Nucleic Acids Res. 42, 12555-12569). Each 20 µg of total RNA derived from the two transduced XS52 sublines was reverse-transcribed using the SuperScript Plus Direct cDNA Labeling System (Invitrogen, Carlbad, CA), with Alexa Fluor 555-aha-dUTP or Alexa Fluor 647-aha-dUTP to differentially label cDNA of either group. Derived fluorescence-labeled cDNA was purified and analysed for quality and yield (Nanodrop ND-100; Peqlab, Erlangen, Germany). Prehybridized (5X SSPE, 0.1% SDS, and 1% BSA) microarrays (Mouse Whole Genome OneArray® Microarray v2; Phalanx Biotech, San Diego) were hybridized with each 20 pmol of either fluorescence-labeled cDNA probe using OneArray Hybridization Buffer with 18% formamide for 16 h (Lucidea SlidePro Hybridizer; GE Healthcare, Munich, Germany) as recommended by the manufacturer. Washed microarrays were analysed using a ScanArray 4000 system equipped with ScanArray 4.0 software (both Perkin-Elmer, Rodgau-Jügesheim, Germany) at 10 pixel size. Laser intensity (90-100%) and photomultiplier tube sensitivity (60-70%) were varied to optimize signal to background ratios. Derived images were analyzed using the ScanArray 4.0 easyquant software module. Intensity data were normalized (loess normalization; MIDAS-2.19) and subjected to significance analysis (MEV 4.3) using appropriate modules of the TM4 microarray software suite (Saeed et al., 2006, Methods Enzymol. 411, 134-193). Changes in gene expression with p-values < 5% and ratios (mmu-miR-223/control) >2 or <0.5 were used for subsequent analyses.
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Project description:For generation of pre-B cell lines, bone marrow from tibias and femurs of 4 to 8 week old C56Bl/6 mice (wt and vav-Gfi1b transgene) was infected with retroviral stocks of different BCR-Abl constructs (MSCV-GFP-IRESp210 or pSRa-p210-tkneo) and plated at a density of 5E+06 per 6 cm dish. All pre-B cells were fed twice a week and analyzed after they became stroma independent. Cells received 1E-08 M dexamethasone or vehicle for 12 hours prior to RNA extraction using TRIzol. mRNAs were converted into doublestranded cDNA using a T7-promoter tagged oligodT primer and transcribed by T7 RNA polymerase in the presence of biotinylated NTPs. Targets were hybridized to Affymetrix MOE430A arrays, washed, stained and scanned (GeneChip scanner 2500) as recommended by the manufacturer. Images were processed either by MAS5.0 (global scaling, TGT=1000) or RMAExpress (quantile normalization and background substraction) software tools. By comparing the gene expression profiles of dexamethasone and non-induced cells, dexamethasone target genes were identified Keywords: ordered
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Project description:Extrahepatic cholestasis leads to complex injury and repair processes that result in bile infarct formation, neutrophil infiltration, cholangiocyte and hepatocyte proliferation, extracellular matrix remodeling, and fibrosis. To identify early molecular mechanisms of injury and repair after bile duct obstruction, microarray analysis was performed on liver tissue 24 hours after bile duct ligation (BDL) or sham surgery. The most upregulated gene identified encodes plasminogen activator inhibitor 1 (PAI-1, Serpine 1), a protease inhibitor that blocks urokinase plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activity. Because PAI-1, uPA, and tPA influence growth factor and cytokine processing as well as extracellular matrix remodeling, we evaluated the role of PAI-1 in cholestatic liver injury by comparing the injury and repair processes in wild-type (WT) and PAI-1-deficient (PAI-1-/-) mice after BDL. PAI-1-/- mice had fewer and smaller bile infarcts, less neutrophil infiltration, and higher levels of cholangiocyte and hepatocyte proliferation than WT animals after BDL. Furthermore, PAI-1-/- mice had higher levels of tPA activation and mature hepatocyte growth factor (HGF) after BDL than WT mice, suggesting that PAI-1 effects on HGF activation critically influence cholestatic liver injury. This was further supported by elevated levels of c-Met and Akt phosphorylation in PAI-1-/- mice after BDL. In conclusion, PAI-1 deficiency reduces liver injury after BDL in mice. These data suggest that inhibiting PAI-1 might attenuate liver injury in cholestatic liver diseases. Total RNA isolated using TRI Reagent (Sigma, St. Louis, MO) was purified with an RNeasy mini kit (Qiagen, Valencia, CA). Twenty micrograms cRNA was hybridized to a mouse GeneChip (U74Av2, Affymetrix, Santa Clara, CA) at the Siteman Cancer Center GeneChip facility as described by the manufacturer. Analyses used one mouse per chip. Gene expression changes were analyzed using Affymetrix MicroArray Suite 4.0 and GeneChip 3.1 Expression Analysis and Statistical Algorithms (Affymetrix). The complete methodology and full data sets for all 6 analyzed chips are available at http://bioinformatics.wustl.edu.beckerproxy.wustl.edu This study compares the injury and repair processed in wild-type mice after BDL.
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