Project description:We were interested in identifying proteins that are regulated in response to the extracellular matrix molecule, fibronectin, in dermal fibroblasts. To determine proteins regulated by exposure to fibronectin, primary murine dermal fibroblasts were cultured on tissue culture plastic or on tissue culture plates coated with a thin layer of fibronectin (2 μg/cm2). Fibroblasts were seeded at 35,000 cells/10 cm2 and were harvested 48 hours later. Cell pellets from these fibroblasts were shipped to Kinexus Bioinformatics Corp. (Vancouver, B.C.) where they were lysed and analyzed using the Kinex TM Antibody Microarray (KAM 1.2), which contains 800 (500 pan-specific and 300 phosphorylation site-specific) antibodies. This enabled us to identify candidate proteins regulated by exposure to fibronectin.
Project description:The current study set to determine the effects of Fibroblast Growth Factor 2 and cell culture surface (tissue culture plastic and glass) on the transcriptome of adult human dermal fibroblasts. Transcriptional profiles of adult human dermal fibroblasts grown in four experimental conditions (glass, glass and addition of FGF2, plastic, plastic and addition of FGF2) were compared. Comparison of the transcriptomes will allow to identify significantly differentially expressed genes due to FGF2 and surface treatment, which in turn will allow for identification of the pathways affected by these factors in the human adult fibroblasts.
Project description:The current study set to determine the effects of Fibroblast Growth Factor 2 and cell culture surface (tissue culture plastic and glass) on the transcriptome of adult human dermal fibroblasts. Transcriptional profiles of adult human dermal fibroblasts grown in four experimental conditions (glass, glass and addition of FGF2, plastic, plastic and addition of FGF2) were compared. Comparison of the transcriptomes will allow to identify significantly differentially expressed genes due to FGF2 and surface treatment, which in turn will allow for identification of the pathways affected by these factors in the human adult fibroblasts. Transcriptome analysis of adult human dermal fibroblasts grown on tissue culture plastic and glass, with and without 4ng/ml FGF2, was performed in two biological replicates and two technical replicates for each treatment condition.
Project description:Primary human dermal fibroblasts were isolated from an individual showing a genetic defect in innate antiviral immunity associated with profound failure of type 1 interferon signaling and absence of the STAT2 protein. The transcriptional response of these cells to a 10h treatment with interferon-alpha was compared with that of control dermal fibroblasts. We used microarrays to detail the global program of gene expression resulting from interferon-alpha treatment in STAT 2 deficient and control fibroblasts. Primary human dermal fibroblasts were grown in the presence or absence of 1,000U/ml interferon-alpha (IFNa) for 10 hours before RNA extraction and hybridization on Affymetrix microarrays.
Project description:In search for factors, overexpression of which in human dermal fibroblasts causes direct conversion to cells similar to keratinocytes, micro RNA expression profiles of human primary keratinocytes and human primary dermal fibroblasts are investigated. Skin samples obtained from 3 different sites of 1 subject were used for establishment of 3 primary keratinocytes and 3 primary dermal fibroblasts. Thus obtained 3 primary keratinocytes and primary dermal fibroblasts underwent micro RNA profiling.
Project description:To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. EMSA confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50X methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. mRNA-seq of primary female mouse dermal fibroblasts with and without thapsigargin identified differentially expressed genes. Genes that are commonly bound by CEBPB and ATF4 to TGAT|GCAA (the best-bound 8-mer in the array) at the promoters were highly expressed and up-regulated or remained unchanged in the thapsigargin treated primary female mouse dermal fibroblasts. RNA-Seq: Examination of whole genome transcriptome profiles (RNA-seq) of primary mouse dermal fibroblasts with and without Thapsigargin treatment ChIP-Seq: Examination of transcription factor binding in dermal fibroblasts with and without Thapsigargin teratment BS-Seq: Determination of whole genome DNA methylation profiles (BS-seq) of primary mouse dermal fibroblasts
Project description:Primary human dermal fibroblasts were isolated from an individual showing a genetic defect in innate antiviral immunity associated with profound failure of type 1 interferon signaling and absence of the STAT2 protein. The transcriptional response of these cells to a 10h treatment with interferon-alpha was compared with that of control dermal fibroblasts.