Project description:We used microarray analysis to investigate if keratinocytes excert an immuno-inflammatory response towards streptococcal M1 protein. Keratinocytes respond to M1 protein by activating pathways to induce the production of inflammatory signaltransducers, AP-1 and NFkB transcription factor subunits, together with inflammatory mediators such as IL-8, CCL20 and IL-1 family proteins etc.
Project description:As part of our study in understanding the role of SP140 in inflammatory pathways in macrophages, we inhibited SP140 mRNA using siRNA. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors (from Sanquin Institute Amsterdam or from GSK Stevenage Blood Donation Unit) by Ficoll density gradient (Invitrogen). CD14+ monocytes were positively selected from PBMCs using CD14 Microbeads according to the manufacturer’s instructions (Miltenyi Biotec). CD14+ cells were differentiated with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) (R&D systems) for 3 days followed by 3 days of polarization into classically activated (inflammatory) M1 macrophages (100 ng/mL IFN-γ; R&D systems). M1 macrophages were transfected with siGENOME human smartpool SP140 siRNA or non-targeting scrambled siRNA for 48h with DharmaFECT™ transfection reagents according to manufacturer’s protocol (Dharmacon). The cells were left unstimulated or stimulated with 100 ng/mL LPS (E. coli 0111:B4; Sigma) for 4h (for qPCR) or 24h (for Elisa). The cells were lysed (ISOLATE II RNA Lysis Buffer RLY-Bioline) for RNA extraction.150 ng total RNA was labelled using the cRNA labelling kit for Illumina BeadArrays (Ambion) and hybridized with Ref8v3 BeadArrays (Illumina). Arrays were scanned on a BeadArray 500GX scanner and data were normalized using quantile normalization with background subtraction (GenomeStudio software; Illumina). This submission only contains processed data
Project description:We used microarrays to investigate if keratinocytes excert an immuno-inflammatory response towards M1 protein. Keratinocytes respond to M1 protein by activating pathways to induce the production of inflammatory signaltransducers, AP-1 and NFkB transcription factor subunits together with inflammatory mediators such as IL-8, CCL20 and IL-1 family proteins etc. HaCaT cells were cultured in serum-free media, without calcium, until 80% confluency. Cells were incubated in absence or presence of M1 protein before RNA extraction. The integrity of RNA quality was analysed by an Bioanalyzer. Out of 12 samples, 3 M1 stimulated and 3 control samples were chosen for hybridization on Affymetrix Human 2.0 ST array.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.