Project description:We previously found that a native lipoprotein mix with a high VLDL+LDL/HDL ratio causes a global de novoDNA methylation in THP-1 macrophages. In the present experiment we assessed the consequences of global lipoprotein-induced de novo DNA methylation on global gene expression in the same cells. Moreover, we sought to use gene expression array data to measure RNA expression levels for candidate factors mediating the epigenetic effects of lipoproteins. Experiment Overall Design: Human native VLDL, LDL and HDL lipoproteins were isolated from buffy coats, fractionated by ultracentrifugation, stored at -80deg., desalted to PBS before usage and kept at 4deg. for a maximum of 7d. THP-1 monocytes were differentiated to macrophages, stimulated for 24h with a mix of 68.8mg/ml VLDL, 32.1mg/ml LDL, 91.1mg/ml HDL or unstimulated (control), in serum-free medium with 2% BSA. Three independently isolated lipoprotein samples were used in triplicate.
Project description:We previously found that a native lipoprotein mix with a high VLDL+LDL/HDL ratio causes a global de novoDNA methylation in THP-1 macrophages. In the present experiment we assessed the consequences of global lipoprotein-induced de novo DNA methylation on global gene expression in the same cells. Moreover, we sought to use gene expression array data to measure RNA expression levels for candidate factors mediating the epigenetic effects of lipoproteins. Keywords: comparison treated vs. control
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: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.