Project description:MiRNAs play pivotal roles in regulating macrophage functions, including differentiation, polarization, recruitment, and activation of inflammation. in this review, to provide novel insight into macrophage differentiation-specific mitomiR signatures and associated mitochondrial pathways, we performed a global miRNA profiling on extracted mitochondria and total cell content of human macrophages. The THP-1 monocytic cell line was treated with phorbol-12-myristate-13-acetate (PMA) to induce differentiation into macrophages The human monocytic cell line THP-1 cells were differentiated into THP-1 derived macrophages with 40 ng/mL phorbol 12-myristate 13-acetate (PMA),The mitochondrial fraction of the cells were enriched and purified using anti-TOM22 microbeads (Miltenyi Biotec). After total RNA extraction, and using Taqman low-density arrays (TLDA), we performed the global miRNA profiling on extracted mitochondria and compared with total cell content
Project description:Eicosanoids are potent regulators of gene expression of inflammatory cells. Pro- (leukotrienes B4 and C4) and anti-indflammatory (lipoxins A4 and B4) eicosanoids have been described in the literature but the detailed impact of these lipid mediators on the gene expression pattern of monocytic cells has not been studied in detail. We cultured the permanent monocytic cell line MonoMac 6 for 12 h in the absence (solvent control) and presence of these eicosanoids and quantified the differential gene expression patterns using the microarray technology. Experiment Overall Design: MonoMac6 cells were grown according to the recommendations of the German Tissue and Cell Culture Collection (Braunschweig) to near confluency. Then 100 nM of the eicosanoids (leukotriene B4, leukotriene C4, lipoxin A4, lipoxin B4) were added to the culture medium as DMSO stock solution and the cells were further cultured for 12 h with the stimuli. Then the cells were harvested, washed and total RNA was extracted according to the Qiagen protocol. Total RNA was used for microarray hybridization.
Project description:Transcriptome analysis of the human monocytic cell line THP1 (ATCC TIB-202) treated or not with phorbol myristate acetate (PMA) +/- lipopolysaccharides-lipopolysaccharides binding protein (LPS-LBP)
Project description:We performed proteomics of mitochondria isolated from leukemia cell line. Digested peptides were labeled with TMT and separated in 9 fractions by High-pH fractionation Kit. Peptides were analyzed by SPS-MS3 mode.
Project description:Treatment with calcitriol, a specific VDR agonist, augmented the heterodimerization between VDR and RXR. We also wanted to investigate its impact on VDR binding to genomic regions. ChIP-seq experiments were carried out with the human monocytic THP-1 cell line under either vehicle (1:1 DMSO-ethanol) or calcitriol (100 nM) treatment conditions. Our finding is that calcitriol may have both a stimulatory or an inhibitory effect on VDR binding depending on the presence of its specific response element (VDRE), a less specific nuclear receptor (NR) half-site or absence thereof ("None"). Upon calcitriol activation, VDRE-containing genomic regions showed considerably higher occupancies on average than in the control, vehicle-treated sample. A similar induction, but to a much lesser extent, was detected for the NR half-site-containing regions. In contrast, genomic regions not containing any of these specific response elements did not show any induction upon calcitriol treatment; they rather showed a decrease of binding in promoter regions. We can conclude that ligand-induced heterodimerization and binding of the NR to its response elements are correlated events.
Project description:The human leukemia cell line U937 is a well-established model for studying monocytic cell differentiation. We used a modified protocol (SADE) of serial analysis of gene expression (SAGE) and developed a SADE linker-anchored PCR assay to investigate the pattern of expression of known genes and to identify new transcripts in proliferating cells and during cell growth arrest and differentiation. We implemented new informatic tools to compare expression profiles before and after exposure of cells to differentiation inducers. From the analysis of 47,388 tags, we identified 13,806 distinct transcripts, 265 of which showed significant variations (P<0.01). Among 1219 well-identified genes, major changes concerned transcription and translation components, cytoskeleton, and macrophage-specific genes. Nearly half of the tags, some of them expressed at high levels, matched partially characterized genes or ESTs, or revealed yet-unknown transcripts, providing a wealth of new candidate genes that may reveal novel aspects of terminal monocytic differentiation. Keywords = 12213207 Keywords: other