Project description:Primary murine hematopoietic cells were retrovirally transduced with Hoxa9 cDNA, expanded in vitro before undergoing secondary infection with the cDNA of Meis1, Prep1 and the Prep1-MC mutant. RNA was extracted and transcriptomes were analyzed. Background corrections and normalisations were performed using RMA in NimbleScan 2.5
Project description:EL4 cells were treated or not with 20nM rapamycin for 48h. Total RNA was extracted from rapamycin-treated and untreated EL4 cells with TRIzol RNA reagent (Invitrogen) as instructed by the manufacturer. Samples were purified using DNase (Qiagen, Mississauga, ON, Canada) and the RNeasy Mini kit (Qiagen), and the overall quality was analyzed with the 2100 Bioanalyzer (Agilent Technologies). Purified RNA (10 ?g/sample) was hybridized on MM8 385K NimbleGen chips according to the manufacturer's instruction. Arrays were scanned using a GenePix4000B scanner (Axon Instruments, Molecular Devices, Sunnyvale, CA) at 5 ?m resolution. Data were extracted and normalized using the NimbleScan 2.4 extraction software (NimbleGen Systems, Madison, WI). Further microarray analyses were performed using GeneSpring GX 7.3.1.
Project description:Global gene expression profiles of primary Baf250a delta/delta and WT FL-derived stromal cell cultures. <br><br>Normalized data files with gene level identifiers are available as additional files on the FTP site for this experiment. Background corrections and normalisations were performed using RMA in NimbleScan 2.5
Project description:Neutrophils are one of the first responders to infection and are a key component of the innate immune system through their ability to phagocytose and kill invading pathogens, secrete antimicrobial molecules and produce extracellular traps. Neutrophils are produced in the bone marrow, circulate within the blood and upon immune challenge migrate to the site of infection. We wanted to understand whether this transition shapes the mouse neutrophil protein landscape, how the mouse neutrophil proteome is impacted by systemic infection and perform a comparative analysis of human and mouse neutrophils. Using quantitative mass spectrometry we reveal tissue-specific, infection-induced and species-specific neutrophil protein signatures. We show a high degree of proteomic conservation between mouse bone marrow, blood and peritoneal neutrophils, but also identify key differences in the molecules that these cells express for sensing and responding to their environment. Systemic infection triggers a change in the bone marrow neutrophil population with considerable impact on the core machinery for protein synthesis and DNA replication along with environmental sensors. We also reveal profound differences in mouse and human blood neutrophils, particularly their granule contents and their receptor repertoires. Our proteomics data provides a valuable resource for understanding neutrophil function and phenotypes across species and model systems.
Project description:This study describes the changes in epigenetic chromatin modifications during murine hematopoietic stem cell differentiation in vivo using a modified miniChIP-chip technology. We have addressed issues including bivalent (H3K4me3/H3K27me3) modifications, lineage priming hypothesis, and stem cell chromatin properties in our study described in Weishaupt et al., 2009 (Blood) Comparison of 4 murine hematopoietic stem, progenitor and mature cell types directly isolated from primary tissues using FACS. HSCs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1+, CD150+, Flk2/Flt3- (LSKCD150+ cells). MPPs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1+, CD150-, Flk2/Flt3+ (LSKCD150- cells). PreMegEs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1-, CD150+, CD105-, FcgRI/IIlo and CD41- cells as described in Pronk et al., 2007. Splenic-derived CD4+ T cells are phentypically identified as CD4+, CD8-, B220-, Nk1.1- cells as described in Rolf et al., 2008.
Project description:we studied the role of exosomes isolated from M.tb infected macrophages in modulating the macrophage response to IFN-γ. Nimblegen microarray gene expression studies were used to compare the suppression of IFN-γ inducible genes by exosomes relative to the virulent strain of M.tuberculosis. Overall our study suggest that exosomes, as carriers of M.tb pathogen associated molecular patterns (PAMPs), may provide a mechanistic link by which M.tb may exert its suppression of host immune response beyond the infected cell, and implies a physiological role for exosomes in immune surveillance of TB. Macrophages were treated with exosomes, infected with M.tb H37Rv or left untreated for 18 hours followed by +/- IFN-γ for an additional 18 hours. Cells were harvested and RNA was isolated and converted to double stranded cDNA and subsequently labeled and hybridized onto Mus musculus 4×72 Nimblegen microarray using Nimblegen Hybridization system 4 according to manufacturer’s instructions (Roche)
Project description:A20 is a negative regulator of NF-κB signaling, crucial to control inflammatory responses and ensure tissue homeostasis. A20 is thought to restrict NF-κB activation both by its ubiquitin-editing activity as by non-enzymatic activities. Besides its role in NF-κB signaling, A20 also acts as a protective factor inhibiting apoptosis and necroptosis. Because of the ability of A20 to both ubiquitinate and deubiquitinate substrates and its involvement in many cellular processes, we hypothesized that deletion of A20 might generally impact on protein levels, thereby disrupting cellular processes. We performed a differential proteomics study of bone marrow derived macrophages (BMDMs) from control and myeloid-specific A20 knockout mice, both in untreated conditions and after LPS and TNF treatment, and demonstrate proteome-wide changes in protein expression upon A20 deletion. Several inflammatory proteins are up-regulated in the absence of A20, even without an inflammatory stimulus. Depending on the treatment and the time, more proteins are regulated. Together these changes may affect multiple signaling pathways disturbing tissue homeostasis and inducing (autoimmune) inflammation, as suggested by genetic studies in patients.
Project description:Mesenchymal stromal cells (MSCs) are used to treat infectious and immune diseases and disorders; however, its mechanism(s) remain incompletely defined. Here we find that MSCs lacking Pinch1/2 proteins display dramatically reduced ability to suppress lipopolysaccharide (LPS)-induced acute lung injury and dextran sulfate sodium (DSS)-induced inflammatory bowel disease in mice. Mice with Pinch loss in MSCs have severe defects in both immune and hematopoietic functions, resulting in premature death, which can be restored by intravenous injection of wild-type but not Pinch-deficient MSCs.