Project description:The CCAAT/enhancer-binding proteins (CEBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. Here, we generated CEBP-beta (CEBPB) and CEBP-epsilon (CEBPE) double-knockout (bbee) mice and compared their phenotypes to those of single-deficient (bbEE and BBee) and wild-type (BBEE) mice. The bbee mice were highly susceptible to fatal infections and died within 2-3 months. Morphologically, their neutrophils were blocked at the myelocytes/metamyelocytes stage, and clonogenic assays of bone marrow cells indicated a significant decrease in the number of myeloid colonies of the bbee mice. In addition, the proportion of hematopoietic progenitor cells [Lin(-)Sca1(+)c-Kit(+)] in the bone marrow of the bbee mice was significantly increased, reflecting the defective differentiation of the myeloid compartment. Furthermore, microarray expression analysis of lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-activated bone marrow-derived macrophages from bbee compared to single knockout mice revealed decreased expression of essential immune response-related genes and networks, including some direct CEBP targets such as Marco and Clec4e. Overall, the phenotype of the bbee mice is distinct from either the bbEE or BBee mice, demonstrating that both transcription factors are crucial for the maturation of neutrophils and macrophages, as well as the innate immune system, and can at least in part compensate for each other in the single knockout mice. To rule out the regulatory influence of both CEBPB and CEBPE on macrophage-related genes, expression analysis of bone marrow-derived macrophages was performed. Macrophages were derived from murine bone marrow with the use of murine M-CSF. The macrophages were stimulated with both LPS (100 ng) and IFN-gamma (100 ng) for 24h, and RNA was extracted for array analysis. Overall, RNA was extracted from stimulated macrophages of one WT mouse, one CEBPB-KO mouse, one CEBPE-KO mouse and one double-KO mouse.
Project description:Gene expression from WT and NFAT5 KO primary macrophage cultures. Keywords: Bone-marrow derived macrophages. We analyzed 4 arrays from each condition: unstimulated WT BMDMs, LPS stimulated WT BMDMs, unstimulated KO BMDMs, LPS stimulated KO BMDMs.
Project description:Purpose: The goal of this study is to identify differential expression profiles between WT and Setd2-KO in BMDMs induced by LPS for a time-course stimulaiton. Methods: We profiled RNA-seq in bone marrow-derived macrophages stimulated with LPS for 0h, 12h, 24h. Results: We find significant up and down regulated genes in Setd2-KO BMDMs under LPS stimulation.
Project description:Conditioned medium (CM) from bone marrow derived macrophages untreated or treated with LPS was collected and filtered through a 0.22-μm filter. The filtered CM was sequentially fractionated with 50-kDa and 100-kDa Amicon filters. The 50–100 kDa fraction of CM was analyzed by mass spectrometry.
Project description:ATAC-seq profiling of Nfat5 KO and wild type macrophages derived from bone marrow (primary cells), treated or not with Lipopolysaccharide (LPS).
Project description:The nuclear orphan receptor Nur77 (NR4A1, TR3, or NGFI-B) has been shown to exhibit an anti-inflammatory function in macrophages. To further elucidate the role of Nur77 in macrophage physiology, we compared the transcriptome of bone marrow-derived macrophages (BMM) from wild-type (WT) and Nur77-knockout (KO) mice both before and after stimulation with IL4 or LPS. Comparison of gene expression in bone marrow-derived macrophages, isolated from 3 wild-type (control) and 3 Nur77-/- mice (case), left untreated or stimulated in triplicate for 8 hours with LPS or IL-4
Project description:Bone marrow-derived macrophages, Unstimulated DMSO Bone marrow-derived macrophages, Unstimulated + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 1h DMSO Bone marrow-derived macrophages, LPS 1h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 2h DMSO Bone marrow-derived macrophages, LPS 2h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 4h DMSO Bone marrow-derived macrophages, LPS 4h + I-BET (GSK525762A) LPS (100 ng/mL) was purchased from Sigma. Bone Marrow-derived macrophages (BMDMs) were differentiated from C57BL/6 bone marrow using 5 ng/mL each of recombinant M-CSF and IL-3 (Peprotech) for 7 days as described (Jeffrey et al, Nature Immunology, 2006). 2 x10^6 BMDMs were treated with DMSO or 1 μM of I-BET for 30 minutes before the addition of LPS (100 ng/mL) for 1, 2 or 4h. Unstimulated control samples were incubated with I-BET only for 1 hour. 500 ng of total RNA from 3 independent samples per group was used to prepare biotin-labeled RNA using Ambion Illumina TotalPrep RNA Amplification Kit (Applied Biosystems) and hybridized to Illumina MouseRef-8 v2.0 expression BeadChip kits. The chips were scanned using Illumina BeadArray Reader. 3 biological replicates and 4 timepoints
Project description:Bone marrow-derived macrophages, Unstimulated DMSO Bone marrow-derived macrophages, Unstimulated + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 1h DMSO Bone marrow-derived macrophages, LPS 1h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 2h DMSO Bone marrow-derived macrophages, LPS 2h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 4h DMSO Bone marrow-derived macrophages, LPS 4h + I-BET (GSK525762A) LPS (100 ng/mL) was purchased from Sigma. Bone Marrow-derived macrophages (BMDMs) were differentiated from C57BL/6 bone marrow using 5 ng/mL each of recombinant M-CSF and IL-3 (Peprotech) for 7 days as described (Jeffrey et al, Nature Immunology, 2006). 2 x10^6 BMDMs were treated with DMSO or 1 μM of I-BET for 30 minutes before the addition of LPS (100 ng/mL) for 1, 2 or 4h. Unstimulated control samples were incubated with I-BET only for 1 hour. 500 ng of total RNA from 3 independent samples per group was used to prepare biotin-labeled RNA using Ambion Illumina TotalPrep RNA Amplification Kit (Applied Biosystems) and hybridized to Illumina MouseRef-8 v2.0 expression BeadChip kits. The chips were scanned using Illumina BeadArray Reader.