Project description:Here we performed a RNA-seq experiment on samples of adherent cultures of mouse neural stem cells (NS5 cell line) under normal growth conditions and after 4 hours of treatment with the gamma-secretase inhibitor LY 411575. This resulted in the generation of a genome-wide mRNA expression pattern and quantification for these cells in the two conditions.
Project description:Myeloid-derived suppressor cells (MDSCs) are increased by tumor-derived factors and suppress anti-tumor immunity. MDSCs obtained at a late time point after tumor injection had stronger suppressive activity than MDSCs obtained at an early time point, as measured by T cell proliferation assays. To find factors in MDSCs that change during tumor growth, we analyzed gene expression profiles from MDSCs at different time points after tumor injection. We found that immune response-related genes were down-regulated, but pro-tumor function-related genes were up-regulated in both Mo-MDSCs and PMN-MDSCs at the late time point. Among differentially expressed genes, FK506 binding protein 51 (FKBP51), which is a member of the immunophilin protein family and plays a role in immunoregulation, was increased in the Mo- and PMN-MDSCs isolated from the late time points. Experiments using siRNA and a chemical inhibitor of FKBP51 revealed that FKBP51 contributes to the regulation of the suppressive function of MDSCs by increasing iNOS, ARG1, and ROS levels and enhancing NF-kappaB activity. Collectively, our data suggest that FKBP51 is a novel molecule that can be targeted to regulate the immunosuppressive function of MDSCs. To identify the factors that licensed MDSCs to be more suppressive as tumors grow, we analyzed gene expression profiles in the two subsets of MDSCs at different time points (3wks, 6wks) during tumor progression. CD11b+Ly-6C(high)Ly-6G(low) Mo-MDSCs and CD11b+Ly-6C(low)Ly-6G(high) PMN-MDSCs were sorted from pooled spleens of naïve mice and Her-2/CT26 tumor-bearing mice. Total RNA was purified and gene expression was analyzed by the Affymetrix GeneChip® Mouse Gene 1.0 ST Array.
Project description:A current paradigm states that monocytes circulate freely and patrol blood vessels, but differentiate irreversibly into dendritic cells or macrophages upon tissue entry. Here we show that bona fide undifferentiated monocytes reside in the spleen and outnumber their equivalents in circulation. The reservoir monocytes are relatively immotile, assemble in clusters in the cords of the subcapsular red pulp, and are distinct from macrophages and dendritic cells. In response to ischemic myocardial injury, splenic monocytes increase their motility, exit the spleen en masse, accumulate in injured tissue and participate in wound healing. These observations uncover a role for the spleen as a site for storage and rapid deployment of monocytes, and identify the splenic monocyte reservoir as a resource that the body exploits to regulate inflammation. The goal of this gene expression study was to compare the gene expression of Ly-6C hi inflammatory monocytes residing in the spleen and their circulating counterparts in the blood. Monocyte subsets of a group of four mice (C57BL/6, 8-12 weeks) were isolated by fluorescence activated cell sorting (FACS Aria, BD biosciences) as CD11bhi (CD90/B220/CD49b/NK1.1/Ly-6G)lo (F4/80/I-Ab/CD11c)lo Ly-6Chi (all antibodies BD biosciences) cells. Samples of 1,000 Ly-6Chi blood and Ly-6Chi splenic monocytes of each mouse were collected directly into 20 M-BM-5l lysis buffer of the PicoPure RNA isolation kit (Arcturus). RNA extraction was subsequently performed according to the manufacturerM-bM-^@M-^Ys instructions (Arcturus). RNA quality was assessed using RNA pico lab chips on the Agilent Bioanalyzer. For all samples a RIN above 8 could be achieved. All further steps were performed at the UCSF Shared Microarray Core Facilities according to standard protocols (http://www.arrays.ucsf.edu and http://www.agilent.com).
Project description:Monocytes and their lineage descendants serve as a central defense system against infection and injury but if uncontrolled can also promote an excessive pathological inflammatory response. Therefore a current research goal is to understand how the organism controls the number and function of monocytes and how these variables can be tailored in therapy. Considering the evidence that monocytes are heterogeneous and exist in at least two subsets committed to divergent functions, we investigated whether distinct factors regulate the balance between monocyte subset responses in vivo. Here we investigate the differential expression of mRNA among murine steady-state monocyte subsets. Blood was drawn from healthy C57BL/6 donors. For each biological replicate 1000 monocytes of the Ly-6Chi and Ly-6Clo phenotype were isolated from the blood sample through fluorescence activated cell sorting.
Project description:We sought to evaluate in an unbiased way the heterogeneity of lung interstitial macrophages and their relationship with alveolar macrophages, lung Ly-6Chi classical monocytes and Ly-6Clo patrolling monocytes, by single cell RNA-Seq.
Project description:The aim of the experiment was to identify genome wide binding sites for retinoic acid receptor beta (RARB) in RARB agonist treated human metastatic pancreatic ductal adenocarcinoma cells (SUIT2). Datasets are prsented for the ChIP-seq analysis for SUIT2 cells after 72 h treatment with either DMSO (vehicle control), 1 µM RAR-β agonist (CD 2314, Tocirs 3824), or 1 µM RAR-β antagonist (LE 135, Tocris 2021).
Project description:The effect of transforming growth factor beta receptor 1 (TGFBR1) inhibitor (LY-364947) treatment was compared to untreated embryos. Experimental details and analyzed data are available in Willaert et al. Human Molecular Genetics 2011; doi: 10.1093/hmg/ddr555 Two-condition experiment, LY-364947 (40microM) treatment vs untreated controls. Biological replicates: 3 LY-364947 treatment replicates compared to a pooled sample of 3 untreated control replicates with dye swap.
Project description:Activation of the serine/threonine kinase Akt contributes to the formation, maintenance, and therapeutic resistance of cancer, which is driving development of compounds that inhibit Akt. Phosphatidylinositol ether lipid analogues (PIAs) are analogues of the products of PI3K that inhibit Akt activation, translocation, and the proliferation of a broad spectrum of cancer cell types. To gain insight into the mechanism of PIAs, time-dependent transcriptional profiling of 5 active PIAs and the PI3K inhibitor LY294002 (LY) was performed in non-small cell lung cancer (NSCLC) cells using high-density oligonucleotide arrays. Gene ontology analysis revealed genes involved in apoptosis, wounding response, and angiogenesis were upregulated by PIAs, while genes involved in DNA replication, repair and mitosis were suppressed. Genes that exhibited early differential expression were partitioned into 3 groups; those induced by PIAs only (DUSP1, KLF6, CENTD2, BHLHB2, PREX1), those commonly induced by PIAs and LY (TRIB1, KLF2, RHOB and CDKN1A), and those commonly suppressed by PIAs and LY (IGFBP3, PCNA, PRIM1, MCM3 and HSPA1B). Increased expression of the tumor suppressors RHOB (RhoB), KLF6 (COPEB) and CDKN1A (p21Cip1/Waf1) was validated as an Akt-independent effect that contributed to PIA-induced cytotoxicity. Despite some overlap with LY, active PIAs have a distinct expression signature that contributes to their enhanced cytotoxicity. H157 cells were plated at 2x10^6 in T-75 flasks in RPMI medium 1640 containing 10% FBS and incubated for 24h. The media was then changed to RPMI medium 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM PIA6 dissolved in DMSO for 0h, 2h, 6h or 12h, and an equal volume of DMSO was added to control samples. For the PIA comparison, 10 microM PIAs (5, 6, 7, 23, 24, 25) or 10 microM LY294002 (LY) were incubated with the cells for 6h. In the PIA comparison experiment, the same concentration of PIA7 (an inert analog consisting of only the lipid side chain) was used as a control. Following incubation, the alterations in cellular morphology were photographed, and cells from 6-well plates were harvested for immunoblot analysis. Total RNA was extracted from cells treated in T-75 flasks using TRIzol reagent (Invitrogen) and chloroform, and purified according to the RNeasy midiprep spin kit protocol (Qiagen). Oligonucleotide microarray was performed with dye-swap. Microarray chips were generated from the 34,580 longmer probe set Human Genome Oligo Set Version 3.0 (Qiagen). Protocols for cDNA labeling, hybridization, and scanning are available through the National Human Genome Research Institute microarray core.
Project description:We developed a simplified flow cytometry strategy in order to discriminate monocytes and macrophages in the lung of C57BL/6 mice. Using this strategy, we identified autofluorescent F4/80+ CD11c+ alveolar macrophages, non-autofluorescent CD64+Ly-6C- interstitial macrophages and Ly-6Chi monocytes residing in the lung of WT mice. A fraction of these Ly-6Chi monocytes corresponded to classical blood monocytes associated with the lung vasculature, but another fraction did not depend on CCR2, the chemokine receptor required for monocytes to egress from the bone marrow, as a population of lung Ly-6Chi monocytes was also present in the lung of Ccr2-/- mice. A remaining question was whether lung monocytes represented a particular population of monocytes that could be distinguishable from the classical CCR2-dependent blood monocytes. To address this issue, we performed a transcriptomic comparison of Ly-6Chi monocytes recovered from flushed lung of WT mice (â60% of CCR2- dependent classical blood monocytes and â40% of lung monocytes) and Ccr2-/- mice (more than 95% of lung monocytes). In addition, we tested whether exposure to TLR ligands would affect interstitial macrophages, and we compared to transcriptome of IM at steady-state and IM 1 week after administration of 50 µg CpG-DNA intratracheally.
Project description:Effect of all trans retinoic acid and the novel retinoid, ST1926, on the profile of gene expression in F9 teratocarcinoma sublines characterized by the presence or absence of the RAR gamma nuclear retinoic acid receptor