Effect of erythropoietin (EPO) and leukemia-inhibitory factor (LIF) on gene expression profiling in differentiating rat CG4-EPOR oligodendrocytes
ABSTRACT: Analysis of the gene expression profile in differentiating rat CG4-EPOR oligodendrocytes treated with EPO, LIF or their combination for 1h or 20h Gene expression was measured undifferentiated CG4-EPOR cells and in differentiating cells at 1h and 20h in 5 conditions: control, EPO 10ng/ml, LIF 0.2ng/ml, LIF 10ng/ml, EPO 10ng/ml+LIF 10ng/ml); 3 or 4 replicates.
Project description:Analysis of the miRNA expression profile in differentiating rat CG4-EPOR oligodendrocytes treated with EPO, LIF or their combination for 1h or 20h Overall design: miRNA expression was measured in undifferentiated CG4-EPOR cells and in differentiating cells at 1h and 20h in 5 conditions: control, EPO 10ng/ml, LIF 0.2ng/ml, LIF 10ng/ml, EPO 10ng/ml+LIF 10ng/ml); 3 or 4 replicates.
Project description:The I/11 and R10 erythroid progenitor cell line was cultivated in serum free medium (StemPro34) supplemented with Epo (0,5U/ml), SCF (100ng/ml) and dexamethasone (10-6M). To identify genes specifically regulated by Epo/SCF-induced polysome recruitment, cells were factor deprived for 4h and subsequently treated for 2h with 5U/ml Epo and 200ng/ml SCF or left untreated. Subsequently we isolated both total and polysome bound mRNA from each condition, which was hybridised to oligonucleotide arrays (Affymetrix). Analysis of data with Rosetta Resolver allowed to identify and compare Epo/SCF induced gene expression in total and polysome bound RNA. To assess gene expression during erythroid differentiation, cells were induced to differentiate in presence of 5U/ml Epo and 0.5mg/ml Fe-loaded transferrin. Polysome bound mRNA was isolated from cells proliferating in presence of Epo, SCF and dexamethasone (renewal conditions), and from cells induced to differentiate for 48h or 60h.
Project description:Erythropoiesis is a tightly regulated process. Development of red blood cells occurs through differentiation of hematopoietic stem cells into more committed progenitors and finally into erythrocytes. Binding of erythropoietin to its receptor (EpoR) is strictly required for erythropoiesis as it promotes survival and late maturation of erythroid progenitors. In vivo and in vitro studies have highlighted the requirement of EpoR signaling through Jak2 tyrosine-kinase and Stat5a/b as a central pathway. Here, we demonstrate that phospholipase C gamma 1 (Plcγ1) is activated downstream of EpoR/Jak2 independently of Stat5. Plcγ1-deficient proerythroblasts and erythroid progenitors exhibited strong impairment in differentiation and colony-forming potential. In vivo, suppression of Plcγ1 in immunophenotypically defined hematopoietic stem cells (Lin-Sca1+KIT+CD48-CD150+) severely reduced erythroid development. To identify Plcγ1 effector molecules involved in regulation of erythroid differentiation we assessed for changes on the level of transcription and DNA methylation after inactivation of Plcγ1. The single common downstream effector was H2AFY2, which encodes for the histone variant macroH2A2 (mH2A2). Suppression of macroH2A2 expression recapitulated the effects of Plcγ1 knockdown on erythroid maturation. Taken together, our findings identify Plcγ1 and its downstream target macroH2A2, as a “non-canonical” signaling pathway essential for erythroid differentiation. MCIP-seq was used to interrogate methylation changes during Epo-induced differentiation of murine I/11 erythroblastic cell line upon knock-down of Plcy1 as compared to a mock control. Two different shRNA constructs that target Plcg1 were investigated at two different time points.
Project description:To gain further insight into the downstream effects of EPO signaling on HCC cells, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to illustrate EPO-induced the proliferation of HCC cells. 1×10e6 7721-EPOR cells were plated in 6 cm dishes and starved for 8 hours and then treated with either NS or EPO (10 IU/ml) for 16 hours. Total RNA was extracted and profiled using oligo microarrays. Differentially expressed genes were then identified through fold change as well as P value calculated with t-test. The threshold set for up- and down-regulated genes was a fold change>= 2.0 and a P value<= 0.05. Afterwards, GO analysis and KEGG analysis were applied to determine the roles of these differentially expressed mRNAs. EPO treated HCC cells up-regulated 14 genes expression and down-regulated 29 gene expression compared to control cells. Overall design: 1×10e6 7721-EPOR cells were plated in 6 cm dishes and starved for 8 hours and then treated with either NS or EPO (10 IU/ml) for 16 hours. Total RNA was extracted from 2 independent experiments.
Project description:To study transcriptional profiles in human CD34+ cell in EPO condition on day 7 and day 14. Experiment Overall Design: After obtaining informed consent, human CD34+ cells were isolated in high purity from the peripheral blood of normal human volunteers. The cells were cultured at a concentration of 104 -105 cells/mL in medium supplemented with 4 U/mL EPO (Amgen, Thousand Oaks, CA) as described previously.1 Cells were enumerated using an electronic cell counter (Coulter, Hialeah, FL). Adult and fetal hemoglobin was analysed by HPLC. RNA was extracted using QIAshredder and Rneasy Minikit.
Project description:Purpose: We found that IFN-g and IL-27 had suppressive effects on ILC2s cultured with IL-33. The goal of this study is to clarify the expressions of RNA induced by IFN-g and IL-27 in ILC2s. Methods: ILC2s were isolated from fat-asociated lymphid clusters (FALC) of wild-type mice. They were cultured with IL-33 (10ng/ml), IL-33 + IFN-g (10ng/ml), or IL-33 + IL-27 (10ng/ml) for 48hrs. RNA was isolated by Allprep DNA/RNA Micro Kit (QIAGEN), and cDNA libraries were prepared by TruSeq RNA Sample Preparation kits v2 (Illumina) according to the manufacturer’s low sample protocol. A HiSeq 1500 system (Illumina) was used for 50 single-end bases (50SE) sequencing. Results: Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to the reference genome (mm9) using Bowtie2 v2.1.0 and TopHat2 v2.0.8. The transcript abundances were estimated as FPKM (fragments per kilobase of exon million fragments mapped) value using Cufflinks v2.1.1. We found that both IL-27 and IFN-g upregulated the expression of STAT1 and IRF1 which are regulated downstream of IFN-g receptor signaling, but there was no difference in the expression of GATA3, a critical transcription factor for ILC2 functions. Conclusions: Our study represents the detailed differences of RNA expressions by RNA-seq technology. RNA-Seq analysis of ILC2s cultured with IL-33 (10ng/ml), IL-33 + IFN-g (10ng/ml), or IL-33 + IL-27 (10ng/ml) for 48hrs.
Project description:Activation of the AKT and ERK signaling pathway is a major contributor to cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell-cycle progression, is poorly understood. Here we study three cell types of hematopoietic origin, in which AKT and ERK signaling is triggered by erythropoietin (Epo). We find that the different cell types exhibit distinct proliferative responses, despite sharing the molecular network for pro-proliferative signaling. Iterating quantitative experiments and mathematical modeling, we show that the cell-type-specific regulation of proliferation emerges from two sources: (1) the protein abundance patterns of signaling components that cause differential flow of signals along the AKT and ERK pathways, and (2) the differential impact of the downstream regulators for protein synthesis and for cell-cycle progression on proliferation. Our integrated mathematical model of Epo-driven proliferation explains cell-type-specific effects of targeted AKT and ERK inhibitors and correctly predicts whether their combined application results in synergy. Overall design: EpoR receptor as well as EpoR and Pten overexpressing BaF3 cells were growth factor deprived for 5 h and resuspended in RPMI supplemented with 1mg/ml BSA. Cells were stimulated with 1U/ml Epo and RNA was extracted at 0, 1, 2, 3, 4, 5, 7 and 18.5 hours after stimulation using the RNeasy Mini Plus Kit (Qiagen, Hilden, Germany).
Project description:Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. We combined mathematical modeling and extensive time-resolved data sets in primary erythroid progenitor cells and dissected the roles of the two transcriptional feedback regulators of the SOCS family, CIS and SOCS3 in JAK2/STAT5 signaling. Our model revealed that both feedbacks are most effective at different ligand concentration ranges. To identify the relevant transcriptional feedback regulators that are involved in attenuation of Epo-induced JAK2-STAT5 signaling in addition to CIS, we performed a time-resolved genome-wide expression profiling of murine erythroid progenitor cells at the colony forming unit erythroid (CFU-E) stage up to 24 and 7 hours after EPO stimulation and in control, repectively. The analysis identified SOCS3 as the only further de novo regulated gene upon Epo-induced JAK2-STAT5 signaling. From subsequent mathematical modeling, we calculated the STAT5 response in previously unobserved Epo concentrations, which provided a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. In conclusion, our combined modeling approach revealed novel insights into the orchestrated action of feedback control to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations. Freshly sorted CFU-E cells were starved for 1 h in Panserin 401 supplemented with BSA and 50 µM β-Mercaptoethanol. Subsequently, cells were stimulated with 0.5 U/ml Epo or left untreated and RNA was extracted at different time points (0,1,2,3,4,5,6,7,8,14,19,24 hrs after Epo stimulation and at 0,1,2,3,4,5,6,7 hrs without Epo treatment) using the RNeasy Mini Plus Kit (Qiagen, Hilden, Germany).