Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Azacytidine time course on MCF7 cells

ABSTRACT: MCF7 cells at 80% confluency were treated with 5 micromolar azacytidine with gene expression profiling determined at 0, 1, 2, 3, 4, 6 and 12 hours of exposure to compound. Keywords: time course Approximately 5,000,000 million cells were plated into 10 cm plates from a common pool. Cells were grown in DMEM supplemented with 10% foetal calf serum until 80% confluency was attained. Cells were then exposed to 5 micromolar azacytidine prepared as a 10 millimolar stock in DMSO. Each time point was represented by three biological replicates. Total RNA was extracted from the cells using the trizol method and RNA samples were validated for integrity throughout the isolation and extraction procedure. All gene expression profiles showed similar distributions with similar raw median intensities.

ORGANISM(S): Homo sapiens

SUBMITTER: George Reid

PROVIDER: E-GEOD-10145 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transient cyclical methylation of promoter DNA.

Kangaspeska Sara S Stride Brenda B Métivier Raphaël R Polycarpou-Schwarz Maria M Ibberson David D Carmouche Richard Paul RP Benes Vladimir V Gannon Frank F Reid George G

Nature 20080301 7183

Methylation of CpG dinucleotides is generally associated with epigenetic silencing of transcription and is maintained through cellular division. Multiple CpG sequences are rare in mammalian genomes, but frequently occur at the transcriptional start site of active genes, with most clusters of CpGs being hypomethylated. We reported previously that the proximal region of the trefoil factor 1 (TFF1, also known as pS2) and oestrogen receptor alpha (ERalpha) promoters could be partially methylated by ...[more]

PMID: 18322535

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Project description:Mouse macrophages J774A.1 were pre-treated with the anti-inflammatory lipid eicosapenaenoic acid (EPA) or the pro-inflammatory lipid ceramide (Cer) for 3h. Macrophages were then infected with Mycobacterium smegmatis for 1h, and total RNA was collected. In a parallel experiment, infected macrophages were infected for 1h, 4h and 24h. At these time points, macrophages were lysed, bacteria was collected and quantified by the Colony Forming Units (CFU) Assay. This provided the kinetics of the killing of Mycobacteria inside mouse macrophages. CFU experiments revealed that cells pre-treated with EPA showed an increased number of bacteria inside macrophages, in contrast to cells pre-treated with Cer. To dissect the molecular mechanisms involved in the survival and killing of mycobacteria infected macrophages, mediated by lipids, gene expression studies were performed. Cultures of Mycobacterium smegmatis mc2155 harbouring a p19-(long lived) EGFP plasmid were grown to exponential growth phase. Bacteria were pelleted, washed in PBS and resuspended in medium DMEM with a multiplicity of infection (MOI) of 10 (10 bacteria per macrophage). Clumps of bacteria were removed by ultrasonic treatment of bacterial suspensions in an ultrasonic water bath for 15 minutes, followed by a low speed centrifugation for 2 minutes. Mouse macrophages J774A.1 were pre-.treated with the anti-inflammatory lipid eicosapenaenoic acid (EPA) or the pro-inflammatory lipid ceramide (Cer), 3h before infection. Mouse macrophages J774A.1 were infected with Mycobacterium smegmatis mc2155 for 1h, at 37M-BM-:C and 5% CO2. After 1h of infection, cells were washed with PBS. After washing, 1 ml Trizol was added per well, to collect total RNA. The experimental condition were: samples 1.1, 1.2, 1.3: Untreated macrophages; samples 2.1, 2.2, 2.3: Mock treated macrophages (ethanol 1ul/ml); samples 3.1, 3.2, 3.3: EPA (15uM) treated macrophages (ethanol 1ul/ml); samples 4.1, 4.2, 4.3: Cer (5ug/ml) treated macrophages (ethanol 1ul/ml); samples 5.1, 5.2, 5.3: Untreated macrophages, infected with Mycobacterium smegmatis mc2155 for 1h; samples 6.1, 6.2, 6.3: Mock treated macrophages (ethanol 1ul/ml) infected with Mycobacterium smegmatis mc2155 for 1h; samples 7.1, 7.2, 7.3: EPA (15uM) treated macrophages (ethanol 1ul/ml) infected with Mycobacterium smegmatis mc2155 for 1h; samples 8.1, 8.2, 8.3: Cer (5ug/ml) treated macrophages (ethanol 1ul/ml) infected with Mycobacterium smegmatis mc2155 for 1h; In a parallel experiment, infected macrophages remained with the bacteria for 1h, 4h and 24h after infection. After these time points, the macrophages were lysed, the bacteria was collected and quantified by the Colony Forming Units (CFU) Assay. This provided the kinetics of the killing of non-pathogenic intracellular bacteria inside mouse macrophages. For the CFU assay, after 1h of infection, cells were washed with PBS and Gentamicin (10ug/ml) in DMEM to kill the extracellular bacteria. At discrete time points, cells were washed with PBS and lysed with sterilized water. Quantitative cultures of bacteria were performed in a 10-fold serial dilutions, inoculated on 7H10 agar plates. 5ul were plated in triplicate and the number of colonies were counted after 48h.

Project description:Background: Epidemiologic associations between acutely increased cardiorespiratory morbidity and mortality and particulate air pollution are well-established, but the effects of acute pollution exposure on human gene expression changes are not well understood. Objectives: In order to identify potential mechanisms underlying epidemiologic associations between air pollution and morbidity, we explored changes in gene expression in humans following inhalation of fresh diesel exhaust (DE), a model for particulate air pollution. Methods: 14 ethnically homogeneous (white males), young, healthy subjects underwent sixty minute inhalation exposures on 2 separate days with clean air (CA) or freshly generated and diluted DE at a concentration of 300 Î¼g/m3 PM2.5. Prior to and 24 hours following each session, whole blood was sampled and fractionated for PBMC isolation, RNA extraction, and generation of cDNA, followed by hybridizationÂ withÂ Agilent Whole Human Genome (4X44K) arrays. Results: Oxidative stress and the ubiquitin proteasome pathway, as well as the coagulation system, were among hypothesized pathways identified by analysis of differentially expressed genes. Nine genes from these pathways were validated using real-time PCR comparing fold change in expression between DE exposed and clean (CA) days. Quantitative gene fold changes generated by real-time PCR were consistent with the directional fold changes from the microarray analysis. Conclusions: Changes in gene expression connected with key oxidative stress, protein degradation, and coagulation pathways are likely to underlie observed physiologic and clinical outcomes and suggest specific avenues and sensitive time points for further physiologic exploration. Diesel exhaust inhalation exposure-induced human gene expression changes were measured in peripheral blood mononuclear cells. 14 white males subjects were seated in a controlled environment facility for 2 separate 60 minute inhalation exposures which occurred at least 1 week apart to either clean air and freshly generate or diluted diesel exhaust (300ug/m3 PM2.5) .Blood collection occurred immediately prior to and 24 hours following exposures. Two microarrays per subject were completed using Agilent Whole Human Genome (4X44K) arrays. Genes that were of interest to our research group were verified using Real Time PCR analysis.

Dataset Information

Azacytidine time course on MCF7 cells

Publications

Transient cyclical methylation of promoter DNA.

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