Changes in gene expression profile in mammalian cells upon Raf activation
ABSTRACT: Comparison of the changes in the gene expression profile of cells in which there is an activation of the Raf oncogene in the absence of any other extracellular signals Keywords: Response to oncogenic activation of the MAPK pathway Overall design: Primary rat Schwann cells containing an inducible RafER allele were plated on poly-L-lysine plus fibronectin coated dishes on DMEM + 3% FCS containing medium. Next day cells were transferred to minimal medium (DMEM containing 100 μg ml-1 transferrin, 100 μg ml-1 bovine serum albumin, 16.1 μg ml-1 putrescine, 39 ng ml-1 selenium) for 24 h. Cells were then treated with either the vehicle (EtOH) or with Tamoxifen for 24 h. Three independent experiments were performed.
INSTRUMENT(S): [Rat230_2] Affymetrix Rat Genome 230 2.0 Array
Project description:Comparison of the changes in the gene expression profile of cells in which there is an activation of the Raf oncogene in the absence of any other extracellular signals Experiment Overall Design: Primary rat Schwann cells containing an inducible RafER allele were plated on poly-L-lysine plus fibronectin coated dishes on DMEM + 3% FCS containing medium. Next day cells were transferred to minimal medium (DMEM containing 100 μg ml-1 transferrin, 100 μg ml-1 bovine serum albumin, 16.1 μg ml-1 putrescine, 39 ng ml-1 selenium) for 24 h. Cells were then treated with either the vehicle (EtOH) or with Tamoxifen for 24 h. Three independent experiments were performed.
Project description:In order to investigate genes regulated by Wnt/Beta-catenin-signaling in immortalized mouse adrenocortical cells, we treated a pair of ATCL7 cell cultures, one with BIO, a small molecule mimicking Wnt/Beta-catenin-signaling, the other with a control treatment. We repeated this 3 additional times resulting in 4 pairs of samples. The Wnt/beta-catenin pathway is not basally active in ATCL7 cells, nor do these cells appear to contain any mutations in the Wnt/Beta-catenin pathway. ATCL7 cells were grown under standard conditions at 37°C in a humidified incubator containing 5% CO2. 250,000 ATCL7 cells per sample were treated with 0.5uM BIO (6-Bromoindirubin-3'-oxime) or 0.01% DMSO (v/v) for 24 hours, in DMEM:F12 growth media containing 100U/mL pencillin/streptomycin, 1X insulin-transferrin-selenium-X, 0.025% fetal bovine serum and 0.025% horse serum. Cells were harvested and RNA was extracted using an RNeasy Plus Mini Kit (Qiagen). Biotinylated cDNA were prepared according to the Ambion WT kit protocol from 250 ng total RNA (GeneAtlas™ WT Expression Kit User Manual P/N 702935 Rev. 3). We assayed the targets with Affymetrix Mouse Gene ST 1.1 strip arrays. We modeled the data using paired T-tests for each probe-set. We also supply a supplementary file holding the data and some statistical analysis, as well as probe-set annotation that we used at that time (users may wish to obtain new annotation though). We analyzed only 28944 probe-sets with category "main", "---", and "flmrna->unmapped" according to Affymetrix annotation. ATCL7 cells were grown under standard growth conditions at 37°C in a humidified incubator containing 5% CO2. 250,000 ATCL7 cells per sample were treated for 24 hours with 0.5uM BIO (6-Bromoindirubin-3'-oxime) or 0.01% DMSO (v/v) in growth media containing 100U/mL pencillin/streptomycin, 1X insulin-transferrin-selenium-X, 0.025% fetal bovine serum and 0.025% horse serum. Cells were harvested and RNA was extracted using an RNeasy Plus Mini Kit (Qiagen). Biotinylated cDNA were prepared according to the Ambion WT kit protocol from 250 ng total RNA (GeneAtlas™ WT Expression Kit User Manual P/N 702935 Rev. 3). We assayed the targets with Affymetrix Mouse Gene ST 1.1 strip arrays.
Project description:Moderate selenium deficiency may lead to an impaired capacity to cope with health challenges. Functional effects of suboptimal selenium intake are not fully known, and biomarkers for an insufficient selenium supply are inadequate. We therefore fed mice diets of moderately deficient or adequate selenium intake for 6 weeks. Changes in global gene expression were monitored by microarray analysis in splenic leukocytes. Genes for four selenoproteins, Sepw1, Gpx1, Selh and Sep15, were the most significantly down-regulated in moderate selenium deficiency, and this was confirmed by quantitative polymerase chain reaction (qPCR). Classification of significantly affected genes revealed that processes related to inflammation, heme biosynthesis, DNA replication and transcription, cell cycle and transport were affected by selenium restriction. Down-regulation by moderate selenium deficiency of specific genes involved in inflammation and heme biosynthesis was confirmed by qPCR. Myeloperoxidase and lysozyme activities were decreased in selenium-restricted leukocytes, providing evidence for functional consequences. Genes for 31 nuclear factor (NF)-κB targets were down-regulated in moderate selenium deficiency, indicating an impaired NF-κB signaling. Together, the observed changes point to a disturbance in inflammatory response. The selenoproteins found here to be sensitive to selenium intake in murine leukocytes might also be useful as biomarkers for a moderate selenium deficiency in humans. Overall design: Male C57BL/6J mice (3–4 wk of age) from Charles River (Sulzfeld, Germany) were randomly assigned to the selenium-deficient or selenium-adequate group (12 mice per group) with free access to food and water. The selenium-adequate diet (Se-adeq) was produced by mixing selenomethionine (Acros, Geel, Belgium) into the selenium-deficient diet (Se-def; No. C1045 with 50% carbohydrates, 17% protein, 5% fat, 4% fibre, and mixture of micronutrients; Altromin, Lage, Germany) containing 0.086mg Se/kg (Riese et al., Endocrinology 2006) to yield a selenium content of 0.15 mg/kg corresponding to the dietary reference intake for mice. Diets were fed as powder for 6wk until mice were killed in the non-fasted state. Animals were anesthetized with isofluran and blood with heparinized capillaries by puncture of the retroorbital plexus. Anesthetized animals were killed by cervical dislocation. Spleens were removed aseptically, placed on a sterile microscope slide and crushed with the end of a 6-ml syringe plunger. Released cells were diluted in 5ml of cell culture medium (RPMI with 5% fetal calf serum; Gibco, Karlsruhe, Germany). Clumps were dispersed by drawing and expelling the suspension repeatedly through a 6-ml syringe with a 20-gauge needle. A 100-μmmesh was used to remove clumps and particles and to receive a single cell suspension. The mesh was rinsed with 5 ml RPMI. After centrifugation (5 min, 200g) and washing with 10 ml RPMI, erythrocytes were lysed for 5 min in 5 ml of ammonium chloride lysis buffer (0.15 mol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L Na2EDTA, pH 7.4). Thereafter, 15 ml of balanced salt solution containing 0.2% bovine serum albumin was added, and the suspension was centrifuged for 5 min at 200g. The pellet was suspended in 1 ml phosphate-buffered saline (PBS) and transferred into a 2-ml tube. Thirty microliters of the suspension was smeared on microscope slides and dried on air for counting. The cells of the remaining suspension were pelleted (5 min, 200g) and washed with 1 ml PBS. The pellet was frozen at−80°C until RNA isolation. As determined with May–Grünwald–Giemsa staining, the leukocyte population was independent of the selenium status and on average was composed of 79% lymphocytes, 18% granulocytes and 2% monocytes.
Project description:Human 143B.206 Rho+ cells (depleted mitochondrial DNA) and cytoplasts (derived from mitochondrial patient fibroblast containing C1624T mutation in mitochondrial tRNAVal ) were previously used to generated transmitochondrial cybrid lines. Hek293T cells,143B cells and cybrid cells were cultured in DMEM supplemented with 10% FCS in 5% CO2 at 37 °C. Cells were treated with Chloramphenicol (100 μg/ ml) for 15 min before harvest. Cells were then lysed and RNase I digested. Lysates were then loaded on a sucrose gradient (7%-47%). Fractions containing mito-monosomes were used to isolated RNA which were used to generate the library for deep sequencing. Mitoribosomal profiling were generated for both control (143B and Hek293T) and cybrid cells to reveal aspects of mitoribosome behaviour.
Project description:Immortalized human corneal, conjunctival, and meibomian gland epithelial cells were pre-treated with dihydrotesteosterone (DHT) or vehicle, then exposed to lipopolysaccharide (LPS) and ligand binding protein (LBP) or vehicle, lysed, and subjected to microarray analysis of RNA expression. Overall design: Cells were cultured in keratinocyte serum-free medium (KSFM) supplemented with bovine pituitary extract (BPE; 25 ug/mL HCEC and HCjE, 50 ug/mL HMGEC), 5 ng/mL epidermal growth factor (EGF), penicillin and streptomycin. After reaching confluence, cells were rinsed with PBS and cultured in medium containing DMEM/F12 with 10% FBS, 10 ng/ml EGF, penicillin and streptomycin and either dihydrotestosterone (DHT, 10 nM) or ethanol control for 2 days. After this time period, cells were incubated in serum-free DMEM/F12 and exposed to vehicle (1% bovine serum albumin [BSA]), or LPS (15 μg/ml) and LBP (150 ng/ml), for six hours. The LPS and LBP were dissolved in DMEM and the BSA in PBS.
Project description:The mouse embryonic stem cell’s differentiation was guided by several treatments, and each stage of differentiation was examined. Keywords: development stage Overall design: EBs were dissociated and plated onto tissue culture dishes in the serum-free medium containing Insulin/Transferrin/Selenium/Fibronectin (ITSF). After 6 days, the cells were expanded and plated onto polyornithine (15 mg ml-1) and laminin-coated plates (Becton Dickinson Labware, Bedford, MA) with the N2 medium (Johe, 1996) supplemented with laminin, bFGF (R&D Systems, Minneapolis, MN), murine N-terminal fragment of SHH and murine FGF8 isoform b (R&D Systems) (stage 4) . Finally the cells were induced to differentiated into DA neurons by removal of bFGF. The differentiation medium consisted of N2 medium supplemented with laminin in the presence or absence of cAMP, AA (Sigma, St. Louis, MO). The cells were incubated under differentiation conditions for 6–15 days.
Project description:Throught an reiterative growth factor and chemical screening, we defined a small molecule and growth factor cocktail, including EGF, glycogen synthase kinase 3 inhibitor (CHIR99021), transforming growth factor β receptor inhibitor (e.g. E-616452), lysophosphatidic acid and sphingosine 1-phosphate, that can sustain long-term self-renewal of murine hepatoblasts under chemically defined conditions. The expandable hepatoblasts (eHBs) by this small molecule and growth factor cocktail expressed a set of genes typical of liver progenitor cells and liver development, and retained the ability to respond to liver developmental cues and produce functional hepatocytes and form bile duct-like structures. Moreover, both early- and late-passage eHBs demonstrated a similar transcriptome profile. Microarray analysis also confirmed that the gene expression of cultured cells resembled hepatoblasts. Overall design: Expandable hepatoblasts (eHBs) were routinely cultured in basal medium (DMEM/F12 medium supplemented with insulin-transferrin-sodium selenite, 5 mM nicotinamide, 30 μg/ml 2-phospho-L-ascorbic acid and 50 μg/ml bovine serum albumin human recombinant albumin and 1% penicillin/streptomycin including 10 ng/ml EGF, 3 μM CHIR99021, 2 μM E-616452, 5 μM LPA and 0.5 μM S1P, on Matrigel or laminin-coated surface. The culture was split 1:6 using Accutase. Total RNA was prepared from passage 5 and passage 20 of eHBs (passage 5 and passage 20 of eHBs were derived from two independent experiments) using the RNeasy Plus Mini Kit.
Project description:Supercritical rosemary extract (containing 16.90% carnosic acid, 1.90% carnosol and 13.59% volatile compounds) showed antitumor activity on colon cancer cells in vitro. We treated colon cancer cells with the extract and we employed whole genome microarray expression profiling to identify genes potentially involved in its antitumor mechanism of action. We analyzed gene expression of colon cancer SW620 cells after treating during 48h with supercritical rosemary extract at concentrations that cause 50% inhibition of cell viability (30 μg/mL), citostatic effect (60 μg/mL) and 50% cell death (100 μg/mL), in comparison to control cells (0 μg/mL). Two independent experiments were performed in triplicate. Each sample is the pool of the triplicates of one of the experiments (a or b) at the indicated concentration.
Project description:We sought to identify Hedgehog-regulated genes in mouse cranial neural crest cells (O9-1) Overall design: Mouse cranial neural crest cells (O9-1) were allowed to attach for 24 hrs and media were replaced with DMEM containing 1% FBS ± SHH-N peptide at 0.4 μg/mL. 48 hrs after treatment, cells were lysed and RNA was collected per the GE Illustra RNAspin kits (GE Healthcare) general protocol for a cell monolayer.