Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human ciliary smooth muscle cells grown culture WITH 200 nM OF either an prostaglandin E2 receptor SUBTYPE EP2 OR SUBTYPE EP4 selective agonist FOR 6 hours IN comparison TO untreated controls

ABSTRACT: Changes of genome-wide mRNA transcription levels of human ciliary smooth muscle (hCSM) cells were determined by treating hCSM cells in culture with 200 nM of either an prostaglandin E2 receptor subtype EP2 or subtype EP4 selective agonist for 6 hours in comparison to untreated controls. This was followed by competitive hybridization of fluorescent Cy3 or Cy5 labeled cRNA probes derived from the treated versus untreated control total RNA samples onto an Agilent Human Whole Genome Expression oligonucleotide microarray. Log 2 (LN) of the intra-slide ratios (RATIO, PRE_VALUE) of treated versus untreated samples was reported as VALUE in the sample files. Experiment Overall Design: Human Ciliary Smooth Muscle Cell Culture: Experiment Overall Design: Human ciliary smooth muscle (hCSM) cells were isolated from a female donor, received from the San Diego Eye BankÂ® (San Diego, CA) and cultured in DMEM with 10% fetal bovine serum and 0.5% penicillin/streptomycin according to the method reported previously by Woldemussie et al. Experiment Overall Design: For transcriptome analysis, cells were used for experiments starting at passage 4. Three independent experiments at consecutive cell passages were performed. For each experiment 0.6 x 106 cells were plated onto 15 cm dishes in MEM D-Valine medium (PromoCell, Germany) supplemented with 10% FCS, L-glutamine and antibiotics/antimycotics. Cells were grown to ~80% confluency for 6 days, followed by starvation in absence of FCS and presence of 100 nM of the cyclooxygenase inhibitor indomethacin, 20 Âµg/ml fatty acid-free BSA and 4 Âµg/ml transferrin for 17 hours. Cells were then exposed for 6 hours to 200 nM of either the EP2 agonist AGN210937 or the EP4 agonist AGN202280 (Allergan, Irvine, CA) or the corresponding amount of DMSO in the absence of FCS and presence of transferrin. Experiment Overall Design: Total RNA Extraction: Experiment Overall Design: Total RNA was isolated using a RNA isolation kit (RNAqueous; Ambion, USA) which was followed by DNase I treatment (Turbo DNA-free; Ambion) and purification, according to the manufacturerâ??s protocols. RNA was quantitated using a spectrophotometer ND-1000 (Nanodrop Technologies, USA). RNA quality was assessed regarding purity and stability using a Bioanalyser 2100 (Agilent Technologies, USA). Extracted total RNA aliquots were snap-frozen in liquid nitrogen and stored at -800C for single use. Experiment Overall Design: cRNA Labelling and Oligonucleotide Microarray Hybridization: Experiment Overall Design: Total RNA was linearly amplified and labelled with Cy3 or Cy5 using a low RNA input Fluor Linear Amp Kit (Agilent Technologies, USA). Internal RNA controls from Agilentâ??s RNA Spike-in Kit were included, comprised of mixtures of 11 in vitro synthesized transcripts derived from the Adenovirus E1A transcriptome at various ratios. The final cRNA concentration of 500 ng total RNA used was typically 590-650 ng/Î¼l and the Cy3- or Cy5-cytidine incorporation was 5.4 -6.0 pmol/Î¼g cRNA as determined using the Nanodrop spectrophotometer ND-1000. Experiment Overall Design: 3.5 Î¼g of Cy3 and Cy5 labeled cRNA (treated vs. untreated samples) were competitively hybridized to high-density DNA arrays from Agilent Technologies (Whole Human Genome Oligo Microarray 44K) for 17 hours at 650C according to the manufacturerâ??s protocol. Each set of three microarray experiments for triplicate analysis contained one dye swap experiment. All microarrays were scanned and the intensities normalized over background as well as to eliminate signal intensity-dependent bias from the ratio of the two channels (Lowess normalization) using a microarray scanner from Agilent Technologies including proprietary software. All microarray data sets were imported into the microarray data analysis software Genespring 7.3 (Agilent Technologies) followed by comparison of normalized intensities. Gene identities were updated according to Agilentâ??s latest list of gene annotations (G4112F, 07Feb07). Experiment Overall Design: Consistency of microarray data quality was assessed by monitoring performance of the spiked internal RNA controls (330 microarray spots/array) at various ratios (Supplementary Data, Fig. S1), by carrying out a global analysis of all absolute fluorescent intensity values, and by analysis of negative control spots (314 microarray spots/array) for determination of detection thresholds.

ORGANISM(S): Homo sapiens

SUBMITTER: Nils Werner Georg Lambrecht

PROVIDER: E-GEOD-10621 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The human glucocorticoid receptor (GR) is expressed as two alternately spliced Cterminal isoforms: Î± and Î². In contrast to the canonical hGRÎ±, hGRÎ² is constitutively nuclear, is not thought to bind ligand, and is believed to affect gene transcription only by acting as a dominant negative to hGRÎ±. Microarray analysis indicated that hGRÎ², expressed in the absence of hGRÎ±, can regulate gene expression, and furthermore, occupation of hGRÎ² with the antagonist RU-486 diminishes that capacity. Experiment Overall Design: RNA preparation: Total RNA was extracted from 5x10 6 U-OFF or U-2 Experiment Overall Design: OSÎ² cells treated with either ethanol vehicle or 1Î¼M RU-486 for 6 hours using the RNAqueous Total RNA Isolation Kit (Ambion Inc. Austin, TX) according to manufacturerâs instructions. RNA was treated with DNase using the DNA-Free DNase Treatment and Removal Reagents (Ambion Inc.) according to manufacturerâs instructions prior to use with the microarray. Four pairs of RNA (vehicle vs. RU-486 treated) were harvested for each cell type to yield four biological replicates for gene expression analysis. Experiment Overall Design: Linear Amplification Label Protocol and Feature Extraction: Gene expression analysis was conducted using Agilent Human1Av2 arrays (Agilent Technologies, Palo Alto, CA). Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturerâs protocol. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol Experiment Overall Design: and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v7.5), using defaults for all parameters.

Project description:Antipsychotic drugs are classified as typical and atypical based on extrapyramidal effects. However, since the frontal cortex is one of the most important regions for antipsychotic actions, this study attempted to classify antipsychotic drugs based on gene expression in the frontal cortex. Chlorpromazine and thioridazine were selected as typical antipsychotics, and olanzapine and quetiapine as atypical antipsychotics. Since these drugs have similar chemical structures, the effect of the basic structure on gene expression can be eliminated. Cluster analysis of microarray experiments showed thioridazine and olanzapine constituted a robust cluster. K-means clustering separated 4-drug-administered mice into chlorpromazine-quetiapine and thioridazine-olanzapine groups. This classification scheme is different from that which is based on criteria currently used to group the typical and atypical drugs and suggests that antipsychotic drugs can be further separated into multiple groups. Experiment Overall Design: Male 13-week-old ddY mice (Japan SLC Co., Hamamatsu, Japan) weighing 38-43 g were used. Animals were housed with free access to standard food in an air-conditioned room under a constant dark-and-light cycle (light: 7:00 a.m. to 7:00 p.m.) at a temperature of 22 to 24Â°C and 60 to 70% relative humidity. Ether was used for anesthesia during decapitation. All efforts were made to minimize animal suffering and to reduce the number of animals used. The present experiments were carried out after obtaining permission from the Committee of Animal Experimentation of the Graduate School of Medical and Dental Sciences at Kagoshima University. Experiment Overall Design: Doses of 25 mg/kg chlorpromazine HCl (Sigma-Aldrich Co., St. Louis, MO), 25 mg/kg thioridazine HCl (Sigma-Aldrich Co.), 1.25 mg/kg olanzapine (gift from Eli Lilly and Company, Indianapolis, IN) and 18.75 mg/kg quetiapine fumarate (gift from AstraZeneca, Macclesfield, UK) were used in the study. The dosages for these drugs were determined from therapeutically equivalent doses previously reported (Lehman et al, 2003; Woods, 2003). The drugs were dissolved in acetic anhydride and diluted with 0.9% saline, resulting in a final concentration of acetic acid of 0.5%. The drugs were injected intraperitoneally once daily for 28 consecutive days in a volume of 0.1 ml/10 g body weight. Experiment Overall Design: Microarray experiments were performed using an Agilent G4121A Mouse Oligo Microarray Kit (Agilent Technologies, Palo Alto, CA) as per the manufacturerâ??s instructions. The frontal cortex was immediately removed and the RNA was stabilized in RNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA) and stored at -80Â°C until use. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). The RNA was amplified and labeled by the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). To synthesize cDNA, 200 ng total RNA was used. Vehicle-injected controls were labeled by cyanine 3 (PerkinElmer Life Sciences, Inc., Boston, MA) and drug-injected mice were labeled by cyanine 5 (PerkinElmer Life Sciences, Inc.). Hybridizations to the microarray were performed using the In situ Hybridization Kit Plus (Agilent). Doses of 750 ng cyanine 3-labeled cRNA, 750 ng cyanine 5-labeled cRNA, and control targets were mixed and fragmented in the kitâ??s fragmentation buffer, and then hybridized to the microarrays for 17 hours at 60Â°C in a hybridization rotator (Agilent) set to rotate at 4 rpm. Microarrays were washed in 6Ã?SSC with 0.005% Triton X-102 at RT for 10 min, followed by 0.1Ã?SSC with 0.005% Triton X-102 at 4Â°C for 5 min. The slides were dried, and then scanned by the Agilent G2565BA Microarray Scanner System. Data were analyzed using the Agilent Feature Extraction Software version 7.1. A rank consistency filter and LOWESS were used for dye normalization. Control mice and drug-injected mice were processed in parallel. The data discussed in this publication are presented in accordance with the MIAME guidelines (http://www.mged.org/Workgroups/MIAME/miame.html) and were deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). They can be accessed using the GEO series accession number GSE1501. Experiment Overall Design: Cluster analysis was performed using free software written by M. Eisen (http://rana.lbl.gov/EisenSoftware.htm). The cclust package in the â??Râ?? statistical software system (www.cran.r-project.org) was used to find the most appropriate number of clusters (i.e. â??kâ?? in the k-means clustering).

Project description:Several recent reports show that the cerebral cortex in humans and animals with altered expressions of Wnt/cadherin network-associate molecules display cytoarchitectural abnormalities reminiscent of cortical dysplasias seen in some (mouse-, rat-, and monkey-based) animal models of prenatal cocaine exposure. Therefore, we employed oligo microarrays followed by real-time RT-PCR to compare expressions of genes involved in Wnt and cadherin systems in the cerebral wall of 18-day-old (E18) fetuses from cocaine-treated (20 mg/kg cocaine, s.c., b.i.d., E8-18) and drug-naive (saline, s.c.) mice. The pregnant mice chronically treated with cocaine in the above-described manner represent one of the animal models producing offspring with widespread cortical dysplasias. Out of more than 150 relevant genes in the arrays, 32 were upregulated and 9 were downregulated in cocaine-exposed fetuses. The majority of these genes (30 out of 41) were similarly affected in the frontal and occipital regions of the cerebral wall. We also used Western immunoblotting to examine the ability of cocaine to regulate the protein levels of beta-catenin, the key functional component of both Wnt and cadherin systems. While the total cell levels of beta-catenin were increased throughout the cerebral wall of cocaine-exposed fetuses, its nuclear (gene-transcription driving) levels remained unaltered. This suggests a transcription-unrelated role for cocaine-induced upregulation of this protein. Overall, our findings point to an intriguing possibility that that cerebral cortical dysplasias observed in several animal models of prenatal cocaine exposure may be at least in part related to alterations in the Wnt/cadherin molecular network. It has been demonstrated that exposure to cocaine increases cell death in the fetal CNS. To examine the molecular mechanisms of this effect, we employed mouse oligo microarrays followed by real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) to compare expressions of apoptosis-related genes in the cerebral wall of 18-day-old (E18) fetuses from cocaine-treated (20 mg/kg cocaine, s.c., b.i.d., E8th-E18th) and drug-naive (saline, s.c.) mice. Out of approximately 400 relevant genes in the arrays, 53 showed alterations in expression in cocaine-exposed fetuses. Upregulation was observed in 35 proapoptotic and 8 antiapoptotic genes; 4 proapoptotic and 6 antiapoptotic genes were down-regulated. The affected genes encode a wide range of apoptosis-related proteins, including death receptors (NTF-R1, NTF-R2, DR3, DR5, LTbeta-R, GITR, P57 TR-1) and their adaptor and regulatory proteins (MASGE-D1, TRAF-2, SIVA, MET, FLIP, FAIM, IAP1, ATFA), members of transcription regulatory pathways (JNK, NF-kappaB, P53), members of BCL-2 family of proteins (BID, BAD, BAX, BIK, NIP21, NIP3, NIX, BCL-2), DNA damage sensor (PARP-1), caspases and their substrates and regulatory proteins (caspases 8, 4, 9, and 3, ACINUS, CIDE-A, CIDE-B, GAS2), mitochondrially released factors (cytochrome c, AIF, PRG3), specific endoplasmic reticulum- and oxidative stress-associated factors (BACH2, ABL1, ALG2, CHOP), members of cell survival AKT and HSP70 pathways (PIK3GA, PTEN, HSP70, BAG1, BAG2), and others. This suggests that cocaine affects survival of developing cerebral cells via multiple apoptosis-regulating mechanisms. Experiment Overall Design: Animals Experiment Overall Design: Timed pregnant Swiss Webster (CFW; Charles River Experiment Overall Design: Lab. Wilmington, MA) dams were maintained in individual Experiment Overall Design: cages in a climate-controlled room on a 12/12-h light/ Experiment Overall Design: dark cycle. They were divided into two groups. The first Experiment Overall Design: (experimental) group received subcutaneous (at the dorsum Experiment Overall Design: of the neck) injections of 20 mg/kg cocaine hydrochloride Experiment Overall Design: (Research Technology Branch, National Institute Experiment Overall Design: of Drug Abuse, Rockville, MD) dissolved in 200 mkl of Experiment Overall Design: 0.9% saline, twice a day (at 8:00 AM and 8:00 PM) from Experiment Overall Design: 8th through 18th day of pregnancy (E8âE18). The second Experiment Overall Design: (control) group was subjected to the same schedule of Experiment Overall Design: 0.9% saline only injections. The cocaine treatment was Experiment Overall Design: designed to replicate the one capable of reducing cerebral Experiment Overall Design: cortical mass in mouse offspring. Also, the Experiment Overall Design: relatively protracted period the chronic treatment was Experiment Overall Design: chosen to maximize the changes in the tissue expression Experiment Overall Design: of cocaine-regulated genes of interest. Throughout the Experiment Overall Design: treatment, all mice were weighed daily, and, from E8, the Experiment Overall Design: control and experimental animals were pair-fed, with the Experiment Overall Design: daily amount of food (Mouse Chow; Ralston Purina Saint Experiment Overall Design: Louis, MO) provided to each control dam being matched Experiment Overall Design: to that consumed by the paired experimental dam. Water Experiment Overall Design: was available ad libitum. We found that this feeding Experiment Overall Design: regiment resulted in similar weight gains from E8 to E18 Experiment Overall Design: in both experimental and control animal groups Experiment Overall Design: (46.41F0.45% and 44.99F0.59% respectively). On E18, Experiment Overall Design: 1 h after the morning treatment, the animals were Experiment Overall Design: anesthetized by peritoneal injection of 50 mg/kg sodium Experiment Overall Design: pentobarbital (Abbott Lab., Abbott Park, IL) and their Experiment Overall Design: uteri were removed. The frontal cerebral wall (containing Experiment Overall Design: cerebral cortex and underlying transient cortical zones Experiment Overall Design: anterior to the striatal level) and the occipital cerebral wall Experiment Overall Design: (containing cerebral cortex and underlying transient Experiment Overall Design: cortical zones posterior to the level of the hippocampus) Experiment Overall Design: were dissected from the fetuses and stored in liquid Experiment Overall Design: nitrogen prior to analysis. The animal experimentations Experiment Overall Design: used in this study were approved by the University of Experiment Overall Design: Maryland Animal Care and Use Committee. Experiment Overall Design: Microarray Experiment Overall Design: Tissue from two control and two experimental fetuses Experiment Overall Design: were used in the processing of a single microarray slide Experiment Overall Design: (Part G4121A, Agilent Technol). Five slides were processed Experiment Overall Design: per region of the fetal cerebral wall. For a given region, the Experiment Overall Design: tissue for each microarray slide was obtained from a Experiment Overall Design: separate set of control and experimental litters. Total RNA Experiment Overall Design: was isolated using TRIzol Reagent (Invitrogen, Carlsbad, Experiment Overall Design: CA). The yield of total RNA was determined by absorbance Experiment Overall Design: at 260 nm on a DU 640 Spectrophotometer (Beckman Experiment Overall Design: Coulter, Fullerton, CA). The 260/280 nm ratios of the Experiment Overall Design: samples were >1.8. For each assay, 5 mkg of RNA from Experiment Overall Design: control tissue and 5 mkg of RNA from experimental tissue Experiment Overall Design: were reverse transcribed using 3DNA Array Detection Kit Experiment Overall Design: (Genesphere, Hatfield, PA) with primers containing different Experiment Overall Design: dcaptureT sequences for control and experimental Experiment Overall Design: samples. SuperScript II Reverse Transcriptase for these Experiment Overall Design: reactions was purchased from Gibco BRL (Gaithersburg, Experiment Overall Design: MD). The resultant cDNAs were hybridized to microarray Experiment Overall Design: slides for 16 h in Agilent Hybridization Buffer (Agilent Experiment Overall Design: Technol) at 60 8C. Upon completion of the hybridization, Experiment Overall Design: the slides were washed for 10 min with 0.005% Triton in Experiment Overall Design: 6xsodium chloride/sodium citrate buffer (pH 7; SSC) at Experiment Overall Design: room temperature and then for five more min with 0.005% Experiment Overall Design: Triton in 0.1xSSC and 1 more minute in 0.1xSSC (pH 7), Experiment Overall Design: both at 4 8C. Washed slides were air-dried for 30 s. For Experiment Overall Design: fluorescent labeling of microarray-bound cDNA, the slides Experiment Overall Design: were incubated for 3 h at 65 8C with Capture Reagent Experiment Overall Design: Hybridization Mixture from 3DNA Array Detection Kit Experiment Overall Design: (Genesphere). In this mixture, Alexa 546 fluorochromeincorporating Experiment Overall Design: dendrimers contained single-stranded arms Experiment Overall Design: complimentary to the dcaptureT sequences used in the Experiment Overall Design: reverse transcription of RNA from control tissue, while Experiment Overall Design: Alexa 647 fluorochrome-incorporating dendrimers contained Experiment Overall Design: arms complimentary to the capture sequences used Experiment Overall Design: in reverse transcription of RNA from experimental tissue. Experiment Overall Design: The labeling reaction was terminated by washing of the Experiment Overall Design: microarray slides at room temperature for 10 min with Experiment Overall Design: 0.005% Triton in 2xSSC and for another 10 min with in Experiment Overall Design: 0.2xSSC. After that, the slides were air-dried for 30 s. The Experiment Overall Design: processed microarray slides were scanned at 10 Am Experiment Overall Design: resolution on a GenePix 4100A scanner (Axon Instr., Union Experiment Overall Design: City, CA) with the laser excitation at 532 nm [emission filter Experiment Overall Design: 575DF35 (green); photomultiplier voltage 550] for Alexa Experiment Overall Design: 546 (control samples) and the laser excitation at 635 nm Experiment Overall Design: [emission filter 670DF40 (red); photomultiplier voltage Experiment Overall Design: 695] for Alexa 647 (experimental samples). The signals Experiment Overall Design: were converted into 16-bits-per pixel resolution images, Experiment Overall Design: providing color depth of 65,536 levels. The densitometry Experiment Overall Design: was performed with GenePix Pro 4.1 software (Axon Instr.). Experiment Overall Design: Background was subtracted using the dlocal background Experiment Overall Design: correctionT procedure available in the software. Quality control utilized 255 positive controls, 161 negative controls Experiment Overall Design: and 646 QC-spots present on Agilentâs arrays. For Experiment Overall Design: replicates within a slide, median signal values were Experiment Overall Design: calculated. For each array, the data were normalized in Experiment Overall Design: Acuity 3.1 software (Axon Instr.) by applying locally Experiment Overall Design: weighted scatterplot smoothing (LOWESS) transformation. Experiment Overall Design: The between array scale normalization Experiment Overall Design: was done using intensity log10 ratio distribution box plots (GeneSight Experiment Overall Design: software, BioDiscovery, El Segundo, CA) for verification. Experiment Overall Design: Statistical analysis of the normalized data was conducted Experiment Overall Design: by the Significant Analyses for Microarray Algorithm Experiment Overall Design: (SAM) using Excel macros available at the Stanford SAM website. Experiment Overall Design: In the analyses of both frontal and occipital regions of the fetal cerebral wall, Experiment Overall Design: the cut-off thresholds were set to identify the maximum Experiment Overall Design: number of cocaine exposure-regulated genes at the minimal Experiment Overall Design: false discovery rate (FDR) allowed by SAM for a given set Experiment Overall Design: of microarray data. For both regions, the FDR rates were Experiment Overall Design: <0.5%. The apoptosis and Wnt pathway-related genes were identified with Pathway Assist 2.0 software (Stratagene, La Jolla, CA). Experiment Overall Design: This was followed by manual review of literature for each Experiment Overall Design: identified gene.

Dataset Information

Transcription profiling of human ciliary smooth muscle cells grown culture WITH 200 nM OF either an prostaglandin E2 receptor SUBTYPE EP2 OR SUBTYPE EP4 selective agonist FOR 6 hours IN comparison TO untreated controls

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