Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene Expression Profile of AGS cell line cultured in 2D and in 3D conditions

ABSTRACT: AGS cell line was cultured in standard T25 flasks (2D condition) and in T25 flasks precoated with 50 M-5L/cm2 of matrigel basement membrane (BD Biosciences) at a 1.5:1 proportion to RPMI serum-free medium (3D condition, overlay method). Cultures were maintained for 14 days with medium change every two days. Transcriptome alterations were then investigated.

ORGANISM(S): Homo sapiens

SUBMITTER: Raquel Almeida

PROVIDER: E-MTAB-1234 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness.

Pereira Bruno B Sousa Sofia S Barros Rita R Carreto Laura L Oliveira Patrícia P Oliveira Carla C Chartier Nicolas T NT Plateroti Michelina M Rouault Jean-Pierre JP Freund Jean-Noël JN Billaud Marc M Almeida Raquel R

Nucleic acids research 20130213 7

The homeobox transcription factor CDX2 plays a crucial role in intestinal cell fate specification, both during normal development and in tumorigenic processes involving intestinal reprogramming. The CDX2 regulatory network is intricate, but it has not yet been fully uncovered. Through genome-wide screening of a 3D culture system, the RNA-binding protein MEX3A was identified as putatively involved in CDX2 regulation; therefore, its biological relevance was addressed by setting up cell-based assay ...[more]

PMID: 23408853

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Project description:In pregnancies involving preeclampsia (PE), there is evidence that the fetal-placental unit is under oxidative stress. Here we examined primary cell lines generated from umbilical cords (UC) delivered by mothers who had either a normal pregnancy or experienced early onset PE to determine whether the two had distinguishable phenotypes. While all UC provided outgrowths when established in 4 % O2, success was less assured for PE cords under ambient (20 % O2) conditions (P < 0.05). Moreover, proliferation rates of established PE lines, although similar to controls in 4 % O2, were significantly lower in 20 % O2. PE lines grown in 4 % O2 were also more susceptible to the pro-oxidant diethylmaleate than control lines, and unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few differentially regulated genes between PE lines and controls in 4 % O2 conditions, but confirmed the more severely stressed phenotype of the PE lines under 20 % O2. We conclude that the primary UC cell lines generated from PE births maintain a susceptibility to oxidative stress that is stable over many cell divisions, but whether the basis of this vulnerability is genetic or epigenetic remains unclear. RNA was isolated from early passages (< p5) of UC fibroblast lines (15 PE and 9 CTL lines, grown in T25 flasks) when they reached ~90% confluency in 4 % O2 conditions. In order to collect RNA from cells under 20 % O2, cell lines at either p 4, 5, or 6 were switched from 4 % O2 conditions to 20 % O2 conditions when they were approximately 20 % confluent. When they reached ~90% confluency (generally 2 days in 4 % O2 and 3-4 days in 20 % O2 conditions), medium was removed and RNA STAT60 (I ml; Tel-Test, Friendswood, TX) was immediately added to each T25 flask and RNA extracted by following the manufacturer’s instructions. The samples of RNA were submitted to University Texas Southwestern Medical Center Microarray Core Facility (https://microarray.swmed.edu/) and microarray analysis performed with Illumina HumanHT-12 v4 expression BeadChips. Raw intensity data were background subtracted by using BeadStudio software and analyzed further by GeneSpring 12.6 software (Agilent Technologies Inc., Santa Clara CA), according to the advanced workflow protocol: percentile shift and filter by flags (detected).

Project description:The incidence of obesity and its related morbidities has rapidly increased over the past twenty years. One consequence of this is the increased probability of having to treat obese individuals for breast cancer. An important co-morbidity associated with obesity are the spectrum of fatty liver diseases. As such, an important question is how treatment of breast cancer with standard therapeutic agents may alter in individuals with fatty liver disease. Such impacts could result in an enhanced adverse effect profile over both acute (e.g. increased cytotoxicity against non-malignant tissues) or chronic (e.g. increased progression through the fatty liver disease spectrum) time periods. In this work we examine if lipid loading in human hepatocytes, leading to a steatotic phenotype, results in an altered toxic liability for the cancer therapeutic doxorubicin. We demonstrate that there is a synergistic impact on cytotoxicity, due to increased oxidative stress. Array analysis reveals the cellular changes associated with hepatocyte adaptation to lipid loading or doxorubicin treatment alone, and how these profiles are significantly altered during doxorubicin treatment of lipid-loaded hepatocytes. Such alterations, we believe, not only underlie the enhanced sensitivity of obese individuals to the acute adverse effects of doxorubicin, but may also potentiate transition along the fatty liver disease spectrum.Experimental design: Huh7 cells were exposed to lipid and/or doxorubicin as required. Huh7 cells were treated with vehicle control or 300 μM FFA mixture for 24 h in T25 culture flasks. The following day, the cells were treated with either 300 μM FFA mixture, 0.1 μM or 3.6 μM DOX and incubated for 4 hrs or 12 hrs. By the end of incubation time, Total RNA was extracted from cells using the RNeasy Plus Mini Kit (QIAGEN-UK) according to the instructions of manufacturer. RNA samples were sent to the Central Biotechnology Services (Cardiff University, UK), for quality control, synthesis of biotin-labeled cRNA, and hybridisation against Illumina Human-HT12 (Illumina, Inc., Hayward, CA) chips. Washed chips were scanned with Bead Station 500x (Illumina) and the signal intensities quantified with BeadStudio (Illumina). To ensure high quality results from the microarray data, RNA samples were extracted from three independent treatments, giving a biological n = 3 per treatment which gives statistical power to increase the confidence of the results/conclusions generated from the microarray experiment.

Dataset Information

Gene Expression Profile of AGS cell line cultured in 2D and in 3D conditions

Publications

CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness.

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