Comment[ArrayExpressAccession] E-GEOD-31315 Public Release Date 2012-08-07 Investigation Title Expression data from SP and non-SP sorted anti-EpCAM treated A549 cells Comment[Submitted Name] Expression data from SP and non-SP sorted anti-EpCAM treated A549 cells Experiment Description Targeted therapies against cancer stem cells, which are enriched in side populations (SP), involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/Î²-catenin signalling was studied. SP of the human lung adenocarcinoma cell line A549 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment. Anti-EpCAM treated and untreated A549 cells were subjected to Hoechst 33342 dye exclusion assay and sorted to SP and non-SP fractions by FACS. Three biological replicates. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name KrÃ¼wel KrÃ¼wel Rohrbeck Person First Name Thomas Thomas Astrid Person Mid Initials Person Email geo@ncbi.nlm.nih.gov Person Affiliation Fraunhofer Institute for Toxicology and Experimental Medicine Person Phone Person Fax Person Address Fraunhofer Institute for Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, Hannover, Germany Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE31315-1 P-GSE31315-6 P-GSE31315-3 P-GSE31315-8 P-GSE31315-7 P-GSE31315-2 P-GSE31315-4 P-GSE31315-5 Protocol Description ID_REF =
VALUE = RMA Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome-U133 Plus 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; PAA, CÃ¶lbe, Germany) supplemented with 10% foetal calf serum, 2 mmol/l L-glutamine, 100 units/ml streptomycin and 100 µg/ml penicillin in a humidified atmosphere containing 5% CO2 at 37°C. Cells were plated in 96well microtiter plates at a density of 5000 cells/well 24 h prior treatment. Data were normalized with the Robust Microarray Averaging (RMA) algorithm. Following experimental normalization signals < 50 were defined as background. Expression values were calculated as the ratio of non-stimulated (control) and anti-EpCAM stimulated SP vs. non-SP signals. Gene expression that was at least 25% increased or decreased in replicate experiments was considered as differentially expressed. GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G Cells were treated with a monoclonal anti-mouse EpCAM antibody at concentrations of 100 µg/ml for 48 h. Subsequently, 10^6 cells/ml were resuspended in warm DMEM containing 2% FCS and 10 mmol/l HEPES buffer (Sigma-Aldrich, Munich, Germany). Hoechst 33342 dye was added to a final concentration of 5 µg/ml in the presence or absence of reserpine (50 µmol/l; Sigma-Aldrich, Munich, Germany) and incubated at 37°C for 90 min with gentle shaking. Afterwards, the cells were centrifuged at 4°C and resuspended in ice-cold Hank's balanced salt solution (HBSS, Invitrogen, Karlsruhe, Germany) containing 2% FCS and 10 mmol/l HEPES. Viable cells were visualized by propidium iodide staining (Invitrogen, Karlsruhe, Germany) and was added to a final concentration of 2 µg/ml. The cells were filtered by a 40 µm cell strainer to obtain a single cell suspension before sorting. Analyses and sorting were carried out on a FACSAria cytometer (Becton Dickinson, Heidelberg, Germany). Emissions of Hoechst 33342 (Hoechst-red and Hoechst-blue) were detected using 695/40 nm band-pass and 450/40 nm band-pass filters, respectively. RNA-extraction was done with the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturerâs instruction. Total RNA (200 ng) was used to generate biotin-labelled cRNA (10 µg) by means of GeneChip 3' IVT Express Kit (Affymetrix). Sample processing was performed exactly as described by the microarray manufacturer (Affymetrix). Quality control of cRNA was performed using a bioanalyzer (Agilent 2001 Biosizing; Agilent Technologies, BÃ¶blingen, Germany). Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation hybridization grow feature_extraction image_aquisition specified_biomaterial_action nucleic_acid_extraction labeling Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name cell type treatment Experimental Factor Type cell type treatment Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Publication Author List PubMed ID Publication DOI Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE31315 Comment[GEOLastUpdateDate] 2012-08-08 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2012-08-06 Comment[ArrayExpressSubmissionDate] 2011-08-09 SDRF File E-GEOD-31315.sdrf.txt