Comment[ArrayExpressAccession] E-GEOD-30364 MAGE-TAB Version 1.1 Public Release Date 2014-06-28 Investigation Title Vectorial secretion of interleukin-8 (IL-8 or CXCL8) mediates homeostatic effects on the intestinal epithelium via apically located CXCR1 Comment[Submitted Name] Vectorial secretion of interleukin-8 (IL-8 or CXCL8) mediates homeostatic effects on the intestinal epithelium via apically located CXCR1 Experiment Description CXCL8 is produced by many cell types including epithelial, endothelial, fibroblasts and macrophages in response to TLR recognition of microbe-associated molecular patterns (MAMPs) or inflammatory cytokines and recruits phagocytes from the vasculature to sites of infection via interaction with its cognate receptors CXCR1 and CXCR2. In the intestine, CXCL8 has been demonstrated to participate in the migration of neutrophils across the epithelium during acute inflammation. Given the well-recognized role of CXCL8 as an initiator of inflammation and the constant presence of commensal bacteria in the intestinal tract, we hypothesized that in the intestinal epithelium, CXCL8 might be secreted in a vectorial fashion depending on the location and type of stimulus as a mechanism to maintain homeostasis. In addition, we hypothesized that the CXCR1 receptor might control specific functions in polarized IECs depending on its location. We tested these hypotheses using microarray gene expression profiling of IL-8 treated and mock-treated Caco-2 cell lines This study was set up according to a one-treatment, one-control design. It contains individual transcriptional profiles from 3 IL-8-treated and 3 buffer control-treated samples. In total, this study includes data from 3 Caco-2 samples x 2 treatments=6 arrays. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name van Baarlen van Baarlen Rossi Wells Person First Name Peter Peter Oriana Jerry Person Mid Initials M Person Email peter.vanbaarlen@wur.nl Person Affiliation Wageningen University Person Address Animal Sciences, Wageningen University, De Elst 1, Wageningen, Netherlands Person Roles submitter Protocol Name P-GSE30364-1 P-GSE30364-5 P-GSE30364-6 P-GSE30364-2 P-GSE30364-3 P-GSE30364-4 P-GSE30364-7 Protocol Description Packages from the Bioconductor project, integrated in an in-house developed on-line management and analysis database for multiplatform microarray experiments, were used for analysing the scanned Affymetrix arrays (Gentleman et al., 2004; Gavai et al, submitted). ID_REF = VALUE = RMA-normalised intensity signals (unlogged scale) Total RNA (500 ng) extracted fromCaco-2 cells was labelled using the GeneChip 3M-bM-^@M-^Y IVT Express kit (cat. no. 901229). The labelled RNA was hybridised to a GeneChip Hu133 Plus2 array (Affymetrix, Santa Clara, CA), washed, and stained. Detailed methods for the labelling and subsequent hybridisations to the arrays are described in the eukaryotic section of the GeneChip Expression Analysis Technical Manual, Revision 3, from Affymetrix. Cell monolayers were treated with 100 pg/ml CXCL8, added to the cells diluted in culture medium. After 6 hours of incubation, Caco-2 cells were incubated with TriZol reagens until lysis was complete, then RNA extraction and purification were performed Caco-2 cells were grown in DMEM (Invitrogen) containing Glutamax and supplemented with 10% heat-inactivated fetal bovine serum (PAA laboratories, Colbe, Germany), 100 U/ml penicillin and 100 M-5g/ml streptomycin (Sigma, St. Louis, MO) in 6-wells microtiter plates (Costar) until polarised (14 days), maintained at 37M-0C in a humidified 5% CO2/95% O2 atmosphere. Total RNA was extracted from Caco-2 cell line samples with TRIzol reagent, purified and DNAse treated using the RNeasy mini kit (Qiagen, Venlo, the Netherlands) and DNase (Qiagen, Venlo, the Netherlands) following the manufacturerM-bM-^@M-^Ys instructions. RNA integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, the Netherlands) with 6000 Nano Chips according to the manufacturerM-bM-^@M-^Ys instructions. RNA was judged as suitable for array hybridization only if samples showed intact bands corresponding to the 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RIN (RNA integrity number) above 8.0. The GeneChip Hu133 Plus2 array was scanned on an Affymetrix GeneChip 3000 7G scanner. Detailed methods are described in the eukaryotic section of the GeneChip Expression Analysis Technical Manual, Revision 3, from Affymetrix Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Comment[SecondaryAccession] GSE30364 Comment[GEOReleaseDate] 2014-06-28 Comment[ArrayExpressSubmissionDate] 2011-07-01 Comment[GEOLastUpdateDate] 2014-06-28 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-30364.sdrf.txt