E-GEOD-11674 - Transcription profiling of mouse VE-cadherin null cell line vs contrik
Released on 20 October 2008, last updated on 27 March 2012
In order to identify genes regulated by VE-cadherin expression, we compared a mouse VE-cadherin null cell line (VEC null) with the same line reconstituted with VE-cadherin wild type cDNA (VEC positive). The morphological and functional properties of these cell lines were described previously [Lampugnani,M.G. et al. Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148. J. Cell Biol. 161, 793-804 (2003)]. By Affymetrix gene expression analysis we found several genes up-regulated by VE-cadherin, among which claudin-5 reached remarkably high levels. The up-regulation of these genes required not only VE-cadherin expression but also cell confluence suggesting that VE-cadherin clustering at junctions was needed. Experiment Overall Design: VEC null or positive cells in the sparse and confluent conditions have been starved in MCDB 131 medium 1% BSA for 36 h. Next, RNA has been extracted with standard guanidinium isothiocyanate lysis buffer and cesium chloride ultracentrifugation. Synthesis of biotinylated cRNA targets, array hybridization (GeneChips MG_U74Av2 and MG_U74Bv2), staining and scanning were performed according to the Affymetrix standard protocols, starting from 15 μg of total RNA. Two copies of the GeneChips were hybridized with each cRNA sample. The MAS5 algorithm was used to determine the expression levels of mRNAs; the absolute analysis was performed using default parameters and scaling factor 500. Report files were extracted for each chip, and performance of labeled target was evaluated on the basis of several values (scaling factor, background and noise values, % present calls, average signal value, etc). Gene expression levels were normalized on the median over all samples. Annotation of Probe Sets is according to Affymetrix Annotation Tables (release May 31, 2007) [Vecchi,M. et al. Gene expression analysis of early and advanced gastric cancers. Oncogene 26, 4284-4294 (2007)]. We selected the genes up-regulated by VE-cadherin expression and clustering at junctions in confluent VEC positive cells.
transcription profiling by array, unknown experiment type
Endothelial adherens junctions control tight junctions by VE-cadherin-mediated upregulation of claudin-5. Andrea Taddei, Costanza Giampietro, Annarita Conti, Fabrizio Orsenigo, Ferruccio Breviario, Valentina Pirazzoli, Michael Potente, Christopher Daly, Stefanie Dimmeler, Elisabetta Dejana.