we used isotope coded affinity tagging analysis coupled with mass spectrometry to quantify PDGF induced protein that alterations in a human visceral SMC sub proteome. In that study we observed marked enrichment in proteins associated with endocytosis and the cytoskeleton in lipid raft microdomains of cells treated with PDGF, consistent with other studies linking PDGF to alterations in cell morphology and the actin cytoskeleton. In this study, we present the first integrated analysis of gene e pression and proteome level alterations in human visceral SMC challenged with PDGF. Results Gene e pression regulated by PDGF In order to interrogate global responses to PDGF BB at both gene and protein levels, we used primary human bladder smooth muscle cells to perform RNA e pression profiling in concert with quantitative analysis of the entire proteome using the SILAC method.
E pres sion of PDGFR and PDGFRB isoforms was verified in pBSMC by real time RT PCR and immunoblot analysis. Cells subjected to triple SILAC labeling were treated with 1 nM PDGF BB for 0, 4 or 24 h. Total protein lysates were analyzed using mass spectrometry, and total RNA was analyzed by e pression profiling. Microarray data were assessed and determined to be of high quality . a high Inhibitors,Modulators,Libraries degree of reproducibility was observed based on inter and intra group variation of the arrays, with Inhibitors,Modulators,Libraries all pairwise correlation coefficients between samples 0. 98. A total of 1695 differentially e pressed genes with overall p 0. 05 were Inhibitors,Modulators,Libraries identified at either Inhibitors,Modulators,Libraries 4 or 24 h using an integrative statistical method previously reported.
Of these, 528 DEGs were significantly changed at both 4 h and 24 h following Dacomitinib PDGF treatment, while 630 and 537 DEGs were significantly changed only at the 4 or 24 h time point, respectively. DEGs were grouped into clusters, based on time dependent differential e pression patterns, by hierarchical cluster analysis. The seven clusters could be sub categorized into those representing up regulated genes and those reflecting down regulated genes. These data showed that 487 of the 528 DEGs identified at both times were consistently up or down regulated, while 63 of the 528 genes perturbed at both times were down regulated at 4 h but up regulated at 24 h.
Functional enrichment analysis of Gene Ontology Biological Processes using Database for Annota tion, Visualization and Integrated Discovery software suggested that cell cycle transit, cell prolifer ation, cell migration and motility, ribosome biogenesis and angiogenesis were the most prominent biological processes Sunitinib c-Kit in the group of genes up regulated by PDGF, whereas cell cycle arrest, chromatin organization and apoptotic pathways were the most prominent processes in the down regulated group. To identify key transcription factors involved in these gene e pression alterations, we collected TF target interaction data from si databases and then identified TFs having significant numbers of DEGs as their targets. Significantly up regulated DEGs were m