Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression data of HUVEC cells after TNF-alpha treatment

ABSTRACT: Tumor necrosis factor a (TNF-aplha) is a potent cytokine that orchestrates the inflammatory response by sequentially activating the expression of many genes in cultured human umbilical vein cells (HUVECs). We apply samples collected every 7.5 min to arrays and monitor the growth of several long transcripts. Keywords: time course Before stimulation, HUVECs were serum-starved in EBM-2 (Clonetics) containing 0.5% FBS for 18 h, and then treated with 10 ng/ml TNF (Peprotec) and samples collected every 7.5 min for 3 h.

ORGANISM(S): Homo sapiens

SUBMITTER: Shuichi Tsutsumi

PROVIDER: E-GEOD-9036 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A wave of nascent transcription on activated human genes.

Wada Youichiro Y Ohta Yoshihiro Y Xu Meng M Tsutsumi Shuichi S Minami Takashi T Inoue Kenji K Komura Daisuke D Kitakami Jun'ichi J Oshida Nobuhiko N Papantonis Argyris A Izumi Akashi A Kobayashi Mika M Meguro Hiroko H Kanki Yasuharu Y Mimura Imari I Yamamoto Kazuki K Mataki Chikage C Hamakubo Takao T Shirahige Katsuhiko K Aburatani Hiroyuki H Kimura Hiroshi H Kodama Tatsuhiko T Cook Peter R PR Ihara Sigeo S

Proceedings of the National Academy of Sciences of the United States of America 20091013 43

Genome-wide studies reveal that transcription by RNA polymerase II (Pol II) is dynamically regulated. To obtain a comprehensive view of a single transcription cycle, we switched on transcription of five long human genes (>100 kbp) with tumor necrosis factor-alpha (TNFalpha) and monitored (using microarrays, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation) the appearance of nascent RNA, changes in binding of Pol II and two insulators (the cohesin subunit RAD21 and the CC ...[more]

PMID: 19826084

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Project description:Metastasizing tumor cells exit the vascular system through dynamic interactions with endothelial cells that line the internal surface of vessels. While extravasation is a key event within the metastatic cascade, the signals regulating tumor cell adhesion to the endothelium and their subsequent transendothelial migration are poorly understood. Here, we combined Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and phosphoproteomic analysis to identify cell-specific signaling pathways regulated between interacting breast cancer cells and endothelial cells (see PRIDE repository PXD001558). Further co-culture experiments were performed alongside the phosphoproteomic analysis in order to control for the protein quantity. These are presented here. Using SILAC, cell-specific labels were introduced into MDA-MB-231-LM2 cells (Human Breast cancer) and HUVECs (Human endothelial cells) to ensure each cell type had a distinct and traceable phosphoproteome when tumor and endothelial cells were co-cultured. To probe regulatory signaling events triggered in cancer cells following contact with endothelial cells, we labeled LM2 cells with medium or heavy isotopomers of arginine and lysine (Arg+6 Da, Lys+4 Da and Arg+10 Da, Lys+8 Da respectively). Heavy-labeled LM2 cells were collected by enzyme-free cell dissociation buffer, thereby preserving membrane proteins and adhesion receptors, and seeded onto a monolayer of light-labeled (Arg+0 Da, Lys+0 Da) HUVECs. Following 15 min of co-culture, non-adherent LM2 cells were gently removed and cancer cells that had attached to the endothelial layer were lysed together with the HUVECs. In parallel, medium-labeled LM2 cells were collected under the same conditions and maintained as suspension cells in monoculture to represent circulating tumor cells prior to any contact with the endothelium. These were then added to the harvested LM2-HUVEC co-culture in a 1:1 ratio of heavy:medium-labeled cells to provide a point of reference. Based on the SILAC labeling of the different cell populations, light-labeled peptides were assigned to HUVECs, medium-labeled peptides to monocultured LM2 cells in suspension, and heavy-labeled peptides to LM2 cells that had made contact with HUVECs. As such, the heavy/medium ratio for each peptide was used to quantify phosphorylation-dependent signaling changes occurring specifically in the LM2 cells upon contact with HUVECs. Conversely, to elucidate signaling events in HUVECs that were initiated by contacting cancer cells, light-labeled LM2 cells were seeded on top of a confluent monolayer of heavy-labeled HUVECs, while medium-labeled HUVECs were maintained in monoculture. Following 15 min of co-culture, the unattached LM2 cells were removed. Cells were lysed, mixed in a 1:1 ratio of heavy:medium HUVECs, followed by membrane fractionation and phosphoproteomic analysis as described above. A variation in phospho-peptide quantity could be due to the experimental difficulties in mixing heavy and medium labels in a 1:1 ratio or a quick regulation of the protein quantity through modification of its expression/degradation balance. Thus, we performed relative quantification of non-phosphorylated peptides in parallel with the phosphoproteomic analysis to account for changes in total protein abundance (data presented here) or correct for experimental bias. Cytoplasmic fractions were analyzed by LC-MS/MS before TiO2 enrichment and their median log2(H/M) were used for normalization of the phosphoproteomic dataset. In order to increase the number of proteins quantified in the LM2 cells, we performed a supplementary co-culture experiment and fractionated the peptides by SDS-PAGE. In order to get a confident relative quantification of Ephrin type-A receptor 2 (EPHA2), we performed 2 independent experiments followed by EPHA2 IP and LC-MSMS.

Dataset Information

Expression data of HUVEC cells after TNF-alpha treatment

Publications

A wave of nascent transcription on activated human genes.

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