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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120176	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Yu X	2012	0%
Study summary Full title Mechanism of TNF-alpha autocrine effects in hypoxic cardiomyocytes: initiated by hypoxia inducible factor 1alpha, presented by exosomes All authors Yu X, Deng L, Wang D, Li N, Chen X, Cheng X, Yuan J, Gao X, Liao M, Wang M, Liao Y Journal J Mol Cell Cardiol Abstract Excessive tumor necrosis factor-? (TNF-?) expression is increasingly thought to be detrimental to ca (show more...)Excessive tumor necrosis factor-? (TNF-?) expression is increasingly thought to be detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-? is mainly released from macrophages, but with persistent ischemia, it can originate from cardiomyocytes and contribute to cardiac remodeling. The initiating factor and exact molecular mechanism of TNF-? release from cardiomyocytes is presently unclear. In this study, we investigated direct effects of hypoxia on TNF-? expression of cardiomyocytes, the role of hypoxia inducible factor-1? (HIF-1?) in TNF-? regulation and potential secretory pathway of TNF-?. Elevated TNF-? expression and HIF-1? activation in primary cultured cardiomyocytes under hypoxia were detected by real-time PCR, Western blotting and immunofluorescence. TNF-? mRNA elevation and protein secretion were obviously inhibited by nucleofection of HIF-1? small interfering RNA (siRNA) and treatment with 2-methoxyestradiol (inhibitor of HIF-1? protein). Similar results were observed in HEK293 and HepG2 cells. Putative hypoxia response elements were identified in the human TNF-? gene promoter. Deletion analysis and site-directed mutagenesis demonstrated that HIF consensus binding sites spanning bp-1295 to bp-1292 relative to the transcription start site were functional for activation of the TNF-? promoter which was confirmed by electrophoretic mobility-shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. Exosomes (vesicles mediating a non-classical route of protein secretion) in supernatants from hypoxic cardiomyocytes were identified by an anti-CD63 antibody in Western blot and observed by electron microscopy. The presence of TNF-? within exosomes precipitated from supernatants of hypoxic cardiomyocytes was verified by immunoelectron microscopy and immunoblotting. Results of this study indicate that under hypoxia, HIF-1? initiates expression of TNF-?, mediated by exosomes in cardiomyocytes. (hide) EV-METRIC 0% (median: 14% of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: AChE/ CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ AChE ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV120176
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	0