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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110002	1/1	Homo sapiens Canis familiaris	NAY	(d)(U)C DG Filtration	Higginbotham JN	2011	67%
Study summary Full title Amphiregulin exosomes increase cancer cell invasion All authors Higginbotham JN, Demory Beckler M, Gephart JD, Franklin JL, Bogatcheva G, Kremers GJ, Piston DW, Ayers GD, McConnell RE, Tyska MJ, Coffey RJ Journal Curr Biol Abstract Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands inclu (show more...)Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands including EGF, TGF-? (TGF?), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin, and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells 4-fold over TGF? or HB-EGF exosomes and 5-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGF? or HB-EGF. An average of 24 AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between nontransformed and transformed cells is unknown. Exosomes from DLD-1 colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, nontransformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes might contribute to diverse cancer phenomena such as field effect and priming of the metastatic niche. (hide) EV-METRIC 67% (94th percentile 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 DG Filtration Protein markers EV: TSG101/ Flotilin1/ ADAM10/ ADAM17/ HSP70/ CD9 non-EV: Cell organelle protein Proteomics no TEM measurements 51.3(median) Show all info Study aim Function Sample Species Homo sapiens / Canis familiaris Sample Type Cell culture supernatant EV-harvesting Medium serum free 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) 150 Density gradient Only used for validation of main results Yes Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Flotilin1/ HSP70/ TSG101/ ADAM10/ ADAM17 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins ADAM10/ ADAM17 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field Report size (nm) 51.3(median)

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EV-TRACK ID	EV110002
species	Homo sapiens Canis familiaris
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG Filtration
Exp. nr.	1
EV-METRIC %	67