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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200071	1/2	Homo sapiens	NPC/HK1	(d)(U)C 16% polyethylene glycol precipitation UF Filtration	Nkosi, Dingani	2020	56%
Study summary Full title Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells All authors Dingani Nkosi, Li Sun, Leanne C Duke, David G Meckes Jr Journal PLoS Pathog Abstract Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal ves (show more...)Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal vesicle content contribute to function and disease initiation or progression. The ability to package a variety of cargo and transmit molecular information between cells renders EVs important mediators of cell-to-cell crosstalk. Latent membrane protein 1 (LMP1) is a chief viral oncoprotein expressed in most Epstein-Barr virus (EBV)-associated cancers and is released from cells at high levels in EVs. LMP1 containing EVs have been demonstrated to promote cell growth, migration, differentiation, and regulate immune cell function. Despite these significant changes in recipient cells induced by LMP1 modified EVs, the mechanism how this viral oncogene modulates the recipient cells towards these phenotypes is not well understood. We hypothesize that LMP1 alters EV content and following uptake of the LMP1-modified EVs by the recipient cells results in the activation of cell signaling pathways and increased gene expression which modulates the biological properties of recipient cell towards a new phenotype. Our results show that LMP1 expression alters the EV protein and microRNA content packaged into EVs. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κB signaling pathways. The LMP1 containing EVs induced transcriptome reprogramming in the recipient cells by altering gene expression of different targets including cadherins, matrix metalloproteinases 9 (MMP9), MMP2 and integrin-α5 which contribute to extracellular matrix (ECM) remodeling. Altogether, our data demonstrate the mechanism in which LMP1-modified EVs reshape the tumor microenvironment by increasing gene expression of ECM interaction proteins. (hide) EV-METRIC 56% (90th 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 Control condition Focus vesicles extracellular vesicle 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 16% polyethylene glycol precipitation UF Filtration Protein markers EV: TSG101/ CD63/ HSC70/ CD81/ Alix/ Flotillin2 non-EV: Calnexin Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells NPC/HK1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Other Name other separation method 16% polyethylene glycol precipitation Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ HSC70/ Flotillin2/ TSG101/ Alix/ CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175.25 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3880000000000

	EV200071	2/2	Homo sapiens	NPC/HK1	(d)(U)C 16% polyethylene glycol precipitation UF Filtration	Nkosi, Dingani	2020	56%
Study summary Full title Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells All authors Dingani Nkosi, Li Sun, Leanne C Duke, David G Meckes Jr Journal PLoS Pathog Abstract Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal ves (show more...)Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal vesicle content contribute to function and disease initiation or progression. The ability to package a variety of cargo and transmit molecular information between cells renders EVs important mediators of cell-to-cell crosstalk. Latent membrane protein 1 (LMP1) is a chief viral oncoprotein expressed in most Epstein-Barr virus (EBV)-associated cancers and is released from cells at high levels in EVs. LMP1 containing EVs have been demonstrated to promote cell growth, migration, differentiation, and regulate immune cell function. Despite these significant changes in recipient cells induced by LMP1 modified EVs, the mechanism how this viral oncogene modulates the recipient cells towards these phenotypes is not well understood. We hypothesize that LMP1 alters EV content and following uptake of the LMP1-modified EVs by the recipient cells results in the activation of cell signaling pathways and increased gene expression which modulates the biological properties of recipient cell towards a new phenotype. Our results show that LMP1 expression alters the EV protein and microRNA content packaged into EVs. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κB signaling pathways. The LMP1 containing EVs induced transcriptome reprogramming in the recipient cells by altering gene expression of different targets including cadherins, matrix metalloproteinases 9 (MMP9), MMP2 and integrin-α5 which contribute to extracellular matrix (ECM) remodeling. Altogether, our data demonstrate the mechanism in which LMP1-modified EVs reshape the tumor microenvironment by increasing gene expression of ECM interaction proteins. (hide) EV-METRIC 56% (90th 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 LMP1 overexpression Focus vesicles extracellular vesicle 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 16% polyethylene glycol precipitation UF Filtration Protein markers EV: TSG101/ CD63/ HSC70/ CD81/ Alix/ Flotillin2 non-EV: Calnexin Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells NPC/HK1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Other Name other separation method 16% polyethylene glycol precipitation Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Flotillin2/ HSC70/ TSG101/ Alix/ CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Other Reported size (nm) 186 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5300000000000

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EV-TRACK ID	EV200071
species	Homo sapiens
sample type	Cell culture
cell type	NPC/HK1
condition	Control condition	LMP1 overexpression
separation protocol	(d)(U)C 16% polyethylene glycol precipitation UF Filtration	(d)(U)C 16% polyethylene glycol precipitation UF Filtration
Exp. nr.	1	2
EV-METRIC %	56	56