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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230950	1/2	Yersinia pseudotuberculosis	ATCC 29833	(d)(U)C DG Filtration UF	Monnappa AK	2018	50%
Study summary Full title The Cytotoxic Necrotizing Factor of Yersinia pseudotuberculosis (CNFy) is Carried on Extracellular Membrane Vesicles to Host Cells. All authors Monnappa AK, Bari W, Seo JK, Mitchell RJ Journal Sci Rep Abstract In this study we show Yersinia pseudotuberculosis secretes membrane vesicles (MVs) that contain diff (show more...)In this study we show Yersinia pseudotuberculosis secretes membrane vesicles (MVs) that contain different proteins and virulence factors depending on the strain. Although MVs from Y. pseudotuberculosis YPIII and ATCC 29833 had many proteins in common (68.8% of all the proteins identified), those located in the outer membrane fraction differed significantly. For instance, the MVs from Y. pseudotuberculosis YPIII harbored numerous Yersinia outer proteins (Yops) while they were absent in the ATCC 29833 MVs. Another virulence factor found solely in the YPIII MVs was the cytotoxic necrotizing factor (CNFy), a toxin that leads to multinucleation of host cells. The ability of YPIII MVs to transport this toxin and its activity to host cells was verified using HeLa cells, which responded in a dose-dependent manner/ nearly 70% of the culture was multinucleated after addition of 5 µg/ml of the purified YPIII MVs. In contrast, less than 10% were multinucleated when the ATCC 29833 MVs were added. Semi-quantification of CNFy within the YPIII MVs found this toxin is present at concentrations of 5 ~ 10 ng per µg of total MV protein, a concentration that accounts for the cellular responses seen. (hide) EV-METRIC 50% (87th 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 MV (membrane 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 (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Protein markers EV: Not applicable non-EV: Not applicable Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Yersinia pseudotuberculosis Sample Type Cell culture supernatant EV-producing cells ATCC 29833 EV-harvesting Medium serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type not specified Pelleting: speed (g) not spec Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0% Highest density fraction 40% Total gradient volume, incl. sample (mL) 11.5 Sample volume (mL) 2 Orientation Bottom-up Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Other 1 SDS-PAGE + Coomassie Blue staining Detected EV-associated proteins Not applicable Not detected EV-associated proteins Not applicable Detected contaminants Not applicable Not detected contaminants Not applicable Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 70-150

	EV230950	2/2	Yersinia pseudotuberculosis	YPIII	(d)(U)C DG Filtration UF	Monnappa AK	2018	50%
Study summary Full title The Cytotoxic Necrotizing Factor of Yersinia pseudotuberculosis (CNFy) is Carried on Extracellular Membrane Vesicles to Host Cells. All authors Monnappa AK, Bari W, Seo JK, Mitchell RJ Journal Sci Rep Abstract In this study we show Yersinia pseudotuberculosis secretes membrane vesicles (MVs) that contain diff (show more...)In this study we show Yersinia pseudotuberculosis secretes membrane vesicles (MVs) that contain different proteins and virulence factors depending on the strain. Although MVs from Y. pseudotuberculosis YPIII and ATCC 29833 had many proteins in common (68.8% of all the proteins identified), those located in the outer membrane fraction differed significantly. For instance, the MVs from Y. pseudotuberculosis YPIII harbored numerous Yersinia outer proteins (Yops) while they were absent in the ATCC 29833 MVs. Another virulence factor found solely in the YPIII MVs was the cytotoxic necrotizing factor (CNFy), a toxin that leads to multinucleation of host cells. The ability of YPIII MVs to transport this toxin and its activity to host cells was verified using HeLa cells, which responded in a dose-dependent manner/ nearly 70% of the culture was multinucleated after addition of 5 µg/ml of the purified YPIII MVs. In contrast, less than 10% were multinucleated when the ATCC 29833 MVs were added. Semi-quantification of CNFy within the YPIII MVs found this toxin is present at concentrations of 5 ~ 10 ng per µg of total MV protein, a concentration that accounts for the cellular responses seen. (hide) EV-METRIC 50% (87th 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 MV (membrane 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 (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Protein markers EV: Not applicable non-EV: Not applicable Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Yersinia pseudotuberculosis Sample Type Cell culture supernatant EV-producing cells YPIII EV-harvesting Medium serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type not specified Pelleting: speed (g) not spec Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0% Highest density fraction 40% Total gradient volume, incl. sample (mL) 11.5 Sample volume (mL) 2 Orientation Bottom-up Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Other 1 SDS-PAGE Coomassie Blue staining Detected EV-associated proteins Not applicable Not detected EV-associated proteins Not applicable Detected contaminants Not applicable Not detected contaminants Not applicable Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 70-150

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EV-TRACK ID	EV230950
species	Yersinia pseudotuberculosis
sample type	Cell culture
cell type	ATCC 29833	YPIII
condition	Control condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration
Exp. nr.	1	2
EV-METRIC %	50	50