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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210021	1/2	Rattus norvegicus	Primary rat hepatocytes	(d)(U)C Filtration DG	Mleczko, J.E.	2021	89%
Study summary Full title Extracellular vesicles released by steatotic hepatocytes alteradipocyte metabolism All authors J.E. Mleczko, F. Royo, I. Samuelson, M. Clos-Garcia, C. Williams, D. Cabrera, M. Azparren-Angulo, E. Gonzalez, C. Garcia-Vallicrosa, S. Carobbio, S. Rodriguez-Cuenca, M. Azkargorta, S. van Liempd, F. Elortza, A. Vidal-Puig, S. Mora, J.M. Falcon-Perez Journal Journal of Extracellular Biology Abstract The composition of extracellular vesicles (EVs) is altered in many pathological condi-tions, and the (show more...)The composition of extracellular vesicles (EVs) is altered in many pathological condi-tions, and their molecular content provides essential information on features of par-ent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome(MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dys-lipidemia and hypertension that increases the risk of cardiovascular disease and type 2diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes bycharacterizing EVs secreted by primary hepatocytes isolated from Zucker rat model,and studied the effect they have on 3T3-L1 adipocytes. We found that steatotic hepa-tocytes secrete EVs with significantly reduced exosomal markers in comparison withtheir lean counterpart. Moreover, proteomic analysis revealed that those EVs reflectthe metabolic state of the parent cell in that the majority of proteins upregulated relateto fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway.In addition, hepatocytes-secreted EVs influenced lipolysis and insulin sensitivity inrecipient 3T3-L1 adipocytes. Untargeted metabolomic analysis detected alterations indifferent adipocyte metabolic pathways in cells treated with hepatic EVs. In summary,our work showed that steatosis has a significant impact in the amount and composi-tion of EVs secreted by hepatocytes. Moreover, our data point to the involvement ofhepatic-EVs in the development of pathologies associated with MetS. (hide) EV-METRIC 89% (99th 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 Obese hepatocytes 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 (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: TSG101/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ HSP70 non-EV: GRP78/ COXIV/ Parp Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells Primary rat hepatocytes EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 300000000 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 45 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 2 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16,5 Sample volume (mL) 5,5 Orientation Top-down Speed (g) 100000 Duration (min) 160 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction Not repo Pelleting: duration (min) 60 Pelleting: rotor type Not reported Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD63/ HSP90/ TSG101/ HSP70/ Alix/ CD81 Not detected EV-associated proteins HSP90/ HSP70/ CD81/ Flotillin1/ TSG101/ CD63/ Alix Detected contaminants COXIV/ GRP78/ Parp Not detected contaminants COXIV/ GRP78/ Parp Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-400 EV concentration Yes Particle yield as number of particles per million cells: 2 EM EM-type Cryo-EM Image type Close-up

	EV210021	2/2	Rattus norvegicus	Primary rat hepatocytes	(d)(U)C Filtration	Mleczko, J.E.	2021	89%
Study summary Full title Extracellular vesicles released by steatotic hepatocytes alteradipocyte metabolism All authors J.E. Mleczko, F. Royo, I. Samuelson, M. Clos-Garcia, C. Williams, D. Cabrera, M. Azparren-Angulo, E. Gonzalez, C. Garcia-Vallicrosa, S. Carobbio, S. Rodriguez-Cuenca, M. Azkargorta, S. van Liempd, F. Elortza, A. Vidal-Puig, S. Mora, J.M. Falcon-Perez Journal Journal of Extracellular Biology Abstract The composition of extracellular vesicles (EVs) is altered in many pathological condi-tions, and the (show more...)The composition of extracellular vesicles (EVs) is altered in many pathological condi-tions, and their molecular content provides essential information on features of par-ent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome(MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dys-lipidemia and hypertension that increases the risk of cardiovascular disease and type 2diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes bycharacterizing EVs secreted by primary hepatocytes isolated from Zucker rat model,and studied the effect they have on 3T3-L1 adipocytes. We found that steatotic hepa-tocytes secrete EVs with significantly reduced exosomal markers in comparison withtheir lean counterpart. Moreover, proteomic analysis revealed that those EVs reflectthe metabolic state of the parent cell in that the majority of proteins upregulated relateto fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway.In addition, hepatocytes-secreted EVs influenced lipolysis and insulin sensitivity inrecipient 3T3-L1 adipocytes. Untargeted metabolomic analysis detected alterations indifferent adipocyte metabolic pathways in cells treated with hepatic EVs. In summary,our work showed that steatosis has a significant impact in the amount and composi-tion of EVs secreted by hepatocytes. Moreover, our data point to the involvement ofhepatic-EVs in the development of pathologies associated with MetS. (hide) EV-METRIC 89% (99th 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 Lean hepatocytes 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ HSP70 non-EV: Grp78/ GRP78/ COXIV/ Parp Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells Primary rat hepatocytes EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 300000000 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: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 45 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 2 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16,5 Sample volume (mL) 5,5 Orientation Top-down Speed (g) 100000 Duration (min) 160 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction Not repo Pelleting: duration (min) 60 Pelleting: rotor type Not reported Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD63/ HSP90/ TSG101/ HSP70/ Alix/ CD81 Not detected EV-associated proteins HSP90/ HSP70/ CD81/ Flotillin1/ TSG101/ CD63/ Alix Detected contaminants COXIV/ GRP78/ Parp Not detected contaminants COXIV/ Grp78/ Parp Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-400 EV concentration Yes Particle yield as number of particles per million cells: 1,8 EM EM-type Cryo-EM Image type Close-up

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EV-TRACK ID	EV210021
species	Rattus norvegicus
sample type	Cell culture
cell type	Primary rat hepatocytes
condition	Obese hepatocytes	Lean hepatocytes
separation protocol	dUC/ Filtration/ Density gradient	dUC/ Filtration
Exp. nr.	1	2
EV-METRIC %	89	89