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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV130137	3/3	Mus musculus Rattus norvegicus/rattus	Cell culture supernatant	0.2 µm filter Density cushion (valid.) dUC Sucrose-DG (valid.)	Royo F	2013	44%
Study summary Full title Transcriptome of extracellular vesicles released by hepatocytes All authors Royo F, Schlangen K, Palomo L, Gonzalez E, Conde-Vancells J, Berisa A, Aransay AM, Falcon-Perez JM Journal PLoS One Abstract The discovery that the cells communicate through emission of vesicles has opened new opportunities f (show more...)The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers. (hide) EV-METRIC 44% (84th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Density cushion (valid.) + dUC + Sucrose-DG (valid.) Protein markers EV: CD81/ Flotilin1/ TSG101/ Aip1 non-EV: None Proteomics no EV density (g/ml) 1.12-1.2;1.19-1.23 Show all info Study aim Omics Sample Species Mus musculus / Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Flotilin1/ TSG101/ Aip1 ELISA Detected EV-associated proteins Aip1 Fluorescent NTA Characterization: Particle analysis NTA EM EM-type cryo EM Image type Close-up

	EV130137	1/3	Mus musculus Rattus norvegicus/rattus	Cell culture supernatant	Commercial	Royo F	2013	0%
Study summary Full title Transcriptome of extracellular vesicles released by hepatocytes All authors Royo F, Schlangen K, Palomo L, Gonzalez E, Conde-Vancells J, Berisa A, Aransay AM, Falcon-Perez JM Journal PLoS One Abstract The discovery that the cells communicate through emission of vesicles has opened new opportunities f (show more...)The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers. (hide) EV-METRIC 0% (median: 22% 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Commercial Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Omics Sample Species Mus musculus / Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Commercial kit ExoQuick Fluorescent NTA Characterization: Particle analysis

	EV130137	2/3	Rattus norvegicus/rattus	Serum	Commercial	Royo F	2013	0%
Study summary Full title Transcriptome of extracellular vesicles released by hepatocytes All authors Royo F, Schlangen K, Palomo L, Gonzalez E, Conde-Vancells J, Berisa A, Aransay AM, Falcon-Perez JM Journal PLoS One Abstract The discovery that the cells communicate through emission of vesicles has opened new opportunities f (show more...)The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers. (hide) EV-METRIC 0% (median: 13% 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Serum Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Commercial Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Omics Sample Species Rattus norvegicus/rattus Sample Type Serum Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Commercial kit ExoQuick Fluorescent NTA Characterization: Particle analysis

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EV-TRACK ID	EV130137
species	Mus musculus Rattus norvegicus/rattus	Mus musculus Rattus norvegicus/rattus	Rattus norvegicus/rattus
sample type	Cell culture	Cell culture	Serum
medium	EV Depleted	EV Depleted
isolation protocol	0.2 µm filter DC (valid.) dUC Sucrose-DG	Commercial	Commercial
case number	3	1	2
EV-METRIC %	44	0	0