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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190055	1/4	Homo sapiens	ARPE-19	DG (d)(U)C	Ferreira JV	2019	78%
Study summary Full title Exosomes and STUB1/CHIP cooperate to maintain intracellular proteostasis. All authors Ferreira JV, Rosa Soares A, Ramalho JS, Ribeiro-Rodrigues T, Máximo C, Zuzarte M, Girão H, Pereira P. Journal PLoS One Abstract Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the ac (show more...)Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material. (hide) EV-METRIC 78% (97th 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 small extracellular vesicles (sEVs) 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 DG (d)(U)C Protein markers EV: CD63/ Hsc70/ Flotillin1/ GAPDH/ CANX non-EV: Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ARPE-19 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 8 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 210000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Size Used subtypes < 200 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ CD63/ GAPDH/ Hsc70 Not detected EV-associated proteins CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Other particle analysis name(1) RhoB-PE staining Report type Not Reported EV-concentration No

	EV190055	3/4	Homo sapiens	ARPE-19	DG (d)(U)C	Ferreira JV	2019	78%
Study summary Full title Exosomes and STUB1/CHIP cooperate to maintain intracellular proteostasis. All authors Ferreira JV, Rosa Soares A, Ramalho JS, Ribeiro-Rodrigues T, Máximo C, Zuzarte M, Girão H, Pereira P. Journal PLoS One Abstract Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the ac (show more...)Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material. (hide) EV-METRIC 78% (97th 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 small extracellular vesicles (sEVs) 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 DG (d)(U)C Protein markers EV: CD63/ HSC70/ Flotillin1/ GAPDH/ CANX non-EV: Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ARPE-19 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 8 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 210000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Size Used subtypes < 200 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ CD63/ GAPDH/ HSC70 Not detected EV-associated proteins CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Other particle analysis name(1) RhoB-PE staining Report type Not Reported EV-concentration No

	EV190055	2/4	Homo sapiens	ARPE-19	DG (d)(U)C	Ferreira JV	2019	67%
Study summary Full title Exosomes and STUB1/CHIP cooperate to maintain intracellular proteostasis. All authors Ferreira JV, Rosa Soares A, Ramalho JS, Ribeiro-Rodrigues T, Máximo C, Zuzarte M, Girão H, Pereira P. Journal PLoS One Abstract Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the ac (show more...)Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material. (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 Control condition Focus vesicles Microvesicles (MVs) 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 DG (d)(U)C Protein markers EV: CD63/ GAPDH/ CANX/ Na/K A1 ATPase non-EV: Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ARPE-19 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 8 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 210000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Size Used subtypes >200 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ GAPDH/ Na/K A1 ATPase/ CANX Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up

	EV190055	4/4	Homo sapiens	ARPE-19	DG (d)(U)C	Ferreira JV	2019	67%
Study summary Full title Exosomes and STUB1/CHIP cooperate to maintain intracellular proteostasis. All authors Ferreira JV, Rosa Soares A, Ramalho JS, Ribeiro-Rodrigues T, Máximo C, Zuzarte M, Girão H, Pereira P. Journal PLoS One Abstract Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the ac (show more...)Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material. (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 Control condition Focus vesicles Microvesicles (MVs) 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 DG (d)(U)C Protein markers EV: CD63/ GAPDH/ CANX/ Na/K A1 ATPase non-EV: Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ARPE-19 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 8 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 210000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Size Used subtypes >200 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins GAPDH/ Na/K A1 ATPase/ CANX/ CD63 Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV190055
species	Homo sapiens
sample type	Cell culture
cell type	ARPE-19
condition	Control condition
separation protocol	DG (d)(U)C	DG (d)(U)C	DG (d)(U)C	DG (d)(U)C
EV subtype	< 200	< 200	>200	>200
vesicle related term	small EVs (sEVs)	small EVs (sEVs)	Microvesicles (MVs)	Microvesicles (MVs)
Exp. nr.	1	3	2	4
EV-METRIC %	78	78	67	67