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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV180017	1/5	Homo sapiens	Cell culture supernatant	DG dUC Filtration Ultrafiltration	Beghein E	2018	63%
Study summary Full title Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function. All authors Beghein E, Devriese D, Van Hoey E, Gettemans J Journal J Cell Sci Abstract Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide) EV-METRIC 63% (95th 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 Sample origin Control condition Focus vesicles extracellular vesicle 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 DG + dUC + Filtration + Ultrafiltration Protein markers EV: CD9/ CD63/ Flotillin-2/ TSG101/ MT1-MMP non-EV: PMP70/ CytochromeC/ Calnexin/ Golgin245 Proteomics yes Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, Flotillin-2, TSG101, MT1-MMP Not detected contaminants PMP70, CytochromeC, Calnexin, Golgin245 Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 131 EV concentration Yes Particle yield 4.95E+09 particles/million cells

	EV180017	2/5	Homo sapiens	Cell culture supernatant	DG dUC Filtration Ultrafiltration	Beghein E	2018	62%
Study summary Full title Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function. All authors Beghein E, Devriese D, Van Hoey E, Gettemans J Journal J Cell Sci Abstract Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide) EV-METRIC 62% (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 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 Sample origin Expressing fascin nanobody Focus vesicles extracellular vesicle 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 DG + dUC + Filtration + Ultrafiltration Protein markers EV: CD9/ CD63 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Expressing fascin nanobody EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63 Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 142 EV concentration Yes Particle yield 2.25E+10 particles/million cells

	EV180017	5/5	Homo sapiens	Cell culture supernatant	DG dUC Filtration Ultrafiltration	Beghein E	2018	62%
Study summary Full title Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function. All authors Beghein E, Devriese D, Van Hoey E, Gettemans J Journal J Cell Sci Abstract Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide) EV-METRIC 62% (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 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 Sample origin Expressing cortactin nanobody (NTA domain) Focus vesicles extracellular vesicle 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 DG + dUC + Filtration + Ultrafiltration Protein markers EV: CD9/ CD63 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Expressing cortactin nanobody (NTA domain) EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63 Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 133 EV concentration Yes Particle yield 1.16E+09 particles/million cells

	EV180017	3/5	Homo sapiens	Cell culture supernatant	DG dUC Filtration Ultrafiltration	Beghein E	2018	50%
Study summary Full title Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function. All authors Beghein E, Devriese D, Van Hoey E, Gettemans J Journal J Cell Sci Abstract Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide) EV-METRIC 50% (88th 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 Sample origin Expressing cortactin nanobody (SH3 domain) Focus vesicles extracellular vesicle 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 DG + dUC + Filtration + Ultrafiltration Protein markers EV: CD9/ CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Expressing cortactin nanobody (SH3 domain) EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63 Characterization: Particle analysis NTA Report type Mean Reported size (nm) 133 EV concentration Yes Particle yield 9.79E+09 particles/million cells

	EV180017	4/5	Homo sapiens	Cell culture supernatant	DG dUC Filtration Ultrafiltration	Beghein E	2018	50%
Study summary Full title Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function. All authors Beghein E, Devriese D, Van Hoey E, Gettemans J Journal J Cell Sci Abstract Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide) EV-METRIC 50% (88th 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 Sample origin Expressing gelsolin nanobody Focus vesicles extracellular vesicle 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 DG + dUC + Filtration + Ultrafiltration Protein markers EV: CD9/ CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Expressing gelsolin nanobody EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63 Characterization: Particle analysis NTA Report type Mean Reported size (nm) 128 EV concentration Yes Particle yield 1.97E+09 particles/million cells

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EV-TRACK ID	EV180017
species	Homo sapiens
sample type	Cell culture
cell type	MDAMB231
condition	Control condition	Expressing fascin nanobody	Expressing cortactin nanobody (NTA domain)	Expressing cortactin nanobody (SH3 domain)	Expressing gelsolin nanobody
isolation protocol	DG dUC Filtration Ultrafiltration	DG dUC Filtration Ultrafiltration	DG dUC Filtration Ultrafiltration	DG dUC Filtration Ultrafiltration	DG dUC Filtration Ultrafiltration
Exp. nr.	1	2	5	3	4
EV-METRIC %	63	62	62	50	50