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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210041	1/5	Homo sapiens	SK-MEL-147	qEV	Suarez, Henar	2021	75%
Study summary Full title CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells All authors Henar Suárez, Zoraida Andreu, Carla Mazzeo, Víctor Toribio, Aldo Emmanuel Pérez‐Rivera, Soraya López‐Martín, Susana García‐Silva, Begoña Hurtado, Esperanza Morato, Laura Peláez, Egoitz Astigarraga Arribas, Tarson Tolentino‐Cortez, Gabriel Barreda‐Gómez, Ana Isabel Marina, Héctor Peinado, María Yáñez‐Mó Journal J Extracell Vesicles Abstract Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundan (show more...)Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. (hide) EV-METRIC 75% (96th 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 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 qEV Protein markers EV: Flotillin1/ CD9/ CD63/ HSP90/ MHC1/ TSG101/ Alix/ CD81 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) >98% Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ HSP90/ MHC1/ TSG101/ Alix/ CD81 Proteomics database Yes: Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield Yes, as number of particles per million cells 1800 TRPS Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120 EV concentration Yes

	EV210041	3/5	Homo sapiens	SK-MEL-147	qEV	Suarez, Henar	2021	75%
Study summary Full title CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells All authors Henar Suárez, Zoraida Andreu, Carla Mazzeo, Víctor Toribio, Aldo Emmanuel Pérez‐Rivera, Soraya López‐Martín, Susana García‐Silva, Begoña Hurtado, Esperanza Morato, Laura Peláez, Egoitz Astigarraga Arribas, Tarson Tolentino‐Cortez, Gabriel Barreda‐Gómez, Ana Isabel Marina, Héctor Peinado, María Yáñez‐Mó Journal J Extracell Vesicles Abstract Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundan (show more...)Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. (hide) EV-METRIC 75% (96th 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 CD9 KO 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 qEV Protein markers EV: Flotillin1/ Alix/ CD9/ CD63/ HSP90/ MHC1/ CD81 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) >98% Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Other Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD9/ CD63/ HSP90/ MHC1/ CD81 Proteomics database Yes: Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 110 EV concentration Yes Particle yield Yes, as number of particles per million cells 16000 TRPS Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 110 EV concentration Yes

	EV210041	4/5	Homo sapiens	SK-MEL-147	qEV	Suarez, Henar	2021	75%
Study summary Full title CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells All authors Henar Suárez, Zoraida Andreu, Carla Mazzeo, Víctor Toribio, Aldo Emmanuel Pérez‐Rivera, Soraya López‐Martín, Susana García‐Silva, Begoña Hurtado, Esperanza Morato, Laura Peláez, Egoitz Astigarraga Arribas, Tarson Tolentino‐Cortez, Gabriel Barreda‐Gómez, Ana Isabel Marina, Héctor Peinado, María Yáñez‐Mó Journal J Extracell Vesicles Abstract Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundan (show more...)Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. (hide) EV-METRIC 75% (96th 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 Peptide treatments 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 qEV Protein markers EV: CD63/ Rac1/ CD81/ Flotillin1/ syntenin/ ERM/ CD9 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) >98% Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ ERM/ Rac1/ syntenin/ CD9/ CD63/ CD81 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield Yes, as number of particles per million cells 1800 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120 EV concentration Yes

	EV210041	2/5	Homo sapiens	SK-MEL-147	(d)(U)C	Suarez, Henar	2021	44%
Study summary Full title CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells All authors Henar Suárez, Zoraida Andreu, Carla Mazzeo, Víctor Toribio, Aldo Emmanuel Pérez‐Rivera, Soraya López‐Martín, Susana García‐Silva, Begoña Hurtado, Esperanza Morato, Laura Peláez, Egoitz Astigarraga Arribas, Tarson Tolentino‐Cortez, Gabriel Barreda‐Gómez, Ana Isabel Marina, Héctor Peinado, María Yáñez‐Mó Journal J Extracell Vesicles Abstract Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundan (show more...)Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. (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. 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 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 (d)(U)C Protein markers EV: CD81/ ERMs/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) >98% 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) 120 Pelleting: rotor type AH-627 Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 30 Wash: time (min) 120 Wash: Rotor Type AH-627 Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ ERMs/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 Particle yield NA NA

	EV210041	5/5	Homo sapiens	SK-MEL-147	(d)(U)C	Suarez, Henar	2021	44%
Study summary Full title CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells All authors Henar Suárez, Zoraida Andreu, Carla Mazzeo, Víctor Toribio, Aldo Emmanuel Pérez‐Rivera, Soraya López‐Martín, Susana García‐Silva, Begoña Hurtado, Esperanza Morato, Laura Peláez, Egoitz Astigarraga Arribas, Tarson Tolentino‐Cortez, Gabriel Barreda‐Gómez, Ana Isabel Marina, Héctor Peinado, María Yáñez‐Mó Journal J Extracell Vesicles Abstract Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundan (show more...)Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. (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. 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 Peptide treatments 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 (d)(U)C Protein markers EV: CD81/ ERM/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) >98% 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) 120 Pelleting: rotor type AH-627 Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 30 Wash: time (min) 120 Wash: Rotor Type AH-627 Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins ERM/ CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120

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EV-TRACK ID	EV210041
species	Homo sapiens
sample type	Cell culture
cell type	SK-MEL-147
condition	Control condition	CD9 KO	Peptide treatments	Control condition	Peptide treatments
separation protocol	qEV	qEV	qEV	(d)(U)C	(d)(U)C
Exp. nr.	1	3	4	2	5
EV-METRIC %	75	75	75	44	44