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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130027	4/4	Mus musculus	NAY	(d)(U)C Gel filtration UF	Hajj GN	2013	38%
Study summary Full title The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles All authors Hajj GN, Arantes CP, Dias MV, Roffé M, Costa-Silva B, Lopes MH, Porto-Carreiro I, Rabachini T, Lima FR, Beraldo FH, Prado MA, Linden R, Martins VR Journal Cell Mol Life Sci Abstract The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neur (show more...)The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling. (hide) EV-METRIC 38% (79th 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 NAY Focus vesicles extracellular vesicles 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 Gel filtration UF Protein markers EV: TSG101/ HSP90/ VPS36/ Tf-receptor/ Flotilin1/ Caveolin/ PrP/ HSP70 non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free 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 Pelleting performed No Other Name other separation method Gel filtration Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ HSP90/ HSP70/ TSG101/ Tf-receptor/ Caveolin/ PrP/ VPS36 ELISA Antibody details provided? No Detected EV-associated proteins Tf-receptor/ Caveolin/ PrP/ VPS36 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV130027	1/4	Mus musculus	NAY	(d)(U)C	Hajj GN	2013	33%
Study summary Full title The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles All authors Hajj GN, Arantes CP, Dias MV, Roffé M, Costa-Silva B, Lopes MH, Porto-Carreiro I, Rabachini T, Lima FR, Beraldo FH, Prado MA, Linden R, Martins VR Journal Cell Mol Life Sci Abstract The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neur (show more...)The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling. (hide) EV-METRIC 33% (75th 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 NAY Focus vesicles extracellular vesicles 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: TSG101/ Flotilin1/ VPS36/ Tf-receptor/ HSP90/ PrP/ HSP70 non-EV: GAPDH/ LAMP1 Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free 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 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ HSP90/ HSP70/ TSG101/ PrP/ Tf-receptor/ VPS36 Detected contaminants "GAPDH/ LAMP1" ELISA Antibody details provided? No Detected EV-associated proteins PrP/ Tf-receptor/ VPS36 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV130027	2/4	Mus musculus	NAY	(d)(U)C	Hajj GN	2013	33%
Study summary Full title The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles All authors Hajj GN, Arantes CP, Dias MV, Roffé M, Costa-Silva B, Lopes MH, Porto-Carreiro I, Rabachini T, Lima FR, Beraldo FH, Prado MA, Linden R, Martins VR Journal Cell Mol Life Sci Abstract The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neur (show more...)The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling. (hide) EV-METRIC 33% (75th 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 NAY Focus vesicles extracellular vesicles 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: TSG101/ Flotilin1/ VPS36/ Tf-receptor/ HSP90/ PrP/ HSP70 non-EV: GAPDH/ LAMP1 Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free 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) 960 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ HSP90/ HSP70/ TSG101/ PrP/ Tf-receptor/ VPS36 Detected contaminants "GAPDH/ LAMP1" ELISA Antibody details provided? No Detected EV-associated proteins PrP/ Tf-receptor/ VPS36 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV130027	3/4	Mus musculus	NAY	(d)(U)C	Hajj GN	2013	33%
Study summary Full title The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles All authors Hajj GN, Arantes CP, Dias MV, Roffé M, Costa-Silva B, Lopes MH, Porto-Carreiro I, Rabachini T, Lima FR, Beraldo FH, Prado MA, Linden R, Martins VR Journal Cell Mol Life Sci Abstract The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neur (show more...)The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling. (hide) EV-METRIC 33% (75th 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 NAY Focus vesicles extracellular vesicles 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: TSG101/ Flotilin1/ VPS36/ Tf-receptor/ HSP90/ PrP/ HSP70 non-EV: GAPDH/ LAMP1 Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free 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) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ HSP90/ HSP70/ TSG101/ PrP/ Tf-receptor/ VPS36 Detected contaminants "GAPDH/ LAMP1" ELISA Antibody details provided? No Detected EV-associated proteins PrP/ Tf-receptor/ VPS36 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV130027
species	Mus musculus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Gel filtration UF	(d)(U)C	(d)(U)C	(d)(U)C
Exp. nr.	4	1	2	3
EV-METRIC %	38	33	33	33