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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210024	3/12	Homo sapiens	Glioblastoma Stem-like cells (GSC)	(d)(U)C DG	André-Grégoire, Gwennan	2022	89%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (hide) EV-METRIC 89% (99th 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma Stem-like cells (GSC) EV-harvesting Medium Serum free medium Cell count 5.00E+08 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: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40 Total gradient volume, incl. sample (mL) 11.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Alix Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 100 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50

	EV210024	8/12	Homo sapiens	Glioblastoma Stem-like cells (GSC)	(d)(U)C DG	André-Grégoire, Gwennan	2022	67%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (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 NSA 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma Stem-like cells (GSC) EV-harvesting Medium Serum free medium Cell count 5.00E+08 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: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40 Total gradient volume, incl. sample (mL) 11.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Alix Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 125 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 100

	EV210024	10/12	Homo sapiens	Differentiated Glioblastoma Stem-like cells (DGC)	(d)(U)C	André-Grégoire, Gwennan	2022	56%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (hide) EV-METRIC 56% (90th 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 NSA 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Differentiated Glioblastoma Stem-like cells (DGC) 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: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63 Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 125 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210024	1/12	Homo sapiens	Glioblastoma Stem-like cells (GSC)	(d)(U)C	André-Grégoire, Gwennan	2022	33%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (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 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma Stem-like cells (GSC) EV-harvesting Medium Serum free medium Cell count 5.00E+08 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 Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 14 Wash: time (min) 30 Wash: Rotor Type SW 41 Ti Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Alix Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 300-1600 EV concentration Yes

	EV210024	2/12	Homo sapiens	Glioblastoma Stem-like cells (GSC)	(d)(U)C	André-Grégoire, Gwennan	2022	33%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (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 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma Stem-like cells (GSC) EV-harvesting Medium Serum free medium Cell count 5.00E+08 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: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Alix Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 300-1600 EV concentration Yes

	EV210024	4/12	Homo sapiens	Glioblastoma Stem-like cells (GSC)	(d)(U)C	André-Grégoire, Gwennan	2022	33%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (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 siRNA MLKL 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 (Differential) (ultra)centrifugation Protein markers EV: CD9 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma Stem-like cells (GSC) EV-harvesting Medium Serum free medium Cell count 5.00E+08 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 Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 14 Wash: time (min) 30 Wash: Rotor Type SW 41 Ti Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9 Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210024	5/12	Homo sapiens	Glioblastoma Stem-like cells (GSC)	(d)(U)C	André-Grégoire, Gwennan	2022	33%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (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 siRNA MLKL 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 (Differential) (ultra)centrifugation Protein markers EV: CD9 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma Stem-like cells (GSC) EV-harvesting Medium Serum free medium Cell count 5.00E+08 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: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9 Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210024	6/12	Homo sapiens	Glioblastoma Stem-like cells (GSC)	(d)(U)C	André-Grégoire, Gwennan	2022	33%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (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 NSA 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma Stem-like cells (GSC) EV-harvesting Medium Serum free medium Cell count 5.00E+08 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 Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 14 Wash: time (min) 30 Wash: Rotor Type SW 41 Ti Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63 Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 300-1600 EV concentration Yes

	EV210024	7/12	Homo sapiens	Glioblastoma Stem-like cells (GSC)	(d)(U)C	André-Grégoire, Gwennan	2022	33%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (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 NSA 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma Stem-like cells (GSC) EV-harvesting Medium Serum free medium Cell count 5.00E+08 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: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63 Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 300-1600 EV concentration Yes

	EV210024	9/12	Homo sapiens	Differentiated Glioblastoma Stem-like cells (DGC)	(d)(U)C	André-Grégoire, Gwennan	2022	33%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (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 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Differentiated Glioblastoma Stem-like cells (DGC) 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: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63 Not detected contaminants GM130 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 300-1600 EV concentration Yes

	EV210024	11/12	Mus musculus	Blood plasma	(d)(U)C	André-Grégoire, Gwennan	2022	13%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (hide) EV-METRIC 13% (30th 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 Blood plasma 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 (Differential) (ultra)centrifugation Protein markers EV: CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Mus musculus Sample Type Blood plasma 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: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210024	12/12	Mus musculus	Blood plasma	(d)(U)C	André-Grégoire, Gwennan	2022	13%
Study summary Full title Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma All authors Gwennan André-Grégoire, Clément Maghe, Tiphaine Douanne, Sara, Rosińska, Fiorella Spinelli, An Thys, Kilian Trillet, Kathryn A.Jacobs, Cyndie Ballu, Aurélien Dupont, Anne-Marie Lyne, Florence M.G.Cavalli, Ignacio Busnelli, Vincent Hyenne, Jacky G.Goetz, Nicolas Bidère, Julie Gavard Journal iScience Abstract Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material fro (show more...)Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. (hide) EV-METRIC 13% (30th 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 Blood plasma Sample origin NSA 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 (Differential) (ultra)centrifugation Protein markers EV: CD63 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Mus musculus Sample Type Blood plasma 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: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210024
species	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	Homo sapiens	Mus musculus	Mus musculus
sample type	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Blood plasma	Blood plasma
cell type	Glioblastoma Stem-like cells (GSC)	Glioblastoma Stem-like cells (GSC)	Differentiated Glioblastoma Stem-like cells (DGC)	Glioblastoma Stem-like cells (GSC)	Glioblastoma Stem-like cells (GSC)	Glioblastoma Stem-like cells (GSC)	Glioblastoma Stem-like cells (GSC)	Glioblastoma Stem-like cells (GSC)	Glioblastoma Stem-like cells (GSC)	Differentiated Glioblastoma Stem-like cells (DGC)	NA	NA
medium	Serum free medium	Serum free medium	EV-depleted medium	Serum free medium	Serum free medium	Serum free medium	Serum free medium	Serum free medium	Serum free medium	EV-depleted medium	NA	NA
condition	Control condition	NSA	NSA	Control condition	Control condition	siRNA MLKL	siRNA MLKL	NSA	NSA	Control condition	Control condition	NSA
separation protocol	dUC/ Density gradient	dUC/ Density gradient	dUC	dUC	dUC	dUC	dUC	dUC	dUC	dUC	dUC	dUC
Exp. nr.	3	8	10	1	2	4	5	6	7	9	11	12
EV-METRIC %	89	67	56	33	33	33	33	33	33	33	13	13

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