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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140018	1/2	Homo sapiens Gallus gallus	NAY	(d)(U)C DG	Vyas N	2014	56%
Study summary Full title Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties All authors Vyas N, Walvekar A, Tate D, Lakshmanan V, Bansal D, Lo Cicero A, Raposo G, Palakodeti D, Dhawan J Journal Sci Rep Abstract Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tiss (show more...)Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3? and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation. (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 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 DG Protein markers EV: TSG101/ CD9 non-EV: Flotillin1/ Flotillin2/ Cell organelle protein Proteomics yes EV density (g/ml) 1.120 Show all info Study aim Function Sample Species Homo sapiens / Gallus gallus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 10 Highest density fraction 85 Orientation Top-down Speed (g) 450000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9/ TSG101 Detected contaminants Cell organelle protein/ "Flotillin1/ Flotillin2" Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein CD63 Image type Close-up, Wide-field

	EV140018	2/2	Homo sapiens Gallus gallus	NAY	(d)(U)C DG	Vyas N	2014	56%
Study summary Full title Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties All authors Vyas N, Walvekar A, Tate D, Lakshmanan V, Bansal D, Lo Cicero A, Raposo G, Palakodeti D, Dhawan J Journal Sci Rep Abstract Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tiss (show more...)Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3? and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation. (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 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 DG Protein markers EV: TSG101/ Flotilin1/ CD9/ Flotillin2 non-EV: Cell organelle protein Proteomics yes EV density (g/ml) 1.120 Show all info Study aim Function Sample Species Homo sapiens / Gallus gallus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted 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) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 10 Highest density fraction 85 Orientation Top-down Speed (g) 150000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9/ Flotilin1/ TSG101/ Flotillin2 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Flotillin2 Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein CD63 Image type Close-up, Wide-field

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EV-TRACK ID	EV140018
species	Homo sapiens Gallus gallus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG	(d)(U)C DG
Exp. nr.	1	2
EV-METRIC %	56	56