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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140025	1/1	Drosophila melanogaster	Drosophila	(d)(U)C DG Filtration	Gradilla AC	2014	56%
Study summary Full title Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion All authors Gradilla AC, González E, Seijo I, Andrés G, Bischoff M, González-Mendez L, Sánchez V, Callejo A, Ibáñez C, Guerra M, Ortigão-Farias JR, Sutherland JD, González M, Barrio R, Falcón-Pérez JM, Guerrero I Journal Nat Commun Abstract The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migrat (show more...)The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen. (hide) EV-METRIC 56% (50th 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 Drosophila Sample origin NAY Focus vesicles exosomes 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 Filtration Protein markers EV: Rab11/ Rab8/ TSG101/ Syntaxin/ HSP70 non-EV: ApoLII Proteomics no EV density (g/ml) 1.12-1.19 Show all info Study aim Function Sample Species Drosophila melanogaster Sample Type Drosophila Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Rotor type TLA110 Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins HSP70/ TSG101/ Rab11/ Rab8/ Syntaxin Detected contaminants ApoLII ELISA Detected EV-associated proteins Rab11/ Rab8/ Syntaxin

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EV-TRACK ID	EV140025
species	Drosophila melanogaster
sample type	Drosophila
condition	NAY
separation protocol	(d)(U)C DG Filtration
Exp. nr.	1
EV-METRIC %	56