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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200059	1/1	Homo sapiens	Primary neurospheres	(d)(U)C qEV	Bertolini, Irene	2020	75%
Study summary Full title Interplay Between V-ATPase G1 and Small EV-miRNAs Modulates ERK1/2 Activation in GBM Stem Cells and Nonneoplastic Milieu All authors Irene Bertolini, Alessandra Maria Storaci, Andrea Terrasi, Andrea Di Cristofori, Marco Locatelli, Manuela Caroli, Stefano Ferrero, Dario C Altieri, Valentina Vaira Journal Mol Cancer Res Abstract The ATP6V1G1 subunit (V1G1) of the vacuolar proton ATPase (V-ATPase) pump is crucial for glioma stem (show more...)The ATP6V1G1 subunit (V1G1) of the vacuolar proton ATPase (V-ATPase) pump is crucial for glioma stem cells (GSC) maintenance and in vivo tumorigenicity. Moreover, V-ATPase reprograms the tumor microenvironment through acidification and release of extracellular vesicles (EV). Therefore, we investigated the role of V1G1 in GSC small EVs and their effects on primary brain cultures. To this end, small EVs were isolated from patients-derived GSCs grown as neurospheres (NS) with high (V1G1HIGH-NS) or low (V1G1LOW-NS) V1G1 expression and analyzed for V-ATPase subunits presence, miRNA contents, and cellular responses in recipient cultures. Our results show that NS-derived small EVs stimulate proliferation and motility of recipient cells, with small EV derived from V1G1HIGH-NS showing the most pronounced activity. This involved activation of ERK1/2 signaling, in a response reversed by V-ATPase inhibition in NS-producing small EV. The miRNA profile of V1G1HIGH-NS-derived small EVs differed significantly from that of V1G1LOW-NS, which included miRNAs predicted to target MAPK/ERK signaling. Mechanistically, forced expression of a MAPK-targeting pool of miRNAs in recipient cells suppressed MAPK/ERK pathway activation and blunted the prooncogenic effects of V1G1HIGH small EV. These findings propose that the GSC influences the brain milieu through a V1G1-coordinated EVs release of MAPK/ERK-targeting miRNAs. Interfering with V-ATPase activity could prevent ERK-dependent oncogenic reprogramming of the microenvironment, potentially hampering local GBM infiltration. IMPLICATIONS: Our data identify a novel molecular mechanism of gliomagenesis specific of the GBM stem cell niche, which coordinates a V-ATPase-dependent reprogramming of the brain microenvironment through the release of specialized EVs. (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 glioblastoma Focus vesicles Other / small 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 Commercial method Protein markers EV: TSG101/ CD63/ CD81/ Clathrin/ ATP6V1G1/ CD9 non-EV: Calnexin/ Argonaute2 Proteomics no EV density (g/ml) 1.13-1.19 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary neurospheres EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 20 Pelleting: duration (min) 120 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 20 Pelleting-wash: duration (min) 120 Pelleting-wash: speed (g) Type 50.2 Ti Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ CD63/ Clathrin/ TSG101 Not detected contaminants Calnexin/ Argonaute2 Flow cytometry specific beads Detected EV-associated proteins CD9/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-250 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.00E+07 EM EM-type Transmission-EM/ Immuno-EM EM protein ATP6V1G1 Image type Close-up Report size (nm) 50-250

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EV-TRACK ID	EV200059
species	Homo sapiens
sample type	Cell culture
cell type	Primary neurospheres
condition	glioblastoma
separation protocol	(d)(U)C qEV
Exp. nr.	1
EV-METRIC %	75