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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140033	1/1	Homo sapiens	NAY	(d)(U)C Filtration UF	Liu C	2014	33%
Study summary Full title Unconventional secretion of synuclein-gamma promotes tumor cell invasion All authors Liu C, Qu L, Lian S, Tian Z, Zhao C, Meng L, Shou C Journal FEBS J Abstract Synuclein-? (SNCG) is a chaperone protein and exists mainly in the cytoplasm. SNCG confers chemoresi (show more...)Synuclein-? (SNCG) is a chaperone protein and exists mainly in the cytoplasm. SNCG confers chemoresistance, and is a potential unfavorable biomarker for multiple types of cancer. Our previous work demonstrated that SNCG could be detected in the serum of cancer patients and the medium of cultured cancer cells, but the mechanism of SNCG secretion and its biological roles are unknown. Here, we showed that SNCG levels in the culture medium were positively correlated with cancer cell densities and the concentrations of fetal bovine serum added. SNCG secretion was unaffected by brefeldin A, an inhibitor of the classic protein transport pathway, but was antagonized by exosome inhibitor, lysosome inhibitor, ABC transporter inhibitor, and phosphatidylinositide 3-kinase inhibitor, and knockdown of Rab27a. Ultracentrifugation fractionation revealed that intracellular SNCG was present as both free and vesicle-associated forms, but that the extracellular SNCG was mainly free. The results of reciprocal coimmunoprecipitation experiments showed an interaction between SNCG and flotillin-2, a marker of exosomes and lipid rafts. Moreover, we demonstrated that SNCG, both secreted from tumor cells and exogenously added, markedly promoted cancer cell migration and invasion, but had no effect on noncancerous cells. These findings suggest that SNCG is actively secreted by cancer cells via an unconventional secretion pathway and contributes to aggressive phenotypes of cancer 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 NAY Focus 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 Filtration UF Protein markers EV: HSP90/ GAPDH/ Alix/ Annexin2/ LAMP1/ Flotillin2/ HSP70 non-EV: Cell organelle protein Proteomics no Show all info Study aim Other/Synuclein-gamma secretion Sample Species Homo sapiens 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ HSP90/ HSP70/ "Flotillin2/ LAMP1/ Annexin2/ GAPDH" Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins "Flotillin2/ LAMP1/ Annexin2/ GAPDH"

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EV-TRACK ID	EV140033
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration UF
Exp. nr.	1
EV-METRIC %	33