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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140255	1/1	Homo sapiens	NAY	(d)(U)C	Morhayim J	2014	33%
Study summary Full title Proteomic signatures of extracellular vesicles secreted by nonmineralizing and mineralizing human osteoblasts and stimulation of tumor cell growth All authors Morhayim J, van de Peppel J, Demmers JA, Kocer G, Nigg AL, van Driel M, Chiba H, van Leeuwen JP Journal FASEB J Abstract Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematop (show more...)Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. We focused on the proteomic characterization of nonmineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic, and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent 5-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to 2-fold increase in PC3 cell growth compared to cell-free culture medium-derived vesicle controls. Our findings elucidate the mineralization stage-specific protein content of osteoblast-secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention. (hide) EV-METRIC 33% (74th 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 Adj. k-factor 253.9 (pelleting) Protein markers EV: Annexin2/ CD63 non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free 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 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Annexin2 ELISA Detected EV-associated proteins Annexin2 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140255
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	33