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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100094	1/1	Homo sapiens	NAY	(d)(U)C DC UF	Xiao WH	2010	11%
Study summary Full title Effect of 5-aza-2'-deoxycytidine on immune-associated proteins in exosomes from hepatoma All authors Xiao WH, Sanren GW, Zhu JH, Li QW, Kang HR, Wang RL, Song LP, Ye M Journal World J Gastroenterol Abstract AIM: To study the effect of 5-aza-2'-deoxycytidine (5-aza-CdR) on heat shock protein 70 (HSP70), hum (show more...)AIM: To study the effect of 5-aza-2'-deoxycytidine (5-aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-I (HLA-I) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METHODS: Exosomes derived from HepG(2) and Hep3B cells treated with or without 5-aza-CdR were isolated and purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under electron microscope. Concentration of proteins in exosomes was measured by bicinchoninic acid protein assay. Expression of HSP70, HLA-I and NY-ESO-1 proteins in exosomes was detected by Western blotting and immunoelectron microscopy. mRNA expression of p53 gene was detected by reverse transcription polymerase chain reaction. RESULTS: The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were increased significantly after treatment with 5-aza-CdR (P < 0.05). After treatment with 5-aza-CdR, immunoelectron microscopy and Western blotting showed that the HSP70, HLA-I and NY-ESO-1 proteins were increased in exosomes produced by both hepatoma cell lines. CONCLUSION: 5-Aza-CdR, an inhibitor of DNA methyltransferase, can increase exosomes produced by hepatoma cells and immune-associated protein component of exosomes, which may be mediated by p53 gene up-regulation and 5-aza-CdR demethylation. (hide) EV-METRIC 11% (30th 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 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 DC UF Protein markers EV: HSP70/ Beta-actin/ MHC1 non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ Beta-actin/ MHC1 ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin/ MHC1 Characterization: Particle analysis EM EM-type immune EM EM protein HSP70 Image type Wide-field

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EV-TRACK ID	EV100094
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DC UF
Exp. nr.	1
EV-METRIC %	11