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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130005	1/1	Homo sapiens	NAY	(d)(U)C	Honegger A	2013	56%
Study summary Full title Silencing of human papillomavirus (HPV) E6/E7 oncogene expression affects both the contents and the amounts of extracellular microvesicles released from HPV-positive cancer cells All authors Honegger A, Leitz J, Bulkescher J, Hoppe-Seyler K, Hoppe-Seyler F Journal Int J Cancer Abstract The human papillomavirus (HPV) E6/E7 oncogenes play a crucial role in the HPV-induced carcinogenesis (show more...)The human papillomavirus (HPV) E6/E7 oncogenes play a crucial role in the HPV-induced carcinogenesis. In this study, the authors investigated whether silencing of endogenous HPV E6/E7 expression may influence the contents or amounts of extracellular microvesicles (eMVs) released from HPV-positive cancer cells. It was found that eMVs secreted from HeLa cells are enriched for Survivin protein. RNA interference studies revealed that maintenance of both intracellular and microvesicular Survivin amounts was strongly dependent on continuous E6/E7 expression. This indicates that intracellular HPV activities are translated into visible alterations of protein contents in eMVs. Besides Survivin, eMVs from HeLa cells contain additional members of the inhibitor of apoptosis protein (IAP) family (XIAP, c-IAP1 and Livin). In contrast, no evidence for the presence of the HPV E6 and E7 oncoproteins in eMVs was obtained. Moreover, it was found that silencing of HPV E6/E7 expression led to a significant increase of exosomes-representing eMVs of endocytic origin-released from HeLa cells. This effect was associated with the reinduction of p53, stimulation of the p53 target genes TSAP6 and CHMP4C that can enhance exosome production and induction of senescence. Taken together, these results show that silencing of HPV E6/E7 oncogene expression profoundly affects both the composition and amounts of eMVs secreted by HPV-positive cancer cells. This indicates that HPVs can induce molecular signatures in eMVs that may affect intercellular communication and could be explored for diagnostic purposes. (hide) EV-METRIC 56% (90th 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 microvesicles 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) / 253.9 (washing) Protein markers EV: TSG101/ Survivin/ CD63/ Hsc70/ Annexin1/ Beta-actin/ CD9 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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) 70 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 253.9 Wash: volume per pellet (ml) 36 Wash: Rotor Type SW28 Wash: adjusted k-factor 253.9 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD9/ TSG101/ Beta-actin/ Annexin1/ Hsc70/ Survivin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin/ Annexin1/ Hsc70/ Survivin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV130005
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	56