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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190105	1/2	Homo sapiens	Human umbilical vein endothelial cells (HUVECs)	DG (d)(U)C	Emanuela Mensà	2020	100%
Study summary Full title Small extracellular vesicles deliver miR-21 and miR-217 as pro-senescence effectors to endothelial cells All authors Emanuela Mensà, Michele Guescini, Angelica Giuliani, Maria Giulia Bacalini, Deborah Ramini, Giacomo Corleone, Manuela Ferracin, Gianluca Fulgenzi, Laura Graciotti, Francesco Prattichizzo, Leonardo Sorci, Michela Battistelli, Vladia Monsurrò, Anna Rita Bonfigli, Maurizio Cardelli, Rina Recchioni, Fiorella Marcheselli, Silvia Latini, Serena Maggio, Mirco Fanelli, Stefano Amatori, Gianluca Storci, Antonio Ceriello, Vilberto Stocchi, Maria De Luca, Luca Magnani, Maria Rita Rippo, Antonio Domenico Procopio, Claudia Sala, Iva Budimir, Cristian Bassi, Massimo Negrini, Paolo Garagnani, Claudio Franceschi, Jacopo Sabbatinelli, Massimiliano Bonafè, Fabiola Olivieri Journal J Extracell Vesicles Abstract The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. H (show more...)The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40–100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication. (hide) EV-METRIC 100% (99th 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 Control condition Focus vesicles extracellular vesicle 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 DG (d)(U)C Protein markers EV: TSG101/ Calnexin/ CD63 non-EV: Albumin Proteomics no EV density (g/ml) 1.08-1.10 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Human umbilical vein endothelial cells (HUVECs) EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 90 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) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 103745 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type Type 90 Ti Wash: speed (g) 109354 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 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 28 Speed (g) 103745 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3.5 Pelleting: duration (min) 70 Pelleting: rotor type Type 90 Ti Pelleting: speed (g) 109354 Pelleting-wash: volume per pellet (mL) 3 Pelleting-wash: duration (min) 70 Pelleting-wash: speed (g) Type 90 Ti Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ TSG101/ Calnexin Not detected contaminants Albumin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 20 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 80 (sEVs) / 120 (lEVs) EV concentration Yes Particle yield Number of particles per cell: 3E4 (sEVs)/1.5E4 (lEVs) EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190105	2/2	Homo sapiens	Human umbilical vein endothelial cells (HUVECs)	(d)(U)C	Emanuela Mensà	2020	43%
Study summary Full title Small extracellular vesicles deliver miR-21 and miR-217 as pro-senescence effectors to endothelial cells All authors Emanuela Mensà, Michele Guescini, Angelica Giuliani, Maria Giulia Bacalini, Deborah Ramini, Giacomo Corleone, Manuela Ferracin, Gianluca Fulgenzi, Laura Graciotti, Francesco Prattichizzo, Leonardo Sorci, Michela Battistelli, Vladia Monsurrò, Anna Rita Bonfigli, Maurizio Cardelli, Rina Recchioni, Fiorella Marcheselli, Silvia Latini, Serena Maggio, Mirco Fanelli, Stefano Amatori, Gianluca Storci, Antonio Ceriello, Vilberto Stocchi, Maria De Luca, Luca Magnani, Maria Rita Rippo, Antonio Domenico Procopio, Claudia Sala, Iva Budimir, Cristian Bassi, Massimo Negrini, Paolo Garagnani, Claudio Franceschi, Jacopo Sabbatinelli, Massimiliano Bonafè, Fabiola Olivieri Journal J Extracell Vesicles Abstract The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. H (show more...)The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40–100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication. (hide) EV-METRIC 43% (81st 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 Replicative senescence Focus vesicles extracellular vesicle 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 Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Human umbilical vein endothelial cells (HUVECs) EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 90 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) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 103745 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type Type 90 Ti Wash: speed (g) 109354 EV-subtype Distinction between multiple subtypes Size Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Not detected contaminants Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 80 (sEVs) / 130 (lEVs) EV concentration Yes Particle yield Number of particles per cell: 1.3E5 (sEVs)/1.5E4 (lEVs) EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV190105
species	Homo sapiens
sample type	Cell culture
cell type	Human umbilical vein endothelial cells (HUVECs)
condition	Control condition	Replicative senescence
separation protocol	DG (d)(U)C	(d)(U)C
Exp. nr.	1	2
EV-METRIC %	100	43