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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210336	1/2	Homo sapiens	THP1	(d)(U)C DG	Driedonks, Tom	2024	63%
Study summary Full title Intracellular localisation and extracellular release of Y RNA and Y RNA binding proteins All authors Tom A. P. Driedonks, Sarah Ressel, Thi Tran Ngoc Minh, Amy H. Buck, Esther N. M. Nolte-‘t Hoen Journal J Extracell Biol Abstract Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-siz (show more...)Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between cells. EVs contain microRNAs and various other types of non-coding RNA, of which Y RNA is among the most abundant types. Studies on how RNAs and their binding proteins are sorted into EVs have mainly focused on comparing intracellular (cytoplasmic) levels of these RNAs to the extracellular levels in EVs. Besides overall transcriptional levels that may regulate sorting of RNAs into EVs, the process may also be driven by local intracellular changes in RNA/RBP concentrations. Changes in extracellular Y RNA have been linked to cancer and cardiovascular diseases. Although the loading of RNA cargo into EVs is generally thought to be influenced by cellular stimuli and regulated by RNA binding proteins (RBP), little is known about Y RNA shuttling into EVs. We previously reported that immune stimulation alters the levels of Y RNA in EVs independently of cytosolic Y RNA levels. This suggests that Y RNA binding proteins, and/or changes in the local Y RNA concentration at EV biogenesis sites, may affect Y RNA incorporation into EVs. Here, we investigated the subcellular distribution of Y RNA and Y RNA binding proteins in activated and non-activated THP1 macrophages. We demonstrate that Y RNA and its main binding protein Ro60 abundantly co-fractionate in organelles involved in EV biogenesis and in EVs. Cellular activation led to an increase in Y RNA concentration at EV biogenesis sites and this correlated with increased EV-associated levels of Y RNA and Ro60. These results suggest that Y RNA incorporation into EVs may be controlled by local intracellular changes in the concentration of Y RNA and their protein binding partners. (hide) EV-METRIC 63% (93rd 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.11 -1.18 Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4 M Highest density fraction 2.0 M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.2 Orientation Bottom-up Speed (g) 192000 Duration (min) 900-1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 4 Pelleting: speed (g) 192000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment according to van der Vlist et al, Nature Protocols 2012 Calibration bead size 0.1/ 0.2 EV concentration Yes

	EV210336	2/2	Homo sapiens	THP1	(d)(U)C DG	Driedonks, Tom	2024	63%
Study summary Full title Intracellular localisation and extracellular release of Y RNA and Y RNA binding proteins All authors Tom A. P. Driedonks, Sarah Ressel, Thi Tran Ngoc Minh, Amy H. Buck, Esther N. M. Nolte-‘t Hoen Journal J Extracell Biol Abstract Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-siz (show more...)Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between cells. EVs contain microRNAs and various other types of non-coding RNA, of which Y RNA is among the most abundant types. Studies on how RNAs and their binding proteins are sorted into EVs have mainly focused on comparing intracellular (cytoplasmic) levels of these RNAs to the extracellular levels in EVs. Besides overall transcriptional levels that may regulate sorting of RNAs into EVs, the process may also be driven by local intracellular changes in RNA/RBP concentrations. Changes in extracellular Y RNA have been linked to cancer and cardiovascular diseases. Although the loading of RNA cargo into EVs is generally thought to be influenced by cellular stimuli and regulated by RNA binding proteins (RBP), little is known about Y RNA shuttling into EVs. We previously reported that immune stimulation alters the levels of Y RNA in EVs independently of cytosolic Y RNA levels. This suggests that Y RNA binding proteins, and/or changes in the local Y RNA concentration at EV biogenesis sites, may affect Y RNA incorporation into EVs. Here, we investigated the subcellular distribution of Y RNA and Y RNA binding proteins in activated and non-activated THP1 macrophages. We demonstrate that Y RNA and its main binding protein Ro60 abundantly co-fractionate in organelles involved in EV biogenesis and in EVs. Cellular activation led to an increase in Y RNA concentration at EV biogenesis sites and this correlated with increased EV-associated levels of Y RNA and Ro60. These results suggest that Y RNA incorporation into EVs may be controlled by local intracellular changes in the concentration of Y RNA and their protein binding partners. (hide) EV-METRIC 63% (93rd 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 Pam3CSK4 treated 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.11 -1.18 Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4 M Highest density fraction 2.0 M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.2 Orientation Bottom-up Speed (g) 192000 Duration (min) 900-1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 4 Pelleting: speed (g) 192000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment according to van der Vlist et al Nature Protocols 2012 Calibration bead size 0.1/ 0.2 EV concentration Yes

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EV-TRACK ID	EV210336
species	Homo sapiens
sample type	Cell culture
cell type	THP1
condition	Control condition	Pam3CSK4 treated
separation protocol	dUC/ Density gradient	dUC/ Density gradient
Exp. nr.	1	2
EV-METRIC %	63	63