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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210165	1/2	Homo sapiens	THP-1	DG DC	Phu TA	2022	75%
Study summary Full title IL-4 polarized human macrophage exosomes control cardiometabolic inflammation and diabetes in obesity. All authors Phu TA, Ng M, Vu NK, Bouchareychas L, Raffai RL Journal Mol Ther Abstract Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macr (show more...)Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macrophages contribute to metabolic inflammation and insulin resistance, those polarized to an M2-like phenotype exert protective properties. Building on our observations reporting M2-like macrophage exosomes in atherosclerosis control, we tested whether they could serve to control inflammation in the liver and adipose tissue of obese mice. In thinking of clinical translation, we studied human THP-1 macrophages exposed to interleukin (IL)-4 as a source of exosomes (THP1-IL4-exo). Our findings show that THP1-IL4-exo polarized primary macrophages to an anti-inflammatory phenotype and reprogramed their energy metabolism by increasing levels of microRNA-21/99a/146b/378a (miR-21/99a/146b/378a) while reducing miR-33. This increased lipophagy, mitochondrial activity, and oxidative phosphorylation (OXPHOS). THP1-IL4-exo exerted a similar regulation of these miRs in cultured 3T3-L1 adipocytes. This enhanced insulin-dependent glucose uptake through increased peroxisome proliferator activated receptor gamma (PPARγ)-driven expression of GLUT4. It also increased levels of UCP1 and OXPHOS activity, which promoted lipophagy, mitochondrial activity, and beiging of 3T3-L1 adipocytes. Intraperitoneal infusions of THP1-IL4-exo into obese wild-type and Ldlr mice fed a Western high-fat diet reduced hematopoiesis and myelopoiesis, and favorably reprogramed inflammatory signaling and metabolism in circulating Ly6C monocytes. This also reduced leukocyte numbers and inflammatory activity in the circulation, aorta, adipose tissue, and the liver. Such treatments reduced hepatic steatosis and increased the beiging of white adipose tissue as revealed by increased UCP1 expression and OXPHOS activity that normalized blood insulin levels and improved glucose tolerance. Our findings support THP1-IL4-exo as a therapeutic approach to control cardiometabolic disease and diabetes in obesity. (hide) EV-METRIC 75% (96th 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 exosome 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 Density gradient Density cushion Protein markers EV: CD81/ CD63/ CD9 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 1,00E+06 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Density cushion Density medium Iodixanol Sample volume 98 Cushion volume 2 Density of the cushion 60% Centrifugation time 180 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0,4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes Particle yield as number of particles per million cells: 6.00e+0

	EV210165	2/2	Homo sapiens	THP-1	DG DC	Phu TA	2022	75%
Study summary Full title IL-4 polarized human macrophage exosomes control cardiometabolic inflammation and diabetes in obesity. All authors Phu TA, Ng M, Vu NK, Bouchareychas L, Raffai RL Journal Mol Ther Abstract Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macr (show more...)Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macrophages contribute to metabolic inflammation and insulin resistance, those polarized to an M2-like phenotype exert protective properties. Building on our observations reporting M2-like macrophage exosomes in atherosclerosis control, we tested whether they could serve to control inflammation in the liver and adipose tissue of obese mice. In thinking of clinical translation, we studied human THP-1 macrophages exposed to interleukin (IL)-4 as a source of exosomes (THP1-IL4-exo). Our findings show that THP1-IL4-exo polarized primary macrophages to an anti-inflammatory phenotype and reprogramed their energy metabolism by increasing levels of microRNA-21/99a/146b/378a (miR-21/99a/146b/378a) while reducing miR-33. This increased lipophagy, mitochondrial activity, and oxidative phosphorylation (OXPHOS). THP1-IL4-exo exerted a similar regulation of these miRs in cultured 3T3-L1 adipocytes. This enhanced insulin-dependent glucose uptake through increased peroxisome proliferator activated receptor gamma (PPARγ)-driven expression of GLUT4. It also increased levels of UCP1 and OXPHOS activity, which promoted lipophagy, mitochondrial activity, and beiging of 3T3-L1 adipocytes. Intraperitoneal infusions of THP1-IL4-exo into obese wild-type and Ldlr mice fed a Western high-fat diet reduced hematopoiesis and myelopoiesis, and favorably reprogramed inflammatory signaling and metabolism in circulating Ly6C monocytes. This also reduced leukocyte numbers and inflammatory activity in the circulation, aorta, adipose tissue, and the liver. Such treatments reduced hepatic steatosis and increased the beiging of white adipose tissue as revealed by increased UCP1 expression and OXPHOS activity that normalized blood insulin levels and improved glucose tolerance. Our findings support THP1-IL4-exo as a therapeutic approach to control cardiometabolic disease and diabetes in obesity. (hide) EV-METRIC 75% (96th 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 IL-4 cytokine treated Focus vesicles exosome 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 Density gradient Density cushion Protein markers EV: CD81/ CD63/ CD9 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 1,00E+06 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Density cushion Density medium Iodixanol Sample volume 98 Cushion volume 2 Density of the cushion 60% Centrifugation time 180 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0,4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 90,5 EV concentration Yes Particle yield as number of particles per million cells: 5.00e+0

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EV-TRACK ID	EV210165
species	Homo sapiens
sample type	Cell culture
cell type	THP-1
condition	Control condition	IL-4 cytokine treated
separation protocol	Density gradient/ DC	Density gradient/ DC
Exp. nr.	1	2
EV-METRIC %	75	75