Journal: eLife

Article Title: The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis

doi: 10.7554/eLife.89141

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , CD63-RFP ( Homo sapiens ) (plasmid) , Addgene , RRID: Addgene_62964 , RFP tag.

Techniques: Recombinant, Dominant Negative Mutation, Plasmid Preparation, Membrane, Luciferase, Expressing, Reporter Assay, Software, Imaging, Microinjection, Inverted Microscopy

Journal: Molecular & Cellular Proteomics : MCP

Article Title: The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions

doi: 10.1016/j.mcpro.2023.100514

Figure Lengend Snippet: Characterization of extracellular vesicle (EVs) subsets, namely, small (S-EVs) or large (L-EVs), isolated from pig seminal plasma (SP) samples (n = 3; three ejaculates per sample; one ejaculate per male pig). A , violin plot displaying the total protein concentration in both SP-EV subsets. The dashed line shows the median and dotted lines the 25 to 75% quartiles. B , representative histogram of particle size distribution of S-EVs and L-EVs assessed by nanoparticle tracking analysis. C , particle size distribution of S-EVs and L-EVs analyzed by dynamic light scattering ( red , S-EVs; blue , L-EVs) in terms of intensity and volume. The black and gray lines represent the average of intensity size distribution of S-EVs and L-EVs, respectively. D , representative images of the morphology of S-EVs and L-EVs assessed by transmission electron microscopy. E , representative histogram of CFSE/CD63/HSP90β/ALB expression in S-EVs and L-EVs assessed by flow cytometry. ALB, albumin; CFSE, carboxyfluorescein succinimidyl ester; CNT, control; HSP90β, heat shock protein 90β.

Article Snippet: The EVs were cytometrically characterized following the International Society of Extracellular Vesicles recommendations (MIFlowCyt-EV, ( )) to identify their enrichment in proteins belonging to the three categories established by MISEV 2018 guidelines ( ): CD63 (Anti-CD63-FITC, Clone REA1055, Miltenyi Biotec) as “Category 1” protein (Transmembrane or GPI-anchored proteins associated to plasma membrane and/or endosomes); HSP90β (anti-HSP90β-PE, ADI-SPA-844PE-050, Enzo Life Sciences) as “Category 2” protein (Cytosolic proteins recovered in EVs), and albumin (Anti-swine Albumin-FITC, CLFAG16140, Cedarlane) as “Category 3” protein (Major components of non-EVs coisolated structures).

Techniques: Isolation, Clinical Proteomics, Protein Concentration, Transmission Assay, Electron Microscopy, Expressing, Flow Cytometry, Control

Journal: Acta Neuropathologica Communications

Article Title: Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

doi: 10.1186/s40478-017-0466-0

Figure Lengend Snippet: Higher levels of exosome-enriched EVs in the brains of DS patients and of Ts2 mice as compared to age-matched diploid controls. a Representative Western-blots of EVs isolated from human brain tissue and purified on a sucrose step gradient column. The sucrose gradient fractions b, c and d showed the presence of the exosomal proteins Alix and CD63, and the EVs proteins Flotillin-1 and Flotillin-2. b Quantification of total protein levels of EVs isolated from the brain extracellular space of DS patients, normalized to brain tissue protein levels, showed higher EVs levels compared to controls. c Higher EVs levels were also found in the brain extracellular space of 12- and 24-month-old Ts2 mice compared to 2N littermates. No significant differences were found in total EVs protein levels of 3- and 8-month-old Ts2 mice compared to controls. Similar results were obtained when AChE activity levels were measured in EVs isolated from the brain extracellular space of DS patients ( d ) and Ts2 mice ( e ) as compared to 2N controls when normalized to brain tissue protein content. AChE activity levels normalized to EVs protein content were not different between brains of DS patients ( f ) and Ts2 mice ( g ) compared to 2N controls. EVs levels are presented as trisomic to 2N ratio. Student t-test, n = 5 (DS and 2N human brains), n = 4 (3- and 24-month-old), n = 5 (8-month-old), and n = 7 (12-month-old) brains of Ts2 and 2N mice (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Taqman qPCR primers for CD63 (Mm01966817_g1) and rab35 (Mm01204416_ml) (Life Technologies) were utilized with samples assayed on a real-time qPCR cycler (7900HT, Life Technologies) in 96-well optical plates with coverfilm as described previously [ – , , ].

Techniques: Western Blot, Isolation, Purification, Activity Assay

Journal: Acta Neuropathologica Communications

Article Title: Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

doi: 10.1186/s40478-017-0466-0

Figure Lengend Snippet: Proteomic analysis of exosome-enriched mouse brain EVs. A Venn diagram shows the overlap between biological replicates within each genotype for 2N ( a ) and Ts2 ( b ) EVs. c 1549 proteins were common to both genotypes. 91 and 18 proteins were unique to Ts2 and 2N samples, respectively. d GO analyses for components of the 1549 proteins common to both genotypes revealed enrichment of extracellular vesicles and exosomal proteins in the EVs preparations. P -values for each cellular category are shown on the right. e Representative Western-blots with anti-CD63 and anti-rab35 antibodies of the 2N and Ts2 sucrose gradient EVs fractions and corresponding quantification. Student t-test, n = 7 (* p < 0.05)

Article Snippet: Taqman qPCR primers for CD63 (Mm01966817_g1) and rab35 (Mm01204416_ml) (Life Technologies) were utilized with samples assayed on a real-time qPCR cycler (7900HT, Life Technologies) in 96-well optical plates with coverfilm as described previously [ – , , ].

Techniques: Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

doi: 10.1186/s40478-017-0466-0

Figure Lengend Snippet: Enhanced expression of the proteins CD63 and rab35 in DS brains. a Representative Western-blots (corresponding to samples number 3, 2, 5 and 4 in Table ) and quantification showing the overexpression of CD63 and rab35 in DS brains compared to 2N. b No differences in the levels of Alix or TSG101 were detected in homogenates of human DS brains compared to 2N controls. β-actin was blotted as an internal control for loading. Student t-test, n = 5 (* p < 0.05)

Article Snippet: Taqman qPCR primers for CD63 (Mm01966817_g1) and rab35 (Mm01204416_ml) (Life Technologies) were utilized with samples assayed on a real-time qPCR cycler (7900HT, Life Technologies) in 96-well optical plates with coverfilm as described previously [ – , , ].

Techniques: Expressing, Western Blot, Over Expression, Control

Journal: Acta Neuropathologica Communications

Article Title: Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

doi: 10.1186/s40478-017-0466-0

Figure Lengend Snippet: Exosome secretion is enhanced by DS fibroblasts as compared to 2N controls. a Representative Western-blots and corresponding quantification showing the higher levels of the exosomal markers CD63, Alix and TSG101 in EVs isolated from the conditioned media of DS fibroblasts compared to 2N controls. The intensity of the bands was normalized to cell protein. b Elevated expression levels of CD63 and rab35 and ( c ) no differences in the levels of Alix and TSG101 in lysates of DS fibroblasts compared to 2N controls, as shown by the representative Western-blots and corresponding quantification. β-actin was blotted as an internal control for loading. Student t-test, n = 5 independent experiments (* p < 0.05; ** p < 0.01)

Article Snippet: Taqman qPCR primers for CD63 (Mm01966817_g1) and rab35 (Mm01204416_ml) (Life Technologies) were utilized with samples assayed on a real-time qPCR cycler (7900HT, Life Technologies) in 96-well optical plates with coverfilm as described previously [ – , , ].

Techniques: Western Blot, Isolation, Expressing, Control

Journal: Acta Neuropathologica Communications

Article Title: Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

doi: 10.1186/s40478-017-0466-0

Figure Lengend Snippet: Effect of CD63 knockdown on exosome secretion and endosomal pathology in DS cells. 2N and DS fibroblasts were transfected with either CD63 or negative control siRNAs. a CD63 knockdown was confirmed by Western-blot analysis of cell lysates. b Over 3 days, exosomes were collected from the cell culture media and quantified by Western-blot analysis for the exosomal markers CD63, TSG101, and Alix. c No significant changes were observed in exosome release by 2N cells following CD63 silencing compared to controls. d DS fibroblasts in which CD63 was silenced showed decreased release of exosomes as seen by lower levels of exosomal TSG101 and Alix as compared to control DS cells. Student t-test, n = 4 independent experiments (* p < 0.05; *** p < 0.001). e Early endosomes were immunolabeled with an anti-EEA1 antibody of transfected 2N and DS cells (calibration bar = 20 μm). f No significant changes in the endosomal number were detected in CD63-reduced 2N fibroblasts compared to control 2N cells, while a significant increase in number of endosomes was observed in DS cells following CD63 knockdown. g No significant differences were found in the area occupied by endosomes in 2N and DS cells after knocking down CD63, however DS fibroblasts showed a trend for an increase. Note that the number and area occupied by endosomes in DS fibroblasts is significantly higher than in 2N under basal (control-siRNA) conditions ( f, g ). Area is expressed in pixels per cell. One-way ANOVA followed by Tukey post-hoc multiple comparison test, n = 4 independent experiments (** p < 0.01; *** p < 0.001; ****p< 0.0001)

Article Snippet: Taqman qPCR primers for CD63 (Mm01966817_g1) and rab35 (Mm01204416_ml) (Life Technologies) were utilized with samples assayed on a real-time qPCR cycler (7900HT, Life Technologies) in 96-well optical plates with coverfilm as described previously [ – , , ].

Techniques: Knockdown, Transfection, Negative Control, Western Blot, Cell Culture, Control, Immunolabeling, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities

doi: 10.1186/s40478-017-0466-0

Figure Lengend Snippet: Schematic representation of the endosomal and exosomal pathways in diploid and DS neurons. Endocytosed material in the cell is transported by early endosomes and late endosomes/multivesicular bodies (MVBs) for either degradation in lysosomes or exosome secretion. The invagination of the MVBs membrane results in the formation of intraluminal vesicles (ILVs), which are released as exosomes into the extracellular space upon fusion of MVBs with the plasma membrane. Our findings show that in DS neurons with endosomal enlargement there is an enhanced exosome release regulated by the tetraspanin CD63

Article Snippet: Taqman qPCR primers for CD63 (Mm01966817_g1) and rab35 (Mm01204416_ml) (Life Technologies) were utilized with samples assayed on a real-time qPCR cycler (7900HT, Life Technologies) in 96-well optical plates with coverfilm as described previously [ – , , ].

Techniques: Membrane, Clinical Proteomics

Journal: BBA Advances

Article Title: Valosin-containing protein (VCP), a component of tumor-derived extracellular vesicles, impairs the barrier integrity of brain microvascular endothelial cells

doi: 10.1016/j.bbadva.2024.100130

Figure Lengend Snippet: Schematic representation of EV isolation protocol and characterization of isolated EVs from MDA-MB-231 parental (MDA-par) and brain-seeking (MDA-br) cells. (A) Differential centrifugation scheme for obtaining EVs. (B) Nano-Tracking Analysis of small EV from MDA-MB-231-parental and (C) MDA-MB-231-brain variant, evidencing the size distribution as a function of particle concentration. (D) Western blot analysis of calnexin, TSG101, CD63, and CD9 in sEV, Large EV and Cell Lysate (CL). Data are presented as the mean ± SEM of three independent experiments ( n = 3).

Article Snippet: Proteins were detected by incubation with the following primary antibodies (1:1000): Calnexin (CST, #2679), CD63 (CST, #55051), CD9 (CST, #98327), TSG101 (CST, #72313), β-actin (Sigma-Aldrich, #A5316), diluted in blocking solution (TBST 1 % in BSA 5 %) for 16h at 4 °C.

Techniques: Isolation, Centrifugation, Variant Assay, Concentration Assay, Western Blot

Journal: Immunity, inflammation and disease

Article Title: Human umbilical cord mesenchymal stem cells improve disease characterization of Sjogren's syndrome in NOD mice through regulation of gut microbiota and Treg/Th17 cellular immunity.

doi: 10.1002/iid3.1139

Figure Lengend Snippet: FIGURE 1 Identification of umbilical cord mesenchymal stem cells (UC‐MSCs), UC‐MSCs and derived exosomes (UC‐MSCs‐exo), and Nonobese Diabetic (NOD) mouse splenic T cells. (A) The morphology of UC‐MSCs was characterized by an optical microscope. Scale bar: 100 μm (left), 50 μm (right). (B) The expressions of the surface antigens (CD34, CD44, CD45, CD73, CD90, CD105, and human leukocyte antigen‐DR isotype) of the third‐generation UC‐MSCs were determined by flow cytometry. (C) The morphology of UC‐MSCs‐exo was characterized by transmission electron microscope. Scale bar: 200 nm (left), 100 nm (right). (D) The expressions of CD9, CD63, TSG101, and Calnexin in UC‐MSCs‐exo were assessed by Western blot. (E) Adipogenic (left) and osteogenic (right) differentiation of UC‐MSCs were assessed by Oil red O staining and Alizarin Red staining, respectively. Scale bar: 25 μm (left), 100 μm (right). (F) The splenic CD4+ T cells of NOD mice were selected by immunomagnetic beads.

Article Snippet: The expression levels of CD9 (1: 2000, 60232‐ 1‐Ig; Proteintech), CD63 (1: 500, 25682‐1‐AP; Proteintech), tumor susceptibility gene 101 (TSG101, 1: 5000, 28283‐1‐AP; Proteintech), Calnexin (1: 10,000, 10427‐2‐ AP; Proteintech) in UC‐MSCs‐exo were detected by Western blot with β‐actin used as the reference protein.

Techniques: Derivative Assay, Microscopy, Flow Cytometry, Transmission Assay, Western Blot, Staining