Journal: Molecular & Cellular Proteomics

Article Title: Proteomics Analysis of Bladder Cancer Exosomes

doi: 10.1074/mcp.m000063-mcp201

Figure Lengend Snippet: FIG. 1. Characterization of HT1376-derived exosomes using Western blotting, flow cytometry, and electron microscopy. Cell (CL) and exosome (Exo) lysates (5 g/well) were compared by Western blotting using a range of antibodies as indicated. This demonstrated relative enrichment of several proteins in exosomes. Some markers, such as gp96, were absent from exosomes, indicating negligible contamination of the preparations by cellular debris (this is representative of three experiments) (A). Exosomes coupled to latex beads were analyzed by flow cytometry, and this revealed positive expression of tetraspanin molecules on the exosome surface. Median fluorescence intensity values (MFI) are shown (representative of 5 experiments) (B). Intentional contamination of purified exosomes with increasing amounts of FBS prior to coupling to latex beads reveals a decrease in signal intensity for CD9 (mean S.E.; n 6; **, p 0.001, one-way analysis of variance with Tukey’s post test) (B, line graph). Material pelleted at 70,000 g from cell-conditioned medium was overlaid on a linear sucrose gradient (0.2–2.02 M) and ultracentrifuged for 18 h at 210,000 g. Collected fractions were analyzed by refractometry to ascertain fraction density and thereafter by Western blot using antibodies to TSG101, which is an exosome marker. TSG101 floats at typical exosome densities of between 1.1 and 1.2 g/ml (representative of four experiments) (C). Transmission electron micrograph of a typical exosome preparation reveals heterogeneous vesicles between 30 and 100 nm in diameter (D). CK, cytokeratin.

Article Snippet: The following primary monoclonal antibodies were used: TSG101, lysosomeassociated membrane protein 1 (LAMP-1), hsp90, calnexin, HLA-G, galectin-3, basigin, hnRNPK, gp96, cytokeratins 18 and 17, and CD44 (Santa Cruz Biotechnology), glyceraldehyde-3-phosphate dehydrogenase (BioChain Institute, Inc.), CD9 (R&D Systems), and CD63 and CD81 (Serotec).

Techniques: Derivative Assay, Western Blot, Flow Cytometry, Electron Microscopy, Expressing, Fluorescence, Purification, Marker, Transmission Assay

Journal: Life Science Alliance

Article Title: Leishmania -infected macrophages release extracellular vesicles that can promote lesion development

doi: 10.26508/lsa.202000742

Figure Lengend Snippet: (A) Workflow for collection of extracellular vesicles from RAW264.7 macrophages infected with L. donovani parasites. Infection was performed in media supplemented with exosome-depleted serum. After 24 h, the culture medium was removed. Cultures were washed to remove uninternalized parasites and replenished with fresh medium supplemented with exosome-depleted serum. After an additional 48 h, culture medium was recovered, pooled, and processed following the centrifugation and filtration steps shown in the figure. (B) Nanoparticle tracking analysis was performed from which particle size distribution and particle concentration was obtained. Plot of particles/cell was calculated using cell count at the end of the infection. Data for graphs were obtained from multiple experiments (ceEV n = 7, LieEV n = 7; * P = 0.0082). (C) Representative image of vesicles in LieEV preparation processed for scanning electron microscopy. (D) Representative transmission electron microscopy image of immunogold CD9-labeled particles in LieEVs. Arrows point to gold particles denoting reactivity of antibody.

Article Snippet: The HM20-embedded samples were sliced into 100 nm thin sections that were placed on nickel grids, which were then immunogold-labeled with an anti-CD9 antibody (1/50 dilution vol/vol) (PB9930; Boster Bio) using a previously described method ( ).

Techniques: Infection, Centrifugation, Filtration, Concentration Assay, Cell Counting, Electron Microscopy, Transmission Assay, Labeling

Journal: Life Science Alliance

Article Title: Leishmania -infected macrophages release extracellular vesicles that can promote lesion development

doi: 10.26508/lsa.202000742

Figure Lengend Snippet: The proteome of LieEVs recovered after 72-h infection was compared with the proteome of ceEVs from uninfected cells. (A) Venn diagram was plotted using proteins identified by mass spectrometry and partitioned according to sample type. The presence and absence of host molecules with and without infection and common to both samples are indicated. (B) The protein content of the preparations was revealed by Ponceau S staining of the blots to normalize for material loaded into each well for each sample type. Approximately 1 × 10 10 particles from EV preparations from three replicate experiments and 50 μg of lysates from infected cells at the 72 h infection point were analyzed. (C) Western blot was performed to confirm the presence of known and novel exosome markers. Uninfected macrophages were treated in an identical manner as infected samples. The blots were then probed with anti-CD9, stripped, and probed with anti-Annexin A3. Identical blots were probed initially with anti-CD63, stripped, and probed with anti-calnexin. (D) Quantification was performed by measuring the mean gray background area using ImageJ software. Background pixel density was subtracted from the inverse of each measurement to obtain relative quantification values. Analysis of the blots showed that CD9 was significantly more abundant in LieEVs than ceEVs and cell lysates (* P = 0.0261, n = 3), whereas levels of CD63 were comparable for all samples. Annexin A3 was significantly more abundant in LieEVs than in ceEVs (* P = 0.0123, n = 3). Calnexin was barely detected in EVs as compared with cell lysates. Statistical test for differences was by ANOVA. Source data are available for this figure.

Article Snippet: The HM20-embedded samples were sliced into 100 nm thin sections that were placed on nickel grids, which were then immunogold-labeled with an anti-CD9 antibody (1/50 dilution vol/vol) (PB9930; Boster Bio) using a previously described method ( ).

Techniques: Infection, Mass Spectrometry, Staining, Western Blot, Software, Quantitative Proteomics

Journal: Journal of dairy science

Article Title: Assessment of isolation strategies to remove caseins for high-quality milk-derived extracellular vesicles.

doi: 10.3168/jds.2024-25162

Figure Lengend Snippet: Figure 3. Comparison of MEVs via UC and HA/UC by RNA, protein. (A) RNA concentration of MEVs via UC and HA/UC in the same milk volume. (B) Protein concentration of MEVs via UC and HA/UC in the same milk volume. (C) RNA concentration of MEVs via UC and HA/UC in the same protein. (D) qRT-PCR analysis of the top expression miRNAs (ssc-miRNA-148a-3p, ssc-miRNA-30–5p, and ssc-let-7a) in MEVs via UC and HA/UC in the same milk volume. (E) Western blotting analysis of MEV from UC and HA/UC. The EVs markers (CD9, CD63, TSG101, and ALIX) were used to compare the expression level in the equal protein. β-actin was used as the internal reference protein. (F) Western blotting analysis of contaminated proteins (ApoA1 and AGO2) in MEVs from UC and HA/UC. Skim milk and supernatant after ultracentrifugation were choose as positive control. *P < 0.05

Article Snippet: The following antibodies were used: Alix (D262028, Sangon Biotech, China), TSG101 (A2216, ABclonal, China), CD9 (AP68965, Abcepta, China), CD63 (D160973, Sangon Biotech, China), AGO2 (D121540, Sangon Biotech, China), ApoA1(3350, CST, USA), PCNA (OriGene, TA800875S, USA), β-actin (AP0060, Bioworld, China), Tubulin (AP0064, Bioworld, China), Goat Anti-Mouse IgG antibody(HRP) (BS12478, Bioworld, China), and Goat Anti-Rabbit IgG antibody (HRP) (BS13278, Bioworld, China).

Techniques: Comparison, Concentration Assay, Protein Concentration, Quantitative RT-PCR, Expressing, Western Blot, Positive Control

Journal: Journal of dairy science

Article Title: Assessment of isolation strategies to remove caseins for high-quality milk-derived extracellular vesicles.

doi: 10.3168/jds.2024-25162

Figure Lengend Snippet: Figure 4. Purity assessment of MEVs via UC and HA/UC. (A) Particles concentration of MEVs via UC and HA/UC in the equal protein. (B) the proportion of membranes-bound MEVs by Triton X-100 treatment at a final concentration of 1% (vol/vol) for 1 h. (C) Representative fluorescence plots showing Anti-CD9 staining of MEVs via UC and HA/UC captured by NanoFCM fluorescence analysis. (D) Relative number of MEVs by quantification of flow cytometry data. *P < 0.05

Article Snippet: The following antibodies were used: Alix (D262028, Sangon Biotech, China), TSG101 (A2216, ABclonal, China), CD9 (AP68965, Abcepta, China), CD63 (D160973, Sangon Biotech, China), AGO2 (D121540, Sangon Biotech, China), ApoA1(3350, CST, USA), PCNA (OriGene, TA800875S, USA), β-actin (AP0060, Bioworld, China), Tubulin (AP0064, Bioworld, China), Goat Anti-Mouse IgG antibody(HRP) (BS12478, Bioworld, China), and Goat Anti-Rabbit IgG antibody (HRP) (BS13278, Bioworld, China).

Techniques: Concentration Assay, Fluorescence, Staining, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Differentially circulating exosomal microRNAs expression profiling in oral lichen planus

doi:

Figure Lengend Snippet: Isolation and validation of plasma-derived exosomes. A. The canonical cup-shaped morphology of exosomes was displayed by TEM. B. Size distribution profile by nanoparticle tracking analysis (NTA) demonstrated a homogeneous distribution of exosomes with peak diameter of 175 nm and an average size of 178.7 ± 68.1 nm. C. Identification of exosomal specific biomarkers. Both CD9 and CD63, the commonly acknowledged exosomal markers, were identified by flow cytometry.

Article Snippet: The exosomal biomarkers CD9 (1:1,000; BD Biosciences Pharmingen, USA) and CD63 (1:1,000; Millipore, Germany) were detected by flow cytometric analysis.

Techniques: Isolation, Derivative Assay, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Differentially circulating exosomal microRNAs expression profiling in oral lichen planus

doi:

Figure Lengend Snippet: Plasma derived exosomal RNA analysis. A. Digital gel electropherograms of RNA from plasma exosome. Lane 1, RNA ladder shows the sizes of the nucleotides. Lane 2 and lane 3 display the size of the exosomal RNA from OLP patient and normal individual respectively. Results demonstrated that small RNAs were dominant in the exosomal RNAs. B. (1) Profile of RNA standard; (2) Total RNA from an OLP patient; (3) Total RNA from a normal individual. The data showed that the samples from OLP patients and normal individuals were enriched in nucleotide < 25 nt, indicating the presence of miRNAs.

Article Snippet: The exosomal biomarkers CD9 (1:1,000; BD Biosciences Pharmingen, USA) and CD63 (1:1,000; Millipore, Germany) were detected by flow cytometric analysis.

Techniques: Derivative Assay

Journal: American Journal of Translational Research

Article Title: Differentially circulating exosomal microRNAs expression profiling in oral lichen planus

doi:

Figure Lengend Snippet: Differentially expressed exosomal miRNAs in OLP patients based on miRNA microarray data analysis. A. The columns and rows of hierarchical cluster indicated samples and specific miRNAs. miRNA cluster tree is shown at the bottom of the figure. Red to green color indicated the magnitude of gene expression change. B and C. Volcano plot and scatter plot depicting the miRNAs expression level. Fold change > 2 and P < 0.05 are shown in yellow; fold change < 0.5 and P < 0.05 are shown in blue. D. Multigroup plot displaying the top five significantly differentially expressed miRNAs: miR-34a-5p (fold change = 4.24, P = 0.000165), miR-130b-3p (fold change = 2.92, P = 0.007933), and miR-29c-3p (fold change = 2.12, P = 0.0426) were significantly increased, while miR-301b-3p (fold change = 0.35, P = 0.040683) and miR-144-3p (fold change = 0.47, P = 0.00414) were significantly decreased.

Article Snippet: The exosomal biomarkers CD9 (1:1,000; BD Biosciences Pharmingen, USA) and CD63 (1:1,000; Millipore, Germany) were detected by flow cytometric analysis.

Techniques: Microarray, Expressing

Journal: American Journal of Translational Research

Article Title: Differentially circulating exosomal microRNAs expression profiling in oral lichen planus

doi:

Figure Lengend Snippet: Correlations between differentially expressed exosomal miRNAs and clinical characteristics of OLP. (A) Differential expression of exosomal miRNAs in OLP patients and normal controls. All the results were analyzed by the 2-ΔΔCt method, and spike-in control cel-miR-39 was used as internal reference. Exosomal miR-34a-5p and miR-130b-3p were upregulated, whereas miR-301b-5p was significantly downregulated in OLP. However, no significant difference was found in the expression of exosomal miR-29c-3p or miR-144-3p. (B-D) The correlation between the expression level of circulating exosomal miRNAs and RAE scores: miR-34a-5p (B), miR-130b-3p (C), and miR-301b-3p (D). Significantly positive correlation was found between the expression of exosomal miR-34a-5p and RAE scores, indicating that exosomal miR-34a-5p was correlated to the severity of OLP. *P < 0.05; **P < 0.01.

Article Snippet: The exosomal biomarkers CD9 (1:1,000; BD Biosciences Pharmingen, USA) and CD63 (1:1,000; Millipore, Germany) were detected by flow cytometric analysis.

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: Differentially circulating exosomal microRNAs expression profiling in oral lichen planus

doi:

Figure Lengend Snippet: KEGG pathway analysis of target genes predicted by exosomal miR-34a-5p. Twenty-seven signaling pathways were selected as significantly enriched (P < 0.05). The -log2 (p-value) is displayed on the x-axis, and specific signalling pathways are shown on the y-axis. Among the top 5 predicted pathways, PI3K/Akt signaling pathway is likely to participate in OLP progression.

Article Snippet: The exosomal biomarkers CD9 (1:1,000; BD Biosciences Pharmingen, USA) and CD63 (1:1,000; Millipore, Germany) were detected by flow cytometric analysis.

Techniques: