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:

Journal: Nature Communications

Article Title: The generation and use of recombinant extracellular vesicles as biological reference material

doi: 10.1038/s41467-019-11182-0

Figure Lengend Snippet: rEV are separated from conditioned medium using density gradient centrifugation. a Schematic representation of rEV showing representative molecular components shared with sample EV, PA phosphatidic acid, PC phosphatidylcholine, PE phosphatidylethanolamine, PI phosphatidylinositol, PS phosphatidylserine, SM sphingomyelin. b Schematic overview of the production of rEV at the cellular level: (1) The gag-EGFP fusion protein inserts in regions of the plasma membrane enriched for tetraspanins CD9, CD63 and CD81 via its N-terminal MA domain containing a myristoyl group. (2) The gag-EGFP fusion protein oligomerizes and recruits ESCRT-1 proteins (TSG101) via the PTAP motive on its p6 domain. (3) Recruitment of ESCRT-2/3 proteins initiates the outward budding of the gag-EGFP containing plasma membrane. (4) ESCRT-3 mediated scission of the membranes finally causes release of rEV into the conditioned medium (CM) . c Schematic overview of the workflow to separate rEV from CM of gag-EGFP transfected HEK293T cells. Seventy-two hour post transfection CM is collected from ~3 x 10 9 cells and concentrated to 1 mL. Concentrated CM is loaded on top of an OptiPrep density gradient (ODG) and centrifuged for 18 h at 100,000 × g . Density fractions of 1.086–1.119 g/mL are collected and pelleted for 3 h at 100,000 × g resulting in ~5 x 10 11 rEV per harvest

Article Snippet: The following primary and secondary antibodies were used for immunostaining: mouse monoclonal anti-ALIX (1:1000, #2171)and rabbit monoclonal anti-CD9 clone D3H4P (1:1000, #13403S) (Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-CD63 clone MEM-259 (1:200, #ab8219) and rabbit monoclonal anti-syntenin-1 (1:1000, #ab133267) Abcam, Cambridge, UK), mouse monoclonal anti-CD81 (1:1000, #SC-166029) and mouse monoclonal anti-TSG101 (1:100, #SC-7964) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-flotillin-1 (1:1000, #610820) and rat anti-mouse CD9 (1:1000, #553758) (BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal anti-green fluorescent protein (GFP) (1:1000, #MAB3580) (Merck Millipore, Billerica, MA, USA), mouse monoclonal anti-α-tubulin (1:4000) (T5168, Sigma, Diegem, Belgium), sheep anti-mouse horseradish peroxidase-linked antibody (1:3000, #NA931V) and donkey anti-rabbit horseradish peroxidase-linked antibody (1:4000, #NA934V) (GE Healthcare Life Sciences, Uppsala, Sweden).

Techniques: Gradient Centrifugation, Transfection

Journal: Nature Communications

Article Title: The generation and use of recombinant extracellular vesicles as biological reference material

doi: 10.1038/s41467-019-11182-0

Figure Lengend Snippet: rEV bear physical and biochemical traits characteristic of sample EV. rEV, separated by ODG centrifugation of medium conditioned by gag-EGFP transfected HEK293T cells, are compared to sample EV separated by ODG centrifugation from different sources, including medium conditioned by breast cancer cells (MCF7, 4T1), mock transfected HEK293T cells (mock) or cancer-associated fibroblasts (CAF) or plasma and urine for a size distribution measured with nanoparticle tracking analysis (NTA) ( n > 6), b zeta potential ( n > 3), c refractive index distribution calculated with NTA and MIE theory (supplementary fig. ), d morphology as imaged by transmission electron microscopy (TEM) and the presence of EV-associated proteins ALIX, TSG101, flotillin-1, syntenin-1, CD81, CD9 and CD63 analysed by e western blot analysis (30 μg protein loaded on gel) and f immune-electron microscopy with a secondary gold labelled antibody against a primary antibody targeting the extracellular loop of the tetraspanin CD63. Images are representative of three biological replicates. Data in a and b are (mean, SD). Source data are provided as a source data file

Article Snippet: The following primary and secondary antibodies were used for immunostaining: mouse monoclonal anti-ALIX (1:1000, #2171)and rabbit monoclonal anti-CD9 clone D3H4P (1:1000, #13403S) (Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-CD63 clone MEM-259 (1:200, #ab8219) and rabbit monoclonal anti-syntenin-1 (1:1000, #ab133267) Abcam, Cambridge, UK), mouse monoclonal anti-CD81 (1:1000, #SC-166029) and mouse monoclonal anti-TSG101 (1:100, #SC-7964) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-flotillin-1 (1:1000, #610820) and rat anti-mouse CD9 (1:1000, #553758) (BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal anti-green fluorescent protein (GFP) (1:1000, #MAB3580) (Merck Millipore, Billerica, MA, USA), mouse monoclonal anti-α-tubulin (1:4000) (T5168, Sigma, Diegem, Belgium), sheep anti-mouse horseradish peroxidase-linked antibody (1:3000, #NA931V) and donkey anti-rabbit horseradish peroxidase-linked antibody (1:4000, #NA934V) (GE Healthcare Life Sciences, Uppsala, Sweden).

Techniques: Centrifugation, Transfection, Transmission Assay, Electron Microscopy, Western Blot

Journal: The Journal of Cell Biology

Article Title: Productive HIV-1 infection of tissue macrophages by fusion with infected CD4 + T cells

doi: 10.1083/jcb.202205103

Figure Lengend Snippet: Related to and . (A–C) Related to . (A) Analysis of Myosin expression and MLC phosphorylation by Western blot: MDMs were transfected with non-targeting siRNA (siCTRL) or targeting Myosin IIA (siMYO). Left: Representative images of Western blot analysis of the expression of Myosin IIA (top) and p-MLC (middle), with actin as loading control (bottom). Right: Quantification of Myosin IIA/actin, normalized to the siRNA control condition ( n = 6 donors, median ± interquartile range). (B) Quantification of p-MLC/actin, normalized to the siCTRL condition ( n = 6 donors, median ± interquartile range). (C) Representative images of actomyosin cytoskeleton of MDMs treated with siCTL or siMYO. F-actin (phalloidin, gray), Myosin IIA (red), and nuclei (DAPI, cyan). Scale bars, 10 µm. (D–G) Related to . (D) Quantification by flow cytometry of infection of MDMs incubated with isotype control (Control) or antibody targeting CD9 (anti-CD9) and co-cultured with infected Jurkat cells, normalized to the isotype control condition ( n = 10 donors, median ± interquartile range). (E) Analysis of CD81 depletion by flow cytometry: MDMs were transfected with non-targeting siRNA (siCTRL) or targeting CD81 (siCD81). Left: Representative dot plot of CD81 signals. Right: Quantification of CD81 expression, normalized to the siCTRL condition ( n = 6 donors, mean ± SD). (F) Flow cytometry analysis of alveolar macrophages infection after a 24 h-co-culture with uninfected Jurkat cells (NI) or Jurkat cells infected for 2 d in presence of anti-CD81 antibody or corresponding isotype control. Representative dot plots of HIV-p24 signals and gating strategy for selection of infected cells. See quantification in . (G) Representative images of actomyosin cytoskeleton of MDMs treated with siCTL or siCD81. F-actin (phalloidin, gray), Myosin IIA (red), and nuclei (DAPI, cyan). (G) This image comes from a mosaic (stitched together by the microscope). Scale bars, 10 µm. Statistical analyses: (A, B, and D) Wilcoxon test and (E) Paired t test. * P ≤ 0.05. Source data are available for this figure: .

Article Snippet: The culture medium was then removed and replaced by a dilution of anti-CD18 (clone TS1/18, mouse IgG1, 302102; Biolegend), anti-CD81 (clone 5A6, mouse IgG1, 349502; Biolegend), anti-CD9 (clone M-L13, mouse IgG1, 555370; BD Biosciences), anti-CD4 (clone Leu3a, mouse IgG1, 344602; Biolegend), or mouse IgG1k isotype control (clone MOPC-21, mouse IgG1, 400102; Biolegend).

Techniques: Expressing, Western Blot, Transfection, Flow Cytometry, Infection, Incubation, Cell Culture, Co-Culture Assay, Selection, Microscopy

Journal: Immunology

Article Title: The CD19/CD81 complex physically interacts with CD38 but is not required to induce proliferation in mouse B lymphocytes

doi: 10.1111/j.1365-2567.2012.03602.x

Figure Lengend Snippet: Co-capping of CD38 with CD63 and CD9. (a) Splenocytes from C57BL/6 mice were incubated with rabbit polyclonal anti-CD38 antibody for 30 min on ice, washed and reacted with a secondary anti-rabbit-Cy3 antibody for 1 hr at 37°. Counterstaining was performed at 4° with anti-CD63 (a) or CD9 (b) and DAPI was used for staining of nuclei. Confocal sections for the merged images of representative cells are shown. Coefficient of correlation (CC) for colocalization was calculated using imagej software. (b) The number of co-caps is presented as percentage of cells analyzed. Confocal sections for the merged images of representative cells are shown.

Article Snippet: Anti-CD81 (104907) and anti-CD19 (115513) were purchase from Biolegend (San Diego, CA), anti-B220-FITC (553088) and anti-CD9 (558749) were purchase from BD Pharmingen (San Diego, CA); and anti-CD63 (sc15363) from Santa Cruz Biotechnology, Inc (CA) and secondary antibodies anti-rabbit-Cy3 (Caltag Laboratories, Burlingame, CA; {"type":"entrez-nucleotide","attrs":{"text":"L42010","term_id":"804648","term_text":"L42010"}} L42010 ), anti-hamster-FITC (BD Pharmingen; 554011), anti-rabbit-horseradish peroxidase (HRP) (Thermo Scientific, Pittsburgh, PA; 1858415), anti-hamster-HRP (BD Pharmingen; 554012), anti-rat-HRP (ZyMax, San Francisco, CA; 81-9520) and anti-mouse-HRP (Thermo Scientific; 1858413).

Techniques: Incubation, Staining, Software

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