Journal: International Journal of Molecular Sciences

Article Title: MircoRNA in Extracellular Vesicles from Patients with Pulmonary Arterial Hypertension Alters Endothelial Angiogenic Response

doi: 10.3390/ijms231911964

Figure Lengend Snippet: ( A ) Schematic representation of EV preparation and experimental workflow for miRNA profiling and EV characterization. ( B ) Purified EVs from healthy control (HC) and pulmonary arterial hypertension (PAH) patients were subjected to flow cytometry to detect expression levels of exosome-specific marker CD63. Results represent marker count/50,000 events and are given as mean ± S.E.M. ( n = 10 per group), comparing PAH versus HC. ( C ) Representative immunoblot of CD63 from EVs from HC and PAH patients. ( D ) Representative transmission electron micrographs of purified EVs from HC and PAH patients with uranyl acetate negative staining (scale bar 200–1000 nm) ( n = 5 per group; 10–20 images were taken per sample).

Article Snippet: For immunoblot analysis, the following antibodies were used: rabbit monoclonal anti-CD63 (D4I1X; Cell Signaling, Frankfurt am Main, Germany), rabbit monoclonal anti- NF-κB p65 (D14E12; Cell Signaling), rabbit monoclonal anti-PTEN (D4.3; Cell Signaling), rabbit monoclonal anti-TAK1 (D94D7; Cell Signaling), goat polyclonal anti-human VEGF-165 (VEGF-A; Cell Signaling), mouse monoclonal anti–β-actin (Sigma Aldrich, St. Louis, MI, USA), and secondary anti-mouse or anti-rabbit fluorescently labeled antibody (LI-COR).

Techniques: Purification, Flow Cytometry, Expressing, Marker, Western Blot, Transmission Assay, Negative Staining

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts in a 3D Engineered Tissue Model Induce Tumor-like Matrix Stiffening and EMT Transition

doi: 10.3390/cancers14153810

Figure Lengend Snippet: The presence of iCAFs in the stroma of 3D vesical models induces the proliferation and infiltration of urothelial cells. ( A ) Representative fluorescent images of the HFs-derived and iCAFs-derived models stained for Ki67 (green). Nuclei were counterstained with Hoechst (blue). ( B ) Corresponding quantification in relative fluorescence units (RFU) of the Ki67 signal. N = 4 independent biological replicates. ( C ) Representative fluorescent images of the HFs-derived and iCAFs-derived models of laminin-332. ( D ) Representative fluorescent images of vimentin- and epithelium-specific cytokeratin marker AE1/AE3 in the HFs-derived and iCAFs-derived constructs. The images show infiltration of epithelial cells within the stroma in the iCAFs-derived stroma. E = epithelium; S = stroma. The data are represented as mean ± SEM. **** p < 0.0001.

Article Snippet: Primary antibodies used were as follows: Rabbit anti-YAP1 (#14074, Cell signaling Technology, Whitby, ON, Canada); Mouse anti-vimentin (#550513), Mouse anti-E-Cadherin (#610181) and Mouse anti-Ki67 (#556003) were from BD BIOSCIENCES (Franklin Lakes, NJ, USA); Rabbit anti-ZEB1 (#ABD53), Rabbit anti-total Fibronectin (#F3648), Mouse anti-laminin-V (#MAB19562), and Mouse pan-anti-Cytokeratin AE1/AE3 (#MAB3412) were from MilliporeSigma (Billerica, MA, USA); Rabbit anti-vimentin (#ab45939), Rabbit anti-MMP9 (#ab76003) were from Abcam (San Francisco, CA, USA).

Techniques: Derivative Assay, Staining, Fluorescence, Marker, Construct

Journal: Stem Cell Research & Therapy

Article Title: Mesenchymal stem cell-derived extracellular vesicles protect retina in a mouse model of retinitis pigmentosa by anti-inflammation through miR-146a-Nr4a3 axis

doi: 10.1186/s13287-022-03100-x

Figure Lengend Snippet: Characterization of MSC-EVs. A – B Flow cytometry results showed 73.4% and 74.5% of EVs were positive for CD63 A and CD81 (B). C Particle size analysis of the EVs showing the distribution of EVs with different sizes. Around 75% of the particles had the size of exosomes, i.e., 30–200 nm. D Morphologies of two typical EVs taken by transmission electron microscopy (indicated by arrows). E. Western blotting showing the expression of EVs’ markers (CD9, CD63, Hsp70 and ALIX) in two batches of exosomes, EVs-1 and EVs-2, with lower concentration

Article Snippet: The primary antibodies used included mouse anti-IL-1β (1:1000, CST, #12,242), mouse anti-IκBα (1:1000, CST, #4814), rabbit anti-Phospho-IκBα (Ser32) (1:1000, CST, #5209), mouse anti-NF-κB p65 (1:1000, CST, #8242), rabbit anti-CD9(1:1000, CST, #13403S), rabbit anti-CD63 (1:1000, CST, #55051S), rabbit anti-Hsp70 (1:1000, CST, #4873S) and rabbit anti-ALIX (1:1000, CST, #92880S).

Techniques: Flow Cytometry, Particle Size Analysis, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mechanisms of Senescence-Related NKG2D Ligands Release and Immune Escape Induced by Chemotherapy in Neuroblastoma Cells

doi: 10.3389/fcell.2022.829404

Figure Lengend Snippet: Inhibition of NKG2D expression by exosomal MICA/B. (A) IMR-32 cells were treated with 2 μM MLN8237 or 0.5 μM DOX for 72 h. No treatment was used as a control, and exosomes were isolated by ultracentrifugation. Western blot was used to evaluate CD63 and TSG101 levels in whole cell lysates of the cells or isolated exosomes from supernatants. (B) Representative EM image of exosomes and showing the typical cup-shaped morphology. Scale bar, 100 nm. (C) Analysis of particle size distribution and concentration in exosomes. (D) IMR-32 cells were treated with 2 μM MLN8237 for 72 h, no treatment was used as a control. The expression of MICA/B in IMR-32 cells and exosomes were determined by flow cytometry, and exosomes were pretreated by coated latex microbeads. (E) Normal human peripheral blood NK cells were used as a control. Exosomes isolated from untreated IMR-32 cells, and MLN8237-treated cells were co-cultured with NK cells for 24 h. Co-cultures were spiked with the MICA/B antibody, and NKG2D expression was detected by flow cytometry.

Article Snippet: For western blot, 20 µg of each protein was electrophoresed and then transferred to a polyvinylidene fluoride membrane, which was incubated with Tris-buffered saline containing Tween-20 plus 5% skim milk powder for 1 h. Protein bands were isolated according to the molecular weight marker (26616, Thermo Fisher Scientific, Waltham, MA, United States) and incubated with the following primary antibodies: anti-CD63 (1:1000, 55051S, Cell Signaling Technology, Danvers, MA, United States), tumor susceptibility gene 101 (TSG101, 1:1000, 28405S, Cell Signaling Technology), disintegrin and metalloproteinase domain-containing 10 (ADAM10, 1:1000, GTX104940, GeneTex, Irvine, CA, United States), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1000, GTX100118, GeneTex).

Techniques: Inhibition, Expressing, Isolation, Western Blot, Concentration Assay, Flow Cytometry, Cell Culture

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Correlation between steroid levels in follicular fluid and hormone synthesis related substances in its exosomes and embryo quality in patients with polycystic ovary syndrome

doi: 10.1186/s12958-021-00749-6

Figure Lengend Snippet: Quality control of extracted follicular fluid exosomes. ( a ) Electron microscopic images of extracted exosomes revealed cup-shaped structures with a diameter of about 30–150 nm. Scale bar: 100 nm. ( b ) Western blotting revealed CD9, CD63 and TSG101 proteins in exosome samples. ( c ) Nanoparticle tracking analysis (NTA) revealed the diameter of isolated extracellular vesicles (EVs) is consistent with that of exosomes

Article Snippet: CD9 antibody: CD9 (D8O1A) Rabbit mAb (#13174, Cell signaling technology); CD63 antibody: CD63 (D4I1X) Rabbit mAb (#55051, Cell signaling technology); TSG101 (ab125011, abcam); Calnexin (#2433, Cell signaling technology).

Techniques: Western Blot, Isolation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Correlation between steroid levels in follicular fluid and hormone synthesis related substances in its exosomes and embryo quality in patients with polycystic ovary syndrome

doi: 10.1186/s12958-021-00749-6

Figure Lengend Snippet: Quality control of extracted follicular fluid exosomes. ( a ) Electron microscopic images of extracted exosomes revealed cup-shaped structures with a diameter of about 30–150 nm. Scale bar: 100 nm. ( b ) Western blotting revealed CD9, CD63 and TSG101 proteins in exosome samples. ( c ) Nanoparticle tracking analysis (NTA) revealed the diameter of isolated extracellular vesicles (EVs) is consistent with that of exosomes

Article Snippet: CD9 antibody: CD9 (D8O1A) Rabbit mAb (#13174, Cell signaling technology); CD63 antibody: CD63 (D4I1X) Rabbit mAb (#55051, Cell signaling technology); TSG101 (ab125011, abcam); Calnexin (#2433, Cell signaling technology).

Techniques: Western Blot, Isolation