Journal: International Journal of Molecular Sciences

Article Title: Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy

doi: 10.3390/ijms22031105

Figure Lengend Snippet: ( A ) Western Blot analysis of proteins present (Hsp90, CD63 and TSG101) or absent (calnexin) in exosomes and abundant in bovine milk (β-casein). Protein evaluation in: bovine skimmed milk (SM); exosomes obtained from SM by one (1U) or two (2U) ultracentrifugation steps; and the cellular fraction (CF). Equal amount of protein was loaded. Elution protein profile (F.1, F.8 to F.40) of bovine exosomes isolated by 1U followed by SEC (1U + SEC) ( B ) or 2U + SEC ( C ). Protein concentration (mg/mL) was estimated by the BCA assay. WB of SEC elution fractions from exosomes isolated by 1U + SEC ( D ) or 2U + SEC ( E ). Evaluation of Hsp90, CD63, TSG101, Calnexin and β-casein levels in each fraction (F.1, F.8 to F.40). Mimic hsa-miRNA-148a-3p elution profile (relative expression) of exosomes isolated from skimmed milk by U + T + SEC ( F ) or U + T + U + SEC ( G ). Mw: Molecular weight marker (Bio-Rad).

Article Snippet: Membranes were then incubated with appropriate primary antibodies: anti-Hsp90 (610418, BD, Madrid, Spain), anti-CD63 (bs-1523R, BIOSS, Woburn, MA, USA), anti-TSG101 (A303-506A, Bethyl, Montgomery, TX, USA), anti-calnexin (A303-694A, Bethyl) or anti-β-casein (ab112595, abcam, Cambridge, UK) at 4 °C (overnight).

Techniques: Western Blot, Isolation, Protein Concentration, BIA-KA, Expressing, Molecular Weight, Marker

Journal: International Journal of Biological Sciences

Article Title: Exosomal Transfer of Vasorin Expressed in Hepatocellular Carcinoma Cells Promotes Migration of Human Umbilical Vein Endothelial Cells

doi: 10.7150/ijbs.11943

Figure Lengend Snippet: VASN protein secretion and localization. (A) Western blot analysis of VASN protein in cell extracts and supernatant of HepG2 cells. (B) Electron micrograph of exosomes isolated from supernatants of HepG2 cells. Bar represents 100 nm. (C) VASN expression in exosomes, supernatant, and exosomes-depleted supernatants. The exosomal marker protein CD63 was also detected. (D) Western blot analysis of VASN protein in exosomes derived from HepG2, L02, and HUVECs. CD63 was used as a loading control. (E) Western blot analysis of VASN protein in cell lysates (top panel) and exosomes (bottom panel) from HepG2 cells transfected with NC or VASN siRNA (siVASN). GAPDH and CD63 were used as loading controls for cell lysates and exosomes, respectively.

Article Snippet: Rabbit polyclonal anti-CD63 antibody was purchased from Abcam, USA.

Techniques: Western Blot, Isolation, Expressing, Marker, Derivative Assay, Control, Transfection

Journal: PLOS ONE

Article Title: Use of extracellular vesicle microRNA profiles in patients with acute myeloid leukemia for the identification of novel biomarkers

doi: 10.1371/journal.pone.0306962

Figure Lengend Snippet: (A) The size and concentration trend of vesicles by fractions are presented. The eluted fractions (11 and 12; 0.5 mL each) were used for vesicle isolation. Samples were diluted 10-fold. (B) The size distribution of the isolated vesicles was determined using nanoparticle tracking analysis (NTA). The average size of BM aspirate serum or plasma-derived vesicles was 115.5 ± 2.7 nm and 101.5 ± 4.4 nm, respectively; these sizes were within the size range of typical EVs. Samples were diluted 10-fold. (C) In transmission electron microscopy (TEM) images, the size of isolated vesicles was <200 nm, and they were visualized as cup-shaped vesicles under high magnification. (D) Western blotting showed that the isolated vesicles were positive for the markers of EVs (CD63 and CD81). EVs, extracellular vesicles; BM, bone marrow; AML, acute myelogenous leukemia.

Article Snippet: After blocking with 5% skim milk (w/v) in 0.1% TBST for 2 h, the membranes were probed overnight at 4°C with 1:1000 dilutions of rabbit anti-CD63 polyclonal antibody and rabbit anti-CD81 polyclonal antibodies (all from Bioss Antibodies, MA, USA).

Techniques: Concentration Assay, Isolation, Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot

Journal: PLoS ONE

Article Title: Verification of the role of exosomal microRNA in colorectal tumorigenesis using human colorectal cancer cell lines

doi: 10.1371/journal.pone.0242057

Figure Lengend Snippet: (A) Cell morphology of HT-29 and SW480 cells in RPMI-1640 with exosome-free 10% FBS (original magnification ×100). (B) Exosomes secreted from HT-29 and SW480 cells were detected by electron microscopy (scale bar, 1 um). (C) Immunoblotting assay of CD9 (22~24 kDa) and CD63 (30~65 kDa) in exosomes from HT-29 and SW480 cells. Beta-actin (42 kDa) blot was used as the loading control. (D) Exosomes from HT-29 and SW480 cells were detected by Nanosight particle tracking analysis. The left column represents batch-to-batch variation, and the right column shows the overall size of the exosomes isolated from HT-29 and SW480, respectively.

Article Snippet: The membranes were probed with anti-CD9 antibody (EPR2949, rabbit monoclonal, ab92726, Abcam, Cambridge, UK), anti-CD63 antibody (MX-49.129.5, mouse monoclonal, ab193349, Abcam), and anti-beta-actin antibody (C4, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Electron Microscopy, Western Blot, Control, Isolation