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. 2016 Jun 27;7(31):49998–50016. doi: 10.18632/oncotarget.10307

Figure 3. Nanoparticle tracking and LC-MS/MS analysis of extracellular vesicles.

Figure 3

a. NTA images of vesicles isolated from MDA-MB-231 cells, MCF-7 cells, and fibroblasts as indicated. Vesicles were quantitated into sizes between 10-120 nm and 120-690 nm by NanoSight technology. Results are shown as the number of vesicles within each size range (mean+SEM, n=3). Single and double asterisks indicate statistically significant differences of p<0.05 and p<0.01, respectively. Scale bar = 5 μm. b. Selected proteins identified in vesicles from MDA-MB-231 cells by trypsin digestion and LC-MS/MS analysis are shown along with their peptide number and relative abundance color coded from green to red, as indicated. c. AHNAK and β-actin protein levels in nontransformed fibroblasts, MCF-7 cells, MDA-MB-231 cells, and in isolated vesicles from MDA-MB-231 cells were examined by Western blotting. Vesicles derived from MDA-MB-231 cells were enriched with AHNAK. d. Protein from exosomes (Exo), microvesicles (MV), whole cell lysates (WCL), and whole cells lysates from MDA-MB-231 cells treated with 1 μM staurosporine for 24 hours (WCL+Stau) to induce apoptosis were examined by Western blotting for AHNAK, flotillin, TSG101, β-actin, Bax, and cleaved caspase 3 as indicated. Microvesicles contained AHNAK but not apoptotic markers (cleaved caspase-3 and BAX). Exosomes were enriched with Flotillin and TSG101, but not AHNAK nor apoptotic markers.