Novel method for extracting exosomes of hepatocellular carcinoma cells

Figure 1 Characterization of the exosomes of Huh-7 cells extracted by two methods. Electron micrographs of exosomes isolated from Huh-7 cells in serum-free concentrated culture medium by the traditional method (A) and the new extraction method based on a nanomaterial (B). Negative-stained images show exosomes with a smooth, saucer-like morphology. The sizes are between 30-100 nm. The scale bar is 50 nm. Direct Mag: 100000 ×, HV = 80.0 kV.

Figure 2 Detection of differences in Huh7 whole cell lysates and exosomal proteins. Approximately 30 μg of Huh-7 whole cell lysates and exosomal proteins isolated by the nanomaterial procedure were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were visualized by Coomassie brilliant blue staining. The marker is a prestained protein marker; Huh7-exo indicates exosomal proteins isolated from the supernatants of human hepatocellular carcinoma Huh-7 cells; Mr. stands for molecular weight.

Figure 3 Exosome protein identification and secretion mechanisms of human liver cancer-associated proteins as analyzed by Western blotting. In total, 30 μg of proteins from Huh-7 whole cell lysates and exosomal proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrotransferred and incubated with antibodies against exosome protein markers (HSP70 and CD63) and hepatocellular carcinoma-associated proteins (TGM2 and annexin A2). β-actin was used as an internal control. Exosome protein markers (HSP70 and CD63) were identified on the surfaces of exosome membranes. Exosomes secreted by Huh-7 hepatocellular carcinoma cells contained the hepatoma-associated protein TGM2 but were negative for annexin A2. Huh7-exo indicates exosomal proteins isolated from the supernatants of human hepatocellular carcinoma Huh-7 cells; Mr. stands for molecular weight.