Figure 7.
Effects of Exosomes from miR-192-Transfected Hepatocytes or Exosomes Packaged with miR-192
(A) Experimental scheme: negative-control miRNA (siNTC) or miR-192 mimic RNA was transfected into Huh-7 cells, and exosomes were purified from the transfected cells (siNTC and miR-192 exo, respectively) and used to treat LX-2 cells. (B) Comparisons of protein levels of the exosome markers LAMP2 and CD63 using western blotting (left) and exosome total protein levels using a BCA assay (right) within exosomes from each transfected cell type are shown. (C) Quantification of miR-192 (left: 3.98 ± 1.71 × 106 and 2.08 ± 2.09 × 108 copies per 20 μL of control siNTC exo and miR-192 exo, respectively) and miR-122 levels (right: 2.04 ± 1.48 × 106 and 1.88 ± 1.27 × 106 copies per 20 μL of siNTC exo and miR-192 exo, respectively) in exosomes from each transfected cell type is shown. (D) Immunoblot analysis of the fibrosis markers COL1A1 and α-SMA, as well as TGF-β1, in LX-2 cells treated with each exosome type is shown. (E) LX-2 cells were treated with each type of exosomes for 72 h, fixed, stained, and visualized via fluorescence microscopy. Representative images of α-SMA and DAPI staining in each cell type (left). Scale bars represent 50 μm. The intensity of α-SMA staining was quantified using more than five fields from at least three independent experiments (right). Data are also shown in Figure S8A. (F) Intracellular miR-192 levels in LX-2 cells treated with each type of exosomes were quantified. (G) Experimental scheme: exosomes were first purified from naive Huh-7 cells and then packaged with siNTC or miR-192 (siNTC- and miR-192-packaged exosomes, respectively). (H) Immunoblot analysis of COL1A1, α-SMA, and TGF-β1 expression in LX-2 cells treated with each packaged exosome type is shown (left). Protein levels were quantified relative to those of tubulin (right). (I) LX-2 cells were treated with each packaged exosome type for 72 h, fixed, stained, and visualized using fluorescence microscopy. Representative images of α-SMA and DAPI staining in each cell are shown (left). Scale bars represent 50 μm. Intensity of α-SMA staining was quantified using more than five fields from at least three independent experiments is shown (right). Data are also shown in Figure S8B. (J) Intracellular miR-192 levels in LX-2 cells treated with each packaged exosome type were quantified. All data are presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent SEM. p values were determined using a one-tailed unpaired Student’s t test. *p < 0.05; **p < 0.01; n.s., non-significant.