Figure 6.
Effects of Blocking Exosomal miR-192 Level on HSCs
Effects of exosomes from JFH-1 stable cells treated with anti-miR-192 (anti-miR-192 exo). Control exosomes from scramble-treated JFH-1 stable cells are referred to as scr exo. (A) Comparison of CD63 (left) and exosome total protein levels (right) using a BCA assay is shown. (B) Levels of miR-192 (left: 3.27 ± 1.85 × 106 and 1.24 ± 0.62 × 106 copies per 20 μL of control scr exo and anti-miR-192 exo, respectively), miR-122 (middle: 2.56 ± 1.71 × 106 and 2.49 ± 1.32 × 106 copies per 20 μL of scr exo and anti-miR-192 exo, respectively), and HCV RNA (right: 1.67 ± 1.03 × 106 and 1.54 ± 1.15 × 106 copies per 20 μL of scr exo and anti-miR-192 exo, respectively) within each exosome type were quantified. (C) Immunoblot analysis of the fibrogenic markers COL1A1 and α-SMA, as well as TGF-β1, in LX-2 cells treated with each exosome type (left) is shown. Protein levels were quantified relative to those of tubulin (right). (D) LX-2 cells were treated with each exosome type for 72 h, fixed, stained, and visualized via 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 from more than five fields in three independent experiments (right). Data are also shown in Figure S7. (E) Intracellular miR-192 levels in LX-2 cells treated with each exosome type were quantified. All data are presented as the means of at least three independent experiments, each performed in triplicate. Error bars represent the SEM. p values were determined using a one-tailed unpaired Student’s t test. *p < 0.05; ***p < 0.001; n.s., non-significant.