Fig 4. The secretions of exosomes and exosomal miRNAs are dependent on the activation of caspase-3 in HCV-infected hepatocytes.

Huh 7.5.1 cells were infected with HCV at 0.1 MOI. After 3 days, the culture medium was replaced with exosome-depleted FBS medium, and 20 μM z-VAD-fmk or z-DQMD-fmk dissolved in DMSO was added. After 24 h, exosomes were isolated from cell supernatants using ExoQuick-TC solution, and miRNAs were extracted from cells and exosomes. (A) HCV RNA level in the cells was analyzed by qRT-PCR and HPRT was used as an endogenous control for normalization. (B) Extracellular ATP levels in cell supernatant were measured using a luminescent cell viability assay and (C) intracellular calcium levels were measured using a fluorometric calcium assay. (D) Protein levels of Panx1, activated caspase-3, CD63, and GAPDH were measured in the cell lysates. (E) Exosome protein contents were measured using the BCA assay. (F) Intracellular miRNAs and (G) extracellular exosomal miRNAs were extracted using a miRNeasy mini kit and miRNA expression was analyzed by qRT-PCR. U18 was used as an endogenous control in cells for normalization, and Caenorhabditis elegans miR-39 was used as an exogenous control for normalization in exosomes. Data are expressed as mean ± SD (n=3). *P < 0.05, ** P < 0.01 and *** P < 0.001 were considered statistically significant, as assessed using an unpaired student t-test.