Fig 5. The secretions of exosomes and exosomal miRNAs are dependent on the activation of Panx1 and P2X4 in HCV-infected hepatocytes.

Huh 7.5.1 cells were infected with HCV at 0.1 MOI and medium (NT, non-treatment), 20 μM carbenoxolone (CBX) (Panx-1 inhibitor) dissolved in medium, DMSO or 5 μM BX430 (P2X4 inhibitor) dissolved in DMSO were added every day. After 3 days, the culture medium was replaced with exosome-depleted FBS medium with CBX or BX430. After 24 h, exosomes were isolated from cell supernatants using ExoQuick-TC solution, and miRNAs were extracted from cells and exosomes. Huh 7.5.1 cells were transfected with a negative control (NC), Panx1 short interfering RNA (siRNA) (siPnax1) or a P2X4 siRNA (siP2X4) at 1 day post infection. The culture medium was replaced with exosome-depleted FBS medium at 3 days post infection. 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 was analyzed by qRT-PCR and HPRT was used as an endogenous control for normalization. (B) Extracellular ATP levels in cell supernatants were measured using a luminescent cell viability assay and (C) intracellular calcium levels were measured using a fluorometric calcium assay. (D) Neutral sphingomyelinase (nSMase) activity and (F) protein levels of Panx1, P2X4, activated caspase-3, CD63, and GAPDH were measured in the cell lysates. (E) TO-PRO-3 uptake was measured by flow cytometry. (G) Exosome protein contents were measured by the BCA assay. (H) Extracellular exosomal miRNAs were extracted using a miRNeasy mini kit and miRNA expression was analyzed by qRT-PCR. 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.