Figure 2

NPD8716 inhibited HBV infection. (A) Schematic representation of the scheme for treating the cells with compounds and HBV. HepG2-hNTCP-C4 cells were treated with HBV in the presence or absence of compounds for 16 h. After washing out free HBV and compounds, the cells were cultured without compounds for an additional 12 days, and HBV infection was evaluated by quantifying HBs antigen secreted into the culture supernatant, HBV DNA and HBc antigen in the cells. Black and white bars show periods of treatment and nontreatment, respectively. (B to E) HepG2-hNTCP-C4 cells inoculated with HBV were treated with or without 100 nM preS1 peptide or 200 μM NPD8716 according to the scheme in (A), and HBs antigen in the culture supernatant (B), HBV DNA (C) and HBc antigen (D) in the cells were detected by ELISA, real-time PCR, and immunofluorescence analyses, respectively. Cell viability was also quantified by MTT assay (E). Red and blue signals in (D) show the detection of HBc antigen and nuclear staining, respectively. (F,G) HepG2-hNTCP-C4 cells were treated with or without various concentrations (25, 50, 100, or 200 μM) of NPD8716 as shown in (A). HBs antigen (F) and cell viability (G) were determined by ELISA and MTT assay. (H) Freshly isolated primary human hepatocytes were continuously treated with or without 60 μM NPD8716 from the time of HBV inoculation (left), or 48 h after HBV inoculation (right). Concentration of NPD8716 was 40 μM at the first three days of treatment and then reduced to 20 μM until day 36, while preS1 peptide was treated at 100 nM in the right panel. At 12, 15, 18, 21, and 24 days (left), or 6, 12, 18, 24, 30, and 36 days (right) postinoculation, HBV DNA secreted into the culture supernatant were detected by real-time PCR (right). SDs are also shown as error bars. Statistical significance was determined by using Student’s t test (*P < 0.05; **P < 0.01).