Figure 8.

Transfection of ATP6V1A or its truncate trans-complements the decrease in ERA replication in cells transfected with RNAi of ATP6V1A. HEK293T cells were transfected with ATP6V1A or scrambled siRNA. At 48 h post transfection of siRNAs, cells were transfected with pA-Myc, and the ATP6V1A protein level was detected by Western blotting at 24 h post transfection (A). At 48 h post transfection of siRNAs, cells were transfected with pA-Myc, and pA163-309, or pMu160-309, and infected with ERA virus at an MOI of 1 (B and C) or 0.01 (D and E) at 24 h post transfection. The culture medium of cells was collected and centrifuged (5000g/min, 10 min) at 4 °C at indicated time points post infection. Viral titers in the culture supernatants of the cells were detected at different times after infection (B and D). ATP6V1A and its truncates in the cell lysates and viral P protein in the culture supernatants and lysates of the HEK293T cells were detected by Western blotting. The ratio of viral P protein in the culture supernatant (purple bar) or cellular lysate (gray bar) of cells transfected with ATP6V1A siRNA and pMyc (left panel) or pA160-309 (right panel) was compared with that of cells transfected with Scrambled siRNA and pMyc. The ratio of viral P protein in the culture supernatant (red bar) or cellular lysate (black bar) of cells transfected with ATP6V1A siRNA and pA-Myc (left panel) or pMu160-309 (right panel) was compared with that of cells transfected with Scrambled siRNA and pMyc (C and E). Statistical analysis of the P protein levels in the upper panel was performed by using ImageJ software. The value for the P protein in the culture supernatants or lysates of the cells was standardized to the β-actin level and normalized to the level of the P protein in the culture supernatants or lysates of the cells transfected with the scrambled siRNA and control vector pMyc, respectively. Data were expressed as means ± SD of triplicate well from one independent experiment. Statistical significance was determined by ANOVA with a multiple comparison correction. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. lysate, lysate of cells; super, supernatants of culture medium of cells.