Figure 5.

Overexpression of ATP1A1 promotes PEDV infection. (A,B) Overexpression of ATP1A1 significantly upregulates PEDV RNA abundance. Transfected with FLAG or FLAG-ATP1A1 vector for 24 h, cells were collected at 12, 24 and 48 h after PEDV at 0.1 MOI infection of target cells for quantitative PCR assay. Data represent means ± SD from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significant difference. (C,D) Overexpression of ATP1A1 remarkably facilitates the PEDV N protein expression. After overexpressing of ATP1A1 for 24 h, target cells were infected with PEDV (CV777-G1 or GDgh-G2) at 0.1 MOI and collected and lysed at different time points for Western blot analysis. (E) ATP1A1 promotes the expression of PEDV N protein in a dose-dependent manner. Cells were transfected with increasing concentrations of a vector expressing FLAG-ATP1A1. At 24 h post-transfections, the cells were infected with PEDV at 0.1 MOI, and the cell lysates were collected for analysis of PEDV N protein expression with Western blotting. (F) PTR2 and DF-1 cells overexpressing of ATP1A1 gene maintained resistance to PEDV infection. Cells overexpressed with ATP1A1 were infected with PEDV at 0.1 MOI for 24 h, and the cell lysates were collected for analysis of PEDV N protein expression with Western blotting. (G,H) Overexpression of ATP1A1 enhances PEDV viral titers. Cells overexpressed with ATP1A1 were infected with PEDV at 0.1 MOI infected for 24 h, and cell supernatants were extracted for TCID₅₀ assay. Data represent means ± SD from three independent experiments. *, p < 0.05; **, p < 0.01. (I,J) Overexpression of ATP1A1 enhances PEDV infection. Cells were transfected with FLAG or FLAG-ATP1A1 vector for 24 h, and then PEDV (0.1 MOI) infected for 24 h. The cells were stained with anti-ATP1A1 mAb (red) and anti-PEDV-N mAb (green). Staining of cell nuclei using DAPI (blue) staining solution. The processed samples were photographed and analyzed using fluorescence microscopy. Scale bars, 200 μm.