FIG 5.

PDCoV entry into IPI-2I cells depends on macropinocytosis. (A and F) Effect of EIPA (A) and wortmannin (F) on PDCoV binding and entry. IPI-2I cells were pretreated with increasing subtoxic doses as described in above Fig. 3A, and lysed cells were subjected to determine viral RNA copy numbers by RT-qPCR. (B and G) RT-qPCR and Western blot analysis for the effect of EIPA (B) and wortmannin (G) on PDCoV infection. Cells were pretreated with increasing subtoxic doses of these drugs or DMSO at 37°C for 1 h and then inoculated with PDCoV at 37°C for 6 h, followed by determination of viral infection by RNA copy numbers and viral protein expression levels via RT-qPCR and Western blot analysis. (C) Viral titer detection for PDCoV in the medium from cells treated as described in panel B. (D and H) IFA for the effects of EIPA (D) and wortmannin (H) on PDCoV infection. Cells were treated as described in panels B and G, respectively. Infected cells were fixed and subjected to IFA using anti-PDCoV N antibody as the primary antibody. Relative fluorescence intensity is quantified by Image‐Pro Plus software as shown in panels D and H on the right. (E and I) CCK-8-based cell viability assay for EIPA (E) and wortmannin (I) as described in Materials and Methods. Target protein expression was quantitatively estimated by ImageJ software and presented as the density value relative to that of the β-actin. The presented results represent the means and standard deviations of data from three independent experiments. **, P < 0.01; ***, P < 0.001.