FIG 3.

Caveolae are not required for PDCoV entry into IPI-2I cells. (A) Effect of nystatin on PDCoV binding and entry. IPI-2I cells were pretreated with increasing subtoxic doses at 37°C for 1 h and infected with PDCoV (MOI of 5) at 4°C for 1 h (binding step) and then shifted to 37°C for 1 h (entry step). Cells were lysed to determine viral RNA copy numbers by RT-qPCR. (B) RT-qPCR and Western blot for inhibition of PDCoV infection by nystatin. Cells were pretreated with increasing subtoxic doses of nystatin or DMSO at 37°C for 1 h and then inoculated with PDCoV (MOI of 5) at 37°C for 6 h. Cells were lysed to determine viral RNA copy numbers via RT-qPCR and N protein levels via Western blot analysis. (C) CCK-8-based cell viability assay for nystatin as described in Materials and Methods. (D to F) Effect of DN caveolin (Cav1) on PDCoV entry (D) and infection (E and F) was determined via RT-qPCR, confocal microscopy, and Western blot analysis. Cells transfected with plasmid constructs encoding Flag-tagged WT and DN Cav1 were infected with PDCoV (MOI of 5). At 1 and 6 hpi at 37°C, cells were harvested and subjected to RT-qPCR, IFA, and Western blot analysis, respectively. Bar, 10 μm. (G and H) Cav1 knockdown failed to inhibit PDCoV entry (G) and infection (H). siCav1- or siCtrl-transfected cells were infected with PDCoV (MOI of 5). At 1 and 6 hpi at 37°C, the cells were lysed to determine the silencing efficiency of Cav1, the viral RNA copy numbers, and N protein expression levels via RT-qPCR and Western blot analysis. 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; ns, nonsignificant difference.