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Figure 3 | Veterinary Research

Figure 3

From: PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

Figure 3

PEDV entry relies on the CME pathway. A Vero cells and IPEC-J2 cells were incubated with mixture of Alexa-594 labeled Trf (red) and PEDV (green) at 4 °C for 1 h, and then shifted to 37 °C for 30 min. The cells were fixed and stained for PEDV using monoclonal antibody against PEDV S protein. The cellular localizations of Trf and PEDV were observed with a confocal fluorescence microscope. Light exposure was avoided throughout this process. B, C. The Vero cells were pre-treated with 10 μM and 30 μM of CPZ, and the IPEC-J2 cells were pre-treated with 30 μM and 50 μM of CPZ, respectively, at 37 °C for 1 h and incubated with GDS01 or GDS09 strains for 1 h. Double-distilled water was used as a negative control. The cells were collected at 6 hpi and 9 hpi for qRT-PCR and Western blotting assay, respectively, to test the invasion efficiency of PEDV. D The Vero cells (left) and IPEC-J2 cells (right) were transfected with GFP-EPS15-WT and GFP-EPS15-M, respectively, and infected with PEDV strains at 24 h after transfection. The cells were fixed at 12 hpi and stained for confocal analysis. EH The Vero cells and IPEC-J2 cells were transfected with siCHC and siEPS 15 and infected with PEDV strains at 24 h after the second transfection. The cells were collected at 6 hpi and 9 hpi for qRT-PCR and Western blotting analysis, respectively. I The cells were pre-cooled at 4 °C for 15 min, incubated with PEDV strains at 4 °C for 1 h, shifted to 37 °C for 5 min to initiate internalization, and washed for three times to remove un-internalized viral particles. The cells were fixed and stained with anti-PEDV-S (red) and anti-CHC (green) primary antibodies. Ctrl means control. Scale bars indicate 50 μm in A, 25 μm in D, and 5 μm in I. **0.05 < P < 0.01; ***0.01 < P < 0.001; ****P < 0.001.

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