Fig 1: Clathrin-mediated endocytosis is involved in PCV3 infection. (A) Cell viability upon CPZ treatment was evaluated by MTT assay. **P < 0.01 indicates statistical significance when compared with the DMSO-treated group. (B,C) Chlorpromazine inhibited PCV3 infection. PK15 cells were exposed to CPZ at the indicated doses or DMSO (mock) for 1 h, followed by PCV3 infection for 1 h. After culturing in a medium supplemented with CPZ and 2% NBCS for 48 h, the cells were probed using anti-PCV3 Cap antibodies and observed under immunofluorescence microscopy. Scale bar, 50 µm. The infection rate of PCV3 in PK15 cells decreased after CPZ pretreatment in a dose-dependent pattern, as determined by the percentage of infected cells. The vertical three pictures were taken from the same cell groups treated with the indicating concentrations of CPZ. **P < 0.01 indicate statistical significance when compared with the mock-treated group (0 µM). (D) Cells were transfected with 20 nM siCtrl or 20 nM siCLTC followed by PCV3 infection. After incubated in a medium supplemented with 2% NBCS for 36 h, the total cell protein was extracted and analyzed by WB targeting PCV3-Cap and CLTC to determine the knockdown efficiency and viral Cap expression. ß-actin served as the loading control for protein detection. (E) EPS15-WT or EPS15-DN transfected cells were exposed to PCV3 and incubated in a medium supplemented with 2% NBCS for 36 h. The extracted protein was examined by WB with anti-Cap antibodies to determine viral Cap expression. ß-actin served as the loading control for protein detection. (F) PK15 cells transfected with 20 nM siCtrl or 20 nM siCLTC were infected with PCV3 and incubated in medium supplemented with 2% NBCS for 36 h. Thereafter, the cell cultures were harvested, and the viral titer were assessed by TCID50 analysis after three freeze-thaw cycles. **P < 0.01 indicated statistical significance compared with the siCtrl groups.