Fig. 4

CPZ facilitates the entry step of EV71 infection. a. A549, RD and HepG2 cells were infected with EV71 pseudovirus or transfected with EV71 replicon RNA for 12 h before CPZ (20 μM) or DMSO treatment. Luciferase activity was quantified at 24 h post infection or transfection. The means with SD from three independent experiments each carried out in duplicate are shown. b. A549, RD and HepG2 cells were incubated with EV71 at an MOI of 50 and treated by CPZ or DMSO at 4 °C for 2 h. Then cells were washed extensively and lysed for viral RNA quantification with normalization to 18 s. Data shown are the means with SD from three independent experiments each carried out in duplicate. c. Immunofluorescence analysis was performed on RD and A549 cells infected with EV71 at an MOI of 10 (in RD) and 50 (in A549) in the presence or absence of 2.5 mM GnHCl. After 2 h incubation at 4 °C, cells were immediately shifted to 37 °C and treated by CPZ (20 μM) or DMSO. At 6 hpi, cells were washed extensively, and then fixed and stained with EV71 VP-1 antibody, and DAPI was used to visualize the nuclei. The images were one representative experiment out of three independent experiments. Arrow heads showed the VP-1 foci inside the cells. Scale bar, 100 μm. d. Frequency of VP-1 foci in each infected A549 and RD cell that were pretreated by GnHCl and then incubated with EV71 in the present of CPZ or DMSO. A paired Student’s t test was performed between the mean values in three independent experiments. *, p < 0.05