FIG 5.

SCAMP3 affects PI4KIIIβ and PI4P recruitment for viral replication during EV-A71 infection. (A) Scramble or SCAMP3 KO RD cells were mock infected or infected with EV-A71-3A4-FLAG at an MOI of 20. At 6 h postinfection, cells were fixed with formaldehyde, washed, and immunostained with antibody against 3A-FLAG, PI4KIIIβ, and PI4P. DAPI was used to stain the nucleus. Images were captured by confocal laser scanning microscopy. (B) Quantification of the FLAG fluorescence intensity of EV-A71-infected scramble and SCAMP3 KO cells. (C) Quantification of the PI4KIIIβ fluorescence intensity of EV-A71-infected cells. PI4KIIIβ level was determined by fluorescence intensity overlap of the 3A-FLAG area of the whole cells. (D) Quantification fluorescence intensity of PI4P of EV-A71-infected cells. PI4P level was determined by fluorescence intensity overlap of the 3A-FLAG area of the whole cells. Bars represent the means from 40 cells from the experiment. Significant differences compared to EV-A71-infected scramble cells are indicated as follows: **, P < 0.01, ***, P < 0.001. (E) Western blot analysis of viral protein and SCAMP3 levels of total cell lysates. (F) Total viral RNA level was measured by RT-qPCR. Scale bar, 10 μm.