Figure 4

Inhibition of actin polymerization and clathrin-mediated endocytosis reduce the DENV-induced increase in microglial migration. BV2 cells were inoculated with (A and C) DENV 2 (MOI = 50) or (B and D) Alexa-594-labeled DENV 2 (MOI = 50) for 12 h in the presence of the actin polymerization inhibitor cytochalasin D (Cyto. D, 5 µM) and the clathrin inhibitor chlorpromazine (CPZ, 10 µg/ml). The numbers of migrated cells and wound area measurements showed cell migration. Flow cytometry were used to measure the percentage of microglial cells carrying fluorescent DENV. (E) Furthermore, confocal microscopy showed the binding/entry of Alexa-594-labeled DENV 2 (MOI = 50, red) in BV2 cells with or without Cyto. D and CPZ. Phalloidin (green) staining indicated actin. DAPI staining indicated the nuclei (blue). (F) BV2 cells were treated with dextran (green) or Alexa-594-labeled DENV 2 (red, MOI = 50) for 6 h. The time-lapsed images showed cell morphology changes. (G) Migrating cell tracking showed the velocity and distance of the migrated cells. For all images, representative data were selectively obtained from three individual experiments. The quantitative data are depicted as the means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. ns, not significant.