Autophagy Facilitates Antibody-Enhanced Dengue Virus Infection in Human Pre-Basophil/Mast Cells (original) (raw)
Figure 2
Co-localization of DENV E protein and LC3 punctation in antibody-enhanced DENV infection of KU812 cells.
(A) KU812 cells were incubated with medium alone (Mock), with DENV alone, with DENV in the presence of sub-neutralizing dengue patient sera, sub-neutralizing dengue patient sera alone, with UV-inactivated DENV (iDENV) alone, or iDENV in the presence of sub-neutralizing dengue patient sera. After infection, cells were fixed, permeabilized, and stained with anti-DENV E protein (red), anti-LC3 (green), and DAPI (blue). Cells were then mounted and observed by confocal microscopy. The square areas are zoomed-in images and shown in the right panels (merge, zoom). Bar: 10 µm. The imaging data were repeated three times and one set of representative results is shown. (B) The quantification of E-positive cells (A, filled arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (C) The quantification of LC3 punctation cells (A, empty arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (D) The percentages of cells with E-positive and LC3 punctation (A, arrows) are shown. The means ± SD of three independent experiments are shown. ***P<0.005. (E) After 24 h post-infection, the protein levels of LC3, p62, and NS4B from total cell lysates were detected by Western blotting. β-actin served as internal control. NC: negative control (nutrient-rich medium); PC: positive control (starvation; Hank's balanced salt solution). (F and G) KU812 cells were pre-treated with or without 5 mM 3-MA for 1 h before incubation with medium alone (Mock), DENV alone, or DENV with sub-neutralizing dengue patient sera. 3-MA was maintained in the medium during DENV infection. After 24 h post-infection, the expression of DENV E protein (F) and NS4B protein (G) was detected by flow cytometry. The means ± SD of three independent experiments are shown. **P<0.01, ***P<0.005.