Figure 3

VLP entry is dependent on fluidity of cholesterol but is not involved with caveolae/raft-dependent endocytosis. A Depletion of cholesterol from the plasma membrane inhibited RBS entry. SB cells were pretreated with MβCD at the indicated doses for 1 h prior to incubation with RBS. VLP entry assays, IFA, fluorescent microscopy and ELISA were performed. B Cholesterol replenishment partially recovered CGV entry. High concentration (5 mM) MβCD-treated cells were incubated without or with exogenous cholesterol for 1 h followed by CGV entry assay. After fixation, fluorescent microscopy was performed directly to determine the CGV location. ELISA was also performed to show the entry efficiencies in different treatments. C, D Block of the tyrosine kinase signal by genistein and PI3/4 K activity by wortmannin did not inhibit RBS entry. Inhibitors of genistein (C) and wortmannin (D) at indicated concentrations were used to treat SB cells followed by RBS entry. For A, C, and D, RBS (green) in cells were visualized by IFA and cell nuclei (blue) were stained by DAPI. The entry efficiency was examined by ELISA and the data shown are the mean ± SD of the results from three independent experiments. E RBS entry is dependent on cholesterol but not on sphingolipids. B-CTB and RBS were simultaneously incubated with SB cells to perform entry assay for 4 h and their subcellular localization were determined by IFA using anti-biotin (α-biotin, green) and anti-VLP (α-VLP, red) as primary antibody. The outline of the cell was indicated by the white line. The merge panel displayed that most of B-CTB and VLP were localized in different subcellular locations. F CGV entry is dependent on fluidity of cholesterol. CGV entry assays were performed for 2 h with different additives as indicated above the panels. After being washed and fixed, cells were observed by confocal microscopy. The bars indicate 25 μm. *, p < 0.05, **, p < 0.01.