Fig. 6.

24HC induces cholesterol accumulation in multivesicular bodies or late endosomes. A. N2a cells were treated with ETOH, 24HC (5 μM), or 25HC (5 μM) for 24 h, and then fixed and stained with filipin (cyan). Images were acquired by confocal microscopy. DIC: Differential interference contrast. Scale bar, 10 μm. B. N2a cells were treated with ETOH, 24HC (5 μM), or 25HC (5 μM) for 24 h, then fixed and stained with filipin. The fluorescence signals of filipin were acquired under 360 nm excitation light, and the mean fluorescence intensities of filipin were compared (n = 3). C. N2a cells were treated with ETOH, 24HC (5 μM), or 25HC (5 μM) for 24 h, then fixed and stained with Nile red. The cells were then analyzed for PE fluorescence intensity by flow cytometry (n = 3). D. N2a cells were treated with ETOH, 24HC (5 μM), or 25HC (5 μM) for 24 h and then fixed and stained with filipin (cyan) and Rab7 (red). Cells were analyzed for the co-localization between filipin and Rab7. White boxes indicate the magnified sections of the images. Scale bar, 10 μm. E. N2a cells were pretreated with ETOH or 24HC (5 μM) for 12 h, and then MβCD (2 mM) was added. After 1 h incubation, the cells were infected with RABV (MOI = 10) for 3 h. Cells were analyzed for the co-localization between RABV nucleoprotein (RABV-N) and Rab7. White boxes indicate the magnified sections of the images. Scale bar, 10 μm. Statistical analysis of comparisons between groups was performed by Student's t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Bar graph shows mean ± SD. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)