FIG 7.

IFITM3 does not affect cholesterol levels in cells or in purified virion particles. (A and B) HEK293T cells were analyzed by confocal fluorescence microscopy following staining of intracellular lipid droplets by Nile Red (0.25 μg/ml). Control cells were treated with either methyl-β-cyclodextrin (1 mM) or oleic acid (100 μM), well known to either deplete or increase the intracellular levels of cholesterol. Representative pictures are presented in panel A, while the number of lipid droplets per cell is presented in panel B for over 75 cells analyzed under each condition. (C) Same as panel A, but the accumulation of Nile Red was quantified by flow cytometry. (D) Virions produced in the presence or absence of WT IFITM3 were produced and purified as described above. In this case, viral preparations were normalized by their membrane content upon R18 labeling. Cholesterol levels were measured according to the cholesterol/cholesteryl ester quantification kit (Abcam). The graph presents averages and SEM of data from three independent experiments.