Fig. 1.
Tracing cholesterol cargo. A: U2OS-SRAshNPC1 cells were incubated in medium containing acLDL reconstituted with d7-acLDL (red oval with chain) to load the lysosomal compartment (LYS). Lalistat was included during loading to prevent cleavage of labeled esters by LAL and release from the lysosome. d9 oleate (blue chain) was included during loading to provide cells with labeled oleate to detect the reesterification product. B: Following the removal of acLDL and lalistat, cells were incubated with CD. Washout of lalistat enables the hydrolysis of cholesteryl esters at the lysosome. Upon arrival at the ER, d7 cholesterol was reesterified with d9 oleate to form d16 cholesteryl ester, which can be distinguished from loaded d7 cholesteryl ester. Arrival of cholesterol at the PM was detected by sphingomyelinase and cholesterol oxidase treatment, which converts d7 cholesterol to d7 cholestenone. Efflux of lysosomal cholesterol was monitored by the appearance of d7 cholesterol in the media.