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Fig. 1. U18666A treatment induces the accumulation of swollen cholesterol-rich, LAMP-2-positive vacuoles. Untransfected Mv1Lu cells (A-D,I-L) and Mgat5-transfected M9 clones (E-H,M-T) were cultured in regular medium for 6 days (A-H,Q-T) and selected cultures incubated with U18666A for the final 24 hours (I-P). The cells were then fixed and triple labelled with anti-LAMP-2 antibody (A,E,I,M,Q), Nile Red (B,F,J,N,R) and filipin (C,G,K,O,S). Merged images (D,H,L,P,T) show LAMP-2 in green, Nile Red in red, and filipin in blue. In untreated M9 cells, filipin labelling is concentrated at the limiting membrane of the vacuoles (Q-T). In the presence of U18666A, the LAMP-2/Nile Red-positive vacuoles (arrowheads) accumulate cholesterol and are all positive for filipin after 24 hours. Bar, 5 µm.

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