Fig. 2.
Human macrophages incubated with SMase-LDL generate lysosomal ceroid.
HMDM cells were incubated without LDL (A), with native LDL (B) or SMase-LDL (C) (200 μg protein/ml) in 45% RPMI medium and 45% buffer (10 mM MgCl2, 5 mM HEPES and 150 mM NaCl, pH 7.4) with 10% (v/v) human serum for 24 h. The cells were then cultured for a further 7 days in RPMI with 10% (v/v) LPDS. The cells were stained for ceroid (A, B and C). Scale bar: 30 μm. Colocalisation of ceroid and lysosomes was observed by confocal microscopy for cells incubated with SMase-LDL. (D) Lysosomes were labelled with an anti-LAMP2 antibody, (E) ceroid was stained with LipidTOX™ Red and (F) an overlaid image is shown. Scale bar: 30 μm. HMDM were incubated with SMase-LDL (200 μg protein/ml) for 24 h. They were then incubated for a further 7 days in the absence of lipoproteins in RPMI with 10% (v/v) LPDS, but in the presence of BHT (10 μM), amifostine (100 μM) or WR-1065 (100 μM). Cells were also incubated without LDL or antioxidants. The ceroid levels in the cells were quantified using ImageJ to show inhibition by BHT, amifostine and WR-1065. Mean ± SEM of 3 independent experiments. *p < 0.001, ANOVA and post-hoc Dunnett's test compared to SMase-LDL with no antioxidant.