Figure 8: ML-SA1 and TRPML1 overexpression rescue the trafficking defects and reduce cholesterol accumulation in NPC cells.
(a) A fluorescent analogue of lactosylceramide, BODIPY-LacCer, was mainly localized in the Golgi-like structures of WT macrophages after a pulse-chase for 1 h, but accumulated in the LEL-like vesicles of NPC macrophages. ML-SA1 (10 μM) treatment of NPC1−/− macrophages for 12 h resulted in a primary Golgi-like localization for LacCer. Scale bar=10 μm. (b) ML-SA1 reduced the number of LacCer puncta in NPC1−/− macrophages. (c,d) ML-SA1 reduced unesterified cholesterol (filipin) staining in ML1, but not ML1-KK-expressing U18666A-treated WT CHO cells. Differential interference contrast (DIC) images are shown for comparison. Scale bar=10 μm. (e) Unesterified cholesterol (filipin) staining in NPC CHO cells was partially reduced in cells expressing DsRed-aSMase. (f) Average filipin staining (intensity normalized to non-transfected control cells) in DsRed-aSMase-transfected cells. (g) Unesterified cholesterol (filipin) staining in NPC CHO cells was partially reduced in cells expressing ML1-Va-EGFP (ML1Va). (h) Average filipin staining (intensity normalized to non-transfected control cells) in ML1Va-transfected cells. Scale bar=10 μm for panels a, c, e and g. For panels b, d, f and h, the results were averaged for multiple randomly taken micrographs from at least three independent experiments. Data are presented as the mean±s.e.m. Statistical comparisons were made using analysis of variance: *P<0.05.
