Fig 1: LAMP-1 is essential for cyclodextrin-mediated cholesterol export from lysosomes. Verification of HeLa cells either overexpressing LAMP-1 (HeLa-LE) or stably carrying shRNA for LAMP-1 (Hela LAMP-1 knockdown or HeLa-kd) by western blot (a). HeLa-LE shows significantly increased levels of LAMP-1, while HeLa-Lkd shows decreased levels of LAMP-1 compared to the HeLa cells transfected with a control plasmid. Control or transfected HeLa cells were treated with U18666A (5 µg/ml) for 48–72 h, and metabolic activity (b) and LDH activity (c) were measured. Data are mean ± S.E.M. and representative of three experiments. None of transfected cell lines showed toxicity after three days of treatment with U18666A. HeLa cells lines were analyzed for cholesterol accumulation by filipin imaging (d). U18666A-induced cholesterol accumulation in control cells; however, LAMP-1 overexpression (HeLa-LE) suppressed U18666A-induced cholesterol accumulation. LAMP-1 knockdown (HeLa-Lkd) did not show any protection against cholesterol accumulation and showed higher accumulation of cholesterol compared to control. Microscope images are a representative of at least three random fields of three experimental replicates. Scale bar = 50 µm
Fig 2: HPßCD or HP?CD treatment induces LAMP-1 expression and causes a change in lysosomal positioning inNPC1 mutant cells. The healthy (wild-type) cells or NPC1-/- cells treated with HPßCD or HP?CD for 72 h were lysed for immunoblotting (a, b) or stained for LAMP-1 (c). a The representative western blot and bar diagram of three experiments show that wild-type or NPC1-/- cells treated with either HPßCD or HP?CD showed significantly higher levels of LAMP-1 expression (p < 0.001). The error bars shows mean ± S.E.M. of fold change calculated by densitometry analysis. b The representative western blot of three experiments shows that neither HPßCD nor HP?CD treatment changed expression of LAMP-2 or LAMP-3 in wild-type or NPC1-/- cells. c Immunostaining micrographs show LAMP-1 (red, a lysosome marker) and DAPI (blue, a nucleus marker) staining. The arrow represents the distribution of LAMP-1 from the center of the nuclei. Data depict that the LAMP-1 protein is mostly confined to the area near the nuclear envelop in control NPC1-/- cells, whereas it is distributed more widely throughout the cytoplasm when cells were treated with either HPßCD or HP?CD. Images are a representative of at least three random fields of three experimental replicates. Scale bar = 100 µm. d The LAMP-1 distribution per cell was quantified by measuring the area of fluorescence. Data are mean ± S.E.M. and representative of three experiments
Fig 3: A hypothetical model of cholesterol egress from lysosome to ER or PM in NPC1 mutant cells. When NPC1 mutant cells are treated with HPßCD or HP?CD, free cholesterol released from LDL is handed-off to LAMP-1 by NPC2 in an NPC2-dependent manner or it directly bind to LAMP-1
Supplier Page from OriGene Technologies for LAMP1 Human shRNA Plasmid Kit (Locus ID 3916)