Fig. 2

sHDLs require ABCA1 to remove accumulated cholesterol from Niemann–Pick C fibroblasts. a–f Primary fibroblasts homozygous for NPC1 I1061T were treated with various sHDL formulations. a, b Accumulation of unesterified cholesterol was visualized by filipin staining (a) following 48-h treatment with increasing doses (representative images of 0.75 mg/ml) of vehicle (Veh), 5A peptide, 5A-POPC, 5A-SM, and 5A-DMPC (quantified below) or (b) with 0.75 mg/ml sHDL at various time points. c Effects of 48-h treatment with sHDL (0.75 mg/ml), 5A peptide, or vehicle (Veh) on total cellular cholesterol were measured using the Amplex Red assay. d The ratio of 5A or 22A peptide to sphingomyelin (SM) was altered during synthesis and the effect of peptide: SM ratio on cholesterol removal was determined by filipin staining (48-h treatment). e Cells were treated for two consecutive days with the following siRNAs: non-targeting (NT), ABCA1, or SR-B1, and concurrently treated with vehicle (Veh) or 5A-SM. Cholesterol storage was determined by filipin staining. f Cells were treated with cyclodextrin (Cyclo), 5A-SM, or 5A-SM preloaded with increasing amount of cholesterol content (5–20% total lipid weight), or human HDL (HuHDL). Cholesterol storage was assessed by filipin staining 48 h after treatment. Data are mean ± s.e.m. from (a, b, e) three, (c) five, (d) 5–8, or (f) 4–6 independent experiments. n.s., not significant, *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001 by a, b two-way ANOVA with Bonferroni post hoc test (F, df = (a) 33.53, df = 4; (b) 32.88, 4), c–f one-way ANOVA with Tukey post hoc test (F, df = (c) 13.98, 4; (d) 6.96, 8; (e) 22.5, 6; (f) 6.94, 5). a Dash lines indicate plasma membrane, scale bar = 20 μm