Efflux of cholesterol from LY-A and LY-A/CERT cells.

<p>LY-A cells or LY-A/CERT cells, preincubated in Nutridoma-BO medium for 40 h, were mock-transfected (open bars) or transfected with ABCG4 (filled bars). After 18 h of the transfection, cell lysates (10 µg) were separated by 10% polyacrylamide gel electrophoresis, and ABCG4 was detected with immunoblotting (A). After 6 h of the transfection, the efflux of cholesterol from cells during 12 h in the presence of 0.02% BSA plus 20 µg/ml HDL was analyzed (B). Average values of three experiments are presented with the SD. *<i>P</i><0.05, significantly different from mock-transfected cells.</p

Distribution of ABCA1, ABCG1, and ABCG4 between Brij 96 raft and non-raft domains.

<p>HEK293, HEK/ABCA1, HEK/ABCG1, HEK/ABCG1-KM, or HEK/ABCG4 cells were incubated with buffer containing 1% Brij 96 on ice. The cell lysates were separated by OptiPrep-gradient ultracentrifugation. Ten fractions of each were separated by 5–20% polyacrylamide gel electrophoresis, and ABCA1, ABCG1, ABCG4, caveolin-1, paxillin, or FAK were detected by immunoblotting.</p

Cholesterol extraction by cold MβCD. HEK293, HEK/ABCA1, HEK/ABCG1, HEK/ABCG1-KM, HEK/ABCG4, or HEK/ABCG4-KM cells were incubated with the medium containing 5 mM MβCD on ice for 1 h.

<p>The amount of cholesterol extracted by MβCD from each cell type is presented relative to the amount extracted from HEK293 cells. Average values of 3–9 experiments are presented with the SD. *<i>P</i><0.05; **<i>P</i><0.01, significantly different from HEK293 cells.</p

Aβ secretion from SH-SY5Y cells.

<p>SH-SY5Y cells were transfected with siRNA against ABCG1, ABCG4, or scrambled siRNA (control), and allowed to differentiate for 3 days. Cells were incubated in DMEM containing 0.02% BSA, 5 μM TO901317 and 5 μM 9-<i>cis</i> retinoic acid for 16 h. Media were collected and Aβ40 (A) and Aβ42 (B) levels were measured using an enzyme-linked immunosorbent assay. Values from three experiments were normalized based on total cellular protein, and means are represented with the SD. ** <i>P</i> < 0.01 compared with control cells.</p

Distribution of ABCA1, ABCG1, and ABCG4 between Triton X-100 raft and non-raft domains.

<p>HEK293, HEK/ABCA1, HEK/ABCG1, HEK/ABCG1-KM, or HEK/ABCG4 cells (A) or SH-SY5Y cells (B) treated with TO901317 and 9-cis retinoic acid for 24 h were incubated with buffer containing 1% Triton X-100 on ice. The cell lysates were separated by OptiPrep-gradient ultracentrifugation. Ten fractions of each were separated by 5–20% polyacrylamide gel electrophoresis, and ABCA1, ABCG1, ABCG4, caveolin-1, paxillin, FAK, or flotillin was detected by immunoblotting.</p

Cholera toxin binding to GM1.

<p>(A) HEK293 cells transiently expressing ABCG1-GFP, ABCG1-KM-GFP, ABCG4-GFP, or ABCG4-KM-GFP were incubated with Alexa555-conjugated cholera toxin on ice and fixed with 4% paraformaldehyde. (B) The intensities of cholera toxin (CTB) fluorescence per cell expressing ABCG1, ABCG1-KM, ABCG4, or ABCG4-KM were calculated using ImageJ software and are showed relative to the signal obtained from a control cell that did not express ABCG proteins. *<i>P</i><0.05, significantly different from control cells.</p

Colocalization of ABCG1 with flotillin-1.

<p>HEK293 cells transiently expressing ABCG1, ABCG1-KM, ABCG4, or ABCG4-KM plus flotillin-GFP were permeabilized with Triton X-100 (ABCG1) or methanol (ABCG4) and reacted with anti-ABCG1 or -ABCG4 antibodies and analyzed by confocal microscopy. Line scans analyzed by ImageJ software indicate the fluorescence intensities of flotillin-1 (green), ABCG1 (red), and ABCG4 (red).</p

ABCG1 and ABCG4 increased cellular and surface levels of APP.

<p>(A) HEK/APPsw cells were transiently transfected with ABCG4, ABCG4-KM, or ABCG1, or mock-transfected. Twenty-four hours after transfection, cells were collected and cellular APP was detected by immunoblotting. Vinculin was used as a loading control. (B) The amounts of mature APP and immature APP detected by immunoblotting were analyzed. The data represent the expression levels of APP normalized by vinculin relative to that in mock-transfected cells. Values are represented with the SD. * <i>P</i> < 0.05, significantly different from mock-transfected cells. (C) HEK/APPsw cells, transiently transfected with ABCG1 or ABCG4, or mock-tansfected, were treated with sulfo-NHS-biotin followed by precipitation of biotinylated surface proteins using streptavidin agarose beads. Cell surface APP was detected by immunoblotting. All experiments were carried out in triplicate. (D) The amounts of cell surface APP detected by immunoblotting were analyzed. The data represent the expression levels of surface APP normalized by total APP proteins relative to that in mock-transfected cells. Values are represented with the SD. ** <i>P</i> < 0.01, significantly different from mock-transfected cells.</p

Suppression of ABCG1 and ABCG4 by siRNA.

<p>(A) SH-SY5Y cells were transfected with siRNA against ABCG1, ABCG4, or scrambled siRNA (control), and allowed to differentiate for 3 days. After 16-h incubation with or without TO901317 (TO) and 9-<i>cis</i> retinoic acid (RA), cells were collected and ABCG1 was detected by immunoblotting. (B) The amount of ABCG1 detected by immunoblotting was analyzed. The data represent the expression levels of ABCG1 normalized by vinculin relative to those in control cells incubated with TO and RA. Values are represented with the SD. (C) Total RNA was extracted from differentiated SH-SY5Y cells. Quantitative RT-PCR was performed and the ABCG4 mRNA expression level was normalized to 18S rRNA. Relative expression levels of ABCG4 mRNA in cells transfected with siRNA are represented against those in control cells treated with TO and RA. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with control cells incubated with TO and RA. All experiments were carried out in triplicate.</p

ABCG1 and ABCG4 suppressed Aβ secretion.

<p>HEK/APPsw cells were transiently transfected with ABCG4, ABCG4-KM, or ABCG1, or mock-transfected. Twenty-four hours after transfection, cells were incubated in DMEM containing 0.02% BSA (open bars) or 0.02% BSA and 20 μg/ml HDL (filled bars) for 24 h. Media were collected, and Aβ40 (A) and Aβ42 (B) levels were measured using an enzyme-linked immunosorbent assay. Values from three experiments were normalized based on total cellular protein, and means are represented with the SD. * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with mock-transfected cells.</p