Journal: The FASEB Journal

Article Title: Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

doi: 10.1096/fj.12-216564

Figure Lengend Snippet: Dose-response effect of mifepristone treatment on ABCA1-mediated cholesterol efflux to apoAI and ABCA1 expression. A ) Mifepristone treatment concentrations and percentages of total cell [ 3 H]cholesterol released to apoAI in ABCA1-mediated fashion correlated linearly ( r 2 =0.73) at the mifepristone doses 0 through 1 nM. To derive ABCA1-mediated cholesterol efflux to apoAI, the average of percentages of [ 3 H]cholesterol efflux in ABCA1-expressing cells in the absence of apoAI was subtracted from individual percentages of [ 3 H]cholesterol efflux in ABCA1-expressing cells in the presence of apoAI at 10 μg/ml. Solid line, best nonlinear fit; dotted line, best fit linear regression for the mifepristone concentrations 0 through 1 nM; error bars = sd , n = 3. B ) ABCA1 expression at the indicated mifepristone treatment concentrations.

Article Snippet: Aliquots of the concentrated cell medium and the isolated larger and smaller nascent HDL particles were normalized to the same amount of cell medium or the same apoAI concentration and resolved on 4–20% Tris-glycine native PAGE (at 90 V for 17.3 h) and 3–12% Bis-Tris blue native PAGE gels (Invitrogen/Life Technologies, Grand Island, NY, USA), blotted, and probed with a goat polyclonal anti-apoAI antibody (NB400-147; Novus Biological, Littleton, CO, USA).

Techniques: Expressing

Journal: The FASEB Journal

Article Title: Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

doi: 10.1096/fj.12-216564

Figure Lengend Snippet: Effects of ABCA1 expression levels on nascent HDL particle heterogeneity. A ) Gel-filtration elution profiles (top and middle panels) of nascent HDL particles formed with incrementally increasing ABCA1 expression. [ 3 H]Choline and [ 14 C]cholesterol-labeled BHK-ABCA1 cells were treated with the indicated concentrations of mifepristone and exposed to 20 μg/ml of apoAI. Bottom panel shows [ 14 C]cholesterol/[ 3 H]choline-phospholipid ratio curves derived from data in the top and middle panels. Results are representative of 4 independent experiments. B ) Cpm values in fractions within the larger particle peaks (8.6–14.7 nm) were added and divided by the sum of cpm values in the peaks for the smaller particles (5.7–8.6 nm) to derive the large/small particle ratios for each isotope. These ratios varied by around ±10% for the same conditioned medium that was split into aliquots that were analyzed on a gel column one after the other and were significantly different from experiment to experiment, likely due to substantial differences in cell density at the time of apoAI addition (see ). C , D ) Correlation between the large/small particle ratios and the total HDL [ 3 H]choline-phospholipid and [ 14 C]cholesterol. Values of [ 3 H]choline-phospholipid and [ 14 C]cholesterol large/small particle ratios from panel A and from another independent experiment were plotted as a function of the total [ 3 H]choline-phospholipid ( C ) and total [ 14 C]cholesterol ( D ) in the nascent HDL elution fractions.

Article Snippet: Aliquots of the concentrated cell medium and the isolated larger and smaller nascent HDL particles were normalized to the same amount of cell medium or the same apoAI concentration and resolved on 4–20% Tris-glycine native PAGE (at 90 V for 17.3 h) and 3–12% Bis-Tris blue native PAGE gels (Invitrogen/Life Technologies, Grand Island, NY, USA), blotted, and probed with a goat polyclonal anti-apoAI antibody (NB400-147; Novus Biological, Littleton, CO, USA).

Techniques: Expressing, Filtration, Labeling, Derivative Assay

Journal: The FASEB Journal

Article Title: Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

doi: 10.1096/fj.12-216564

Figure Lengend Snippet: HDL particle heterogeneity as a function of apoAI availability. A ) [ 3 H]Choline-phospholipid (left panel) and [ 3 H]cholesterol (right panel) gel-filtration profiles of nascent HDL particles generated by BHK-ABCA1 cells expressing ABCA1 at the maximum level with the indicated amounts of apoAI. Unlike in , in this case cells were labeled with only one of the radiochemicals per experiment. These profiles are representative of 4 independent experiments, 2 with each isotope. B ) [ 3 H]Choline-phospholipid and [ 14 C]cholesterol large/small particle ratios calculated from the gel-filtration profiles shown in panel A increased with lower availabilities of apoAI and hence higher cell lipid:apoAI ratios.

Article Snippet: Aliquots of the concentrated cell medium and the isolated larger and smaller nascent HDL particles were normalized to the same amount of cell medium or the same apoAI concentration and resolved on 4–20% Tris-glycine native PAGE (at 90 V for 17.3 h) and 3–12% Bis-Tris blue native PAGE gels (Invitrogen/Life Technologies, Grand Island, NY, USA), blotted, and probed with a goat polyclonal anti-apoAI antibody (NB400-147; Novus Biological, Littleton, CO, USA).

Techniques: Filtration, Generated, Expressing, Labeling

Journal: The FASEB Journal

Article Title: Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

doi: 10.1096/fj.12-216564

Figure Lengend Snippet: Native PAGE analysis of nascent HDL generated at different available cell lipid:apoAI ratios. A ) Gel-filtration profile of nascent HDL particles that were produced by BHK-ABCA1 cells at the maximum ABCA1 expression and 10 μg/ml apoAI. B ) Elution fractions containing the larger and smaller particles (shaded portions of the profile) were combined, concentrated, and further resolved on 4–20% Tris-glycine native and 3–12% Bis-Tris blue native PAGE gels. C , D ) Tris-glycine native and Bis-Tris blue native gels of nascent HDL particles generated in BHK-ABCA1 cells at the indicated available cell lipid:apoAI ratios. When loaded at the same volume of medium, the lanes with particles assembled at the low and medium available cell lipid:apoAI ratios have ∼7 times more apoAI than the lane with HDL that formed at the high value of the ratio. Representative gels from 3 independent experiments are shown.

Article Snippet: Aliquots of the concentrated cell medium and the isolated larger and smaller nascent HDL particles were normalized to the same amount of cell medium or the same apoAI concentration and resolved on 4–20% Tris-glycine native PAGE (at 90 V for 17.3 h) and 3–12% Bis-Tris blue native PAGE gels (Invitrogen/Life Technologies, Grand Island, NY, USA), blotted, and probed with a goat polyclonal anti-apoAI antibody (NB400-147; Novus Biological, Littleton, CO, USA).

Techniques: Clear Native PAGE, Generated, Filtration, Produced, Expressing, Blue Native PAGE

Journal: The FASEB Journal

Article Title: Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

doi: 10.1096/fj.12-216564

Figure Lengend Snippet: Cross-linking and lipid mass analysis of nascent HDL particles formed at different available cell lipid:apoAI ratios. A ) ApoAI was cross-linked at an undiluted and a 10×-diluted concentration of nascent HDL particles in order to distinguish between inter- and intraparticle cross-linking products . The former products would diminish at the lower particle concentration. The absence of notable differences in relative band intensities between the right and left panels indicates that all products were a result of intraparticle cross-linking. Certain apoAI cross-link dimers migrate on SDS-PAGE gels in the size range of apoAI trimers ( , ). This likely accounts for the appearance of the apoAI trimer band among cross-linking products of the smaller particles (6.8–8.3 nm), which lack requisite size to accommodate >2 molecules of apoAI . The AI ladder lanes contain cross-linking products of self-associated lipid-free apoAI . The number of apoAI molecules in each cross-linking product is indicated on the right. Representative gels from 2 independent experiments are shown. B ) Choline-phospholipid:apoAI and cholesterol:apoAI molar ratios for the larger particle species of nascent HDL formed at the indicated cell lipid:apoAI ratios in BHK-ABCA1 cells. Error bars = sd for 3 independent experiments.

Article Snippet: Aliquots of the concentrated cell medium and the isolated larger and smaller nascent HDL particles were normalized to the same amount of cell medium or the same apoAI concentration and resolved on 4–20% Tris-glycine native PAGE (at 90 V for 17.3 h) and 3–12% Bis-Tris blue native PAGE gels (Invitrogen/Life Technologies, Grand Island, NY, USA), blotted, and probed with a goat polyclonal anti-apoAI antibody (NB400-147; Novus Biological, Littleton, CO, USA).

Techniques: Concentration Assay, SDS Page

Journal: The FASEB Journal

Article Title: Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

doi: 10.1096/fj.12-216564

Figure Lengend Snippet: ABCA1 mutagenesis and size heterogeneity of nascent HDL particles. BHK cells expressing wild-type ABCA1, ABCA1(W590S), or ABCA1(C1477R) were plated at the same density, labeled with [ 3 H]cholesterol, treated with 10 nM mifepristone, and exposed to 10 μg/ml of human apoAI to generate nascent HDL. The decreased size of gel-filtration elution peaks indicates that W590S and especially C1477R mutations dramatically reduced the ability of ABCA1 to facilitate nascent HDL assembly. As nascent HDL production diminished, so did the size of the larger particles (>8.9 nm) and the [ 3 H]cholesterol large/small particle ratio (inset).

Article Snippet: Aliquots of the concentrated cell medium and the isolated larger and smaller nascent HDL particles were normalized to the same amount of cell medium or the same apoAI concentration and resolved on 4–20% Tris-glycine native PAGE (at 90 V for 17.3 h) and 3–12% Bis-Tris blue native PAGE gels (Invitrogen/Life Technologies, Grand Island, NY, USA), blotted, and probed with a goat polyclonal anti-apoAI antibody (NB400-147; Novus Biological, Littleton, CO, USA).

Techniques: Mutagenesis, Expressing, Labeling, Filtration

Journal: The FASEB Journal

Article Title: Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

doi: 10.1096/fj.12-216564

Figure Lengend Snippet: Available cell lipid:apoAI ratio affects heterogeneity of nascent HDL particles produced by RAW 264.7 cells. A ) Gel-filtration profiles of nascent HDL particles generated by RAW 264.7 cells at the indicated concentrations of cAMP and human apoAI. B ) [ 3 H]Choline-phospholipid and [ 14 C]cholesterol large/small particle ratios calculated from the gel-filtration profiles shown in panel A . Representative results from 2 independent experiments are shown.

Article Snippet: Aliquots of the concentrated cell medium and the isolated larger and smaller nascent HDL particles were normalized to the same amount of cell medium or the same apoAI concentration and resolved on 4–20% Tris-glycine native PAGE (at 90 V for 17.3 h) and 3–12% Bis-Tris blue native PAGE gels (Invitrogen/Life Technologies, Grand Island, NY, USA), blotted, and probed with a goat polyclonal anti-apoAI antibody (NB400-147; Novus Biological, Littleton, CO, USA).

Techniques: Produced, Filtration, Generated

Journal: The FASEB Journal

Article Title: Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

doi: 10.1096/fj.12-216564

Figure Lengend Snippet: Heterogeneity of nascent HDL as a function of the available cell lipid:apoAI ratio in HepG2 cells. A ) Gel-filtration profiles of nascent HDL particles synthesized by HepG2 cells treated with the indicated concentrations of T0901317, an LXR agonist, in the presence of human apoAI. B ) [ 3 H]Choline-phospholipid and [ 14 C]cholesterol large/small particle ratios calculated from the gel-filtration profiles shown in panel A .

Article Snippet: Aliquots of the concentrated cell medium and the isolated larger and smaller nascent HDL particles were normalized to the same amount of cell medium or the same apoAI concentration and resolved on 4–20% Tris-glycine native PAGE (at 90 V for 17.3 h) and 3–12% Bis-Tris blue native PAGE gels (Invitrogen/Life Technologies, Grand Island, NY, USA), blotted, and probed with a goat polyclonal anti-apoAI antibody (NB400-147; Novus Biological, Littleton, CO, USA).

Techniques: Filtration, Synthesized

Journal: The FASEB Journal

Article Title: Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

doi: 10.1096/fj.12-216564

Figure Lengend Snippet: Heterogeneity of nascent HDL particles generated with mouse apoAI reflects the available cell lipid:apoAI ratio. A ) Gel-filtration profiles of nascent HDL particles produced in BHK-ABCA1 cells with mouse apoAI at the indicated mifepristone and apoAI concentrations. B ) [ 3 H]Choline-phospholipid and [ 14 C]cholesterol large/small particle ratios calculated from the gel-filtration profiles shown in panel A .

Article Snippet: Aliquots of the concentrated cell medium and the isolated larger and smaller nascent HDL particles were normalized to the same amount of cell medium or the same apoAI concentration and resolved on 4–20% Tris-glycine native PAGE (at 90 V for 17.3 h) and 3–12% Bis-Tris blue native PAGE gels (Invitrogen/Life Technologies, Grand Island, NY, USA), blotted, and probed with a goat polyclonal anti-apoAI antibody (NB400-147; Novus Biological, Littleton, CO, USA).

Techniques: Generated, Filtration, Produced