26 research outputs found

    Bile acids and a non-steroidal FXR agonist reduce HDL endocytosis.

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    <p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours. Gene expression was analyzed by qRT-PCR and expression levels were normalized to GAPDH expression (n = 2). The increase in SHP mRNA indicates FXR activation. (b) HepG2 cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 50 µg/ml HDL-Alexa<sup>488</sup> for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: HDL; blue: nucleus; bar = 10 µm. (c) Quantification of fluorescence intensities of (b). (d) HepG2 cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 20 µg/ml <sup>125</sup>I-HDL for 1 hour. Uptake was determined after displacing cell surface bound HDL by a 100-fold excess at 4°C for 1 hour (n = 3).</p

    GW4064 and CDCA reduce CD36 expression and function.

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    <p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours and gene expression was analyzed by qRT-PCR (n = 3). (b) Cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hrs and protein expression was determined by western blot analysis and results were quantitated by densitometry (n = 3). (c) Fatty-acid uptake was determined after treatment with 10 µM GW4064 or 100 µM CDCA as described in the methods section (n = 3).</p

    GW4064 and CDCA reduce HDL endocytosis independently of SR-BI.

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    <p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours and gene expression was analyzed by qRT-PCR (n = 3). (b) Cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hrs and protein expression was determined by western blot analysis and results were quantitated by densitometry (n = 3). HepG2 cells transfected with scrambled shRNA (c) or SR-BI shRNA (d) were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 20 µg/ml double labeled <sup>125</sup>I/<sup>3</sup>H-CE-HDL for 1 hr. Selective cholesteryl-ester uptake was calculated by subtracting <sup>125</sup>I-HDL uptake from <sup>3</sup>H-CE-HDL uptake (n = 3).</p

    Modification of HDL by taurocholate does not alter endocytosis.

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    <p>(a) HDL was incubated with or without 1 mM taurocholate in media in the absence of cells for 1 hour. HDL size was then analyzed by size exclusion chromatography. HDL incubated with taurocholate is eluted earlier, indicating increased size. (b) HDL-Alexa<sup>488</sup> was incubated with or without 1 mM taurocholate in media in the absence of cells for 1 hour. Free taurocholate was then removed using gel filtration and HepG2 cells were incubated with this modified HDL-Alexa<sup>488</sup> for 1 hour. Cells were fixed, counterstained with DAPI and imaged. (c) Quantification of fluorescence intensities from (b); n = 3. Green: HDL; blue: nucleus; bar = 10 µm.</p

    Taurocholate neither exerts cytotoxic effects, nor inhibits transferrin or LDL endocytosis in HepG2 cells.

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    <p>(a) Cells were incubated with the indicated concentrations of taurocholate for 1 hour. No release of LDH into the cell culture supernatant was detected. 0.1% Triton-X100 was used as a positive control. (b) Cells were incubated with 20 µg/ml transferrin-Alexa<sup>488</sup> (b) or 50 µg/ml LDL-Alexa<sup>568</sup> (c) with or without 1 mM taurocholate at 37°C for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: transferrin; red: LDL; blue: nucleus; bar = 10 µm. Neither transferrin nor LDL uptake were altered. Quantifications of fluorescent signals are depicted next to the images. (d) Cells were incubated with or without 1 mM taurocholate for 1 hour. Cells were fixed, stained with Filipin and imaged. Bar = 10 µm. Representative images of 3 independent experiments are shown.</p

    Bile acids reduce HDL endocytosis.

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    <p>HepG2 (a) and HuH7 (b) cells were incubated with 50 µg/ml HDL-Alexa<sup>488</sup> with or without 1 mM taurocholate at 37°C for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: HDL; blue: nucleus; bar = 10 µm. Representative images of 3 independent experiments are shown. (c) Quantification of fluorescence intensities of (a) and (b). (d) HepG2 cells were incubated in media containing 20 µg/ml <sup>125</sup>I-HDL with or without 1 mM taurocholate at 37°C for 1 hour. Uptake was determined after displacing cell surface bound HDL by a 100-fold excess at 4°C for 1 hour (n = 3). (e) Cells were incubated with 20 µg/ml <sup>125</sup>I-HDL with the indicated concentrations of taurocholate for 1 hour (n = 3). (f) Cells were incubated with 20 µg/ml <sup>125</sup>I-HDL together with different bile acids for 1 hour (n = 3). Of note taurodeoxycholate, deoxycholate and chenodeoxycholate were cytotoxic at 1 mM and were therefore used at 0.5 mM.</p

    Taurocholate reduces HDL endocytosis SR-BI-dependently.

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    <p>(a) HepG2 cells were incubated with or without 1 mM taurocholate and ATP hydrolysis was measured as a decrease in extracellular ATP. One representative experiment out of three independent experiments is shown. (b) SR-BI knockdown efficiency in HepG2 cells transfected with scrambled shRNA and HepG2 cells transfected with SR-BI shRNA (n = 3). Selective lipid uptake analysis using double labeled <sup>125</sup>I/<sup>3</sup>H-CE-HDL in scrambled control (c) or SR-BI knockdown (d) HepG2 cells (n = 3). Selective cholesteryl-ester uptake was calculated by subtracting <sup>125</sup>I-HDL uptake from <sup>3</sup>H-CE-HDL uptake.</p

    Appearance of EPCs after 3 hour of HDL-HRP internalization.

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    <p><b>A</b>) The population of positively stained endosomal vesicles increase and could be observed throughout the cytoplasm. Positive MVBs (MVB) in various sizes and shapes with tubular membranous extensions as well as prominent Golgi stacks (G) are visible. <b>B</b>) This figure shows the appearance of a tubular endosome. <b>C</b>) Arrows point to an endosome alignment forming “strings of pearl-like structures” (arrowheads). <b>D</b>) A large secondary lysosome shows intraluminal HDL-HRP particles. <b>E</b>) Numerous autophagosomes (white thick arrows) with included endosomal vesicles are noted. (Scale bar: A, D, E = 1 µm; B, C = 0.5 µm).</p

    Ultrastructural detection of the internalization of EPCs with HDL-bodipy-cholesteryl oleate between 30 and 240 min.

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    <p><b>A</b>) MVBs at the cell periphery are filled with numerous tightly packed positively stained microvesicles; the membrane invagination are full filled with the reaction products also present on the other side of cell (inset). <b>B</b>), <b>D</b>) The TGN and stacked Golgi cisternae are positively stained. <b>C</b>) Mitochondria (M) and lipid droplet (Li) are labeled. <b>E</b>), <b>F</b>) A close association between labeled lipid droplets and RER could be demonstrated. (Scale bar: A = 1 µm; A inset = 0.5 µm; B, C, D, E, F = 0.25 µm).</p
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