Nt of radioactivity detected using a 40-fold excess from total activity measured. Cell protein was quantitated making use of the Bradford Process (Biorad, Vienna, Austria) and HDL uptake was expressed as ng HDL per mg cell protein. Selective cholesteryl ester uptake was calculated by subtracting 125I-HDL uptake from 3H-CE-HDL uptake. To distinguish in between HDL binding and uptake, cell surfacebound HDL was displaced with a 100-fold excess of unlabelled HDL in media containing 2 mg/ml fafBSA and 10 mM Hepes at 4uC for two hours. For HDL degradation evaluation, media had been collected after the incubation with 125I-HDL and proteins were precipitated working with 50 TCA. The supernatant was extracted with chloroform, oxidized with 30 hydrogen peroxide and counted to establish the amount of acid-soluble material formed by the cells [19].Uptake experiments with fluorescently labeled lipoproteins and transferrinCells Androgen Receptor Inhibitor supplier seeded on cover-slides were incubated with 50 mg/ml HDL-Alexa488, LDL-Alexa568 or 20 mg/ml transferrin-Alexa488 diluted in MEM containing two mg/ml faf-BSA at 37uC for 1 hour. Cells were washed and fixed in 4 formaldehyde in PBS at 4uC for 30 minutes. Samples had been counterstained with DAPI, washed, mounted and visualized with an Axiovert microscope (Zeiss, Jena, Germany).PLOS 1 | plosone.orgBile Acids Cut down HDL EndocytosisFigure 5. Bile acids and a Adiponectin Receptor Agonist Compound non-steroidal FXR agonist reduce HDL endocytosis. (a) HepG2 cells were treated with all 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 have been normalized to GAPDH expression (n = two). The boost in SHP mRNA indicates FXR activation. (b) HepG2 cells have been incubated with ten mM GW4064 or 100 mM CDCA in media containing lpds for 24 hours. Cells were then incubated with 50 mg/ml HDL-Alexa488 for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: HDL; blue: nucleus; bar = ten mm. (c) Quantification of fluorescence intensities of (b). (d) HepG2 cells had been incubated with ten mM GW4064 or one hundred mM CDCA in media containing lpds for 24 hours. Cells were then incubated with 20 mg/ml 125I-HDL for 1 hour. Uptake was determined just after displacing cell surface bound HDL by a 100-fold excess at 4uC for 1 hour (n = 3). doi:10.1371/journal.pone.0102026.gCytotoxicityCytotoxicity was analyzed by measuring release of lactate dehydrogenase (LDH) in to the media. Cell culture supernatants were assayed for LDH activity by addition of pyruvate and NADH (both from Sigma; final concentration: 1 mM and 0.2 mM, respectively). Turnover of NADH was measured photometrically at 340 nm.Extracellular ATP hydrolysisATP is secreted by hepatic cells beneath physiological conditions [10]. HepG2 cells had been seeded in 24-well plates on day 0 and incubated with media containing ten lpds on day 2. On day 3, cells had been re-fed with media containing 2 mg/ml faf-BSA within the presence or absence of 1 mM taurocholate. The exchange of cell culture media triggers ATP release [11]. Aliquots of the supernatant had been collected just after 10, 30 and 60 minutes and ATP hydrolysis was measured as a lower in extracellular ATP by luminescence using the ATP-lite kit (Perkin Elmer).Filipin StainingCells were seeded on cover-slips and right after bile acid treatment they had been fixed with 4 formaldehyde in PBS at 4uC for 30 minutes. Samples had been stained with 50 mg/ml Filipin III (Sigma) diluted in PBS containing 10 lpds at RT for.