Ur. A 40-fold excess of unlabeled HDL was added to each forth information point. Media have been recovered and cell monolayers were washed twice with cold Tris HCl (pH = 7.four), 0.9 NaCl and 0.2 BSA and twice with no BSA. Cells were lyzed with 0.1 M NaOH. Radioactivity was determined using a c-counter for 125I-HDL or a b-counter for 125 3 I/ H-CE-HDL. Precise cell association was calculated by subtracting the quantity of radioactivity detected using a 40-fold excess from total activity measured. Cell protein was quantitated making use of the Bradford Approach (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 having a 100-fold excess of unlabelled HDL in media containing two mg/ml fafBSA and 10 mM Hepes at 4uC for 2 hours.H-Glu-OtBu web For HDL degradation evaluation, media were collected immediately after the incubation with 125I-HDL and proteins had been precipitated working with 50 TCA. The supernatant was extracted with chloroform, oxidized with 30 hydrogen peroxide and counted to identify the volume of acid-soluble material formed by the cells [19].Uptake experiments with fluorescently labeled lipoproteins and transferrinCells 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 have been 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 Minimize HDL EndocytosisFigure 5. Bile acids as well as a non-steroidal FXR agonist minimize HDL endocytosis.1279032-69-7 Purity (a) HepG2 cells had been treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours.PMID:25558565 Gene expression was analyzed by qRT-PCR and expression levels have been normalized to GAPDH expression (n = 2). The boost in SHP mRNA indicates FXR activation. (b) HepG2 cells were incubated with ten mM GW4064 or one hundred mM CDCA in media containing lpds for 24 hours. Cells had been then incubated with 50 mg/ml HDL-Alexa488 for 1 hour. Cells had been fixed, counterstained with DAPI and imaged. Green: HDL; blue: nucleus; bar = ten mm. (c) Quantification of fluorescence intensities of (b). (d) HepG2 cells have been incubated with ten mM GW4064 or one hundred mM CDCA in media containing lpds for 24 hours. Cells had been 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 = three). doi:10.1371/journal.pone.0102026.gCytotoxicityCytotoxicity was analyzed by measuring release of lactate dehydrogenase (LDH) into the media. Cell culture supernatants have been assayed for LDH activity by addition of pyruvate and NADH (each 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 under physiological circumstances [10]. HepG2 cells were seeded in 24-well plates on day 0 and incubated with media containing 10 lpds on day two. On day three, cells have been re-fed with media containing two mg/ml faf-BSA inside the presence or absence of 1 mM taurocholate. The exchange of cell culture media triggers ATP release [11]. Aliquots from the supernatant we.
