Foxa1 inhibits the expression of genes involved in TG and VLDL synthesis.

<p>Diagrams of TG (<b>A</b>) and VLDL (<b>B</b>) synthesis pathways in the liver. Human hepatocytes (<b>C</b>) and HepG2 cells (<b>D</b>) were infected with Ad-Foxa1 or Ad-Control (12 MOI for human hepatocytes and 24 MOI for HepG2) for 48 h, and the mRNA level of TAG and VLDL genes was measured by real-time Q-RT-PCR and normalized to the expression of the housekeeping PBGD mRNA. Data represent the mean±SD of 4–5 independent experiments. * p<0.05; ** p<0.01; *** p<0.001 in relation to Ad-Control infected cells. <b>GPAT</b>: glycerol-3 phosphate acyltransferase. <b>AGPAT</b>: 1-acylglycerol-3-phosphate-O-acyltransferase. <b>DGAT</b>: diacylglycerol acyltransferase. <b>MTP</b>: microsomal triglyceride transfer protein. <b>ACAT</b>: acyl coenzyme A: cholesterol acyltransferase. <b>APOB</b>: apolipoprotein B.</p

Adenovirus-mediated expression of Foxa1 in human liver cell cultures.

<p>HepG2 cells and cultured human hepatocytes were infected with Ad-Foxa1 or Ad-Control, and Foxa1 or Foxa2 mRNA levels were measured by Q-RT-PCR and normalized to the expression of the housekeeping PBGD mRNA. (<b>A</b>) Time-course analysis of Foxa1 mRNA after transduction of human hepatocytes and HepG2 cells with 5 and 24 MOI of adenoviral vectors, respectively. Dose-response analysis of Foxa1 (<b>B</b>) and Foxa2 (<b>D</b>) in human liver cultured cells after different MOI of adenovirus for 48 h. Data represent the mean±SD of 3 independent experiments. (<b>C</b>) Protein levels of Foxa1 determined by immunoblotting (sc-6553 antibody) in representative samples of human liver cultured cells after 12 (HH) or 24 (HepG2) MOI of adenovirus for 48 h (Ad-C: Ad-Control; Ad-F: Ad-Foxa1; HH: human hepatocytes). Equal loading was verified by Ponceau S staining.</p

Foxa1 up-regulates UCP1 and decreases MMP.

<p>Cultured cells were infected with Ad-Foxa1 or Ad-Control (48 h), the mRNA level of the mitochondrial uncoupling protein 1 (UCP1) was measured by real-time Q-RT-PCR (<b>A</b>), and the Mitochondrial Membrane Potential (MMP) was measured using the TMRM probe in an Olympus ScaR Screening Station (<b>B</b>). Data represent the mean±SD of 4–5 independent experiments. ** p<0.01; *** p<0.001. (<b>C</b>) Representative microphotographs of HepG2 cells and human cultured hepatocytes after 48 h of adenoviral transfection. Cells were stained with TMRM (red) for mitochondrial membrane potential and Hoechst 33342 (blue) for nuclei.</p

Foxa1 is more abundant than Foxa-2 and -3 in human liver, but its expression declines in liver cell cultures.

<p>Foxa-1, -2 and -3 mRNA concentrations were measured in human livers (n = 15), human hepatocyte cultures (48 h of culture, n = 8) and HepG2 cultures (n = 6) by real-time Q-RT-PCR. Foxa1 expression was assessed with two different primer pairs (Foxa1-p1 and Foxa1-p2). Absolute cDNA quantification was performed by interpolation in DNA standard curves with a known input concentration.</p

Anthropometric and analytical parameters of steatotic and non-steatotic liver donors and patients.

1<p>Liver donors showed normal analytical blood test and, apart of the hepatic lipid concentration and the BMI, no major differences were found between steatotic and non-steatotic.</p>2<p>Patients with NAFL (grade 1 or 2) showed a higher BMI, a slight elevation of transaminases and a statistically higher insulin level and HOMA (Homeostasis Model Assessment) index.</p><p>*p<0.05;</p><p>***p<0.001; m, male; f, female; ND, not determined.</p

Foxa1 expression is reduced in human and rat fatty liver.

<p>(<b>A</b>) Liver samples (100 mg) from the Human Liver Bank HLaFe-CIBERehd were homogenized, extracted with methanol-chloroform and evaporated under nitrogen. Total lipids and TG were measured in the lipid residue using colorimetric kits and livers were classified as fatty livers (closed circles) when total lipids were >600 µg/mg prot and TG>500 µg/mg prot (n = 20), or as nonsteatotic livers (open circles) when total lipids were <400 µg/mg prot and TAG<300 µg/mg prot (n = 30). (<b>B</b>) Total RNA was purified from matching Liver Bank samples and the mRNA level of Foxa1 was determined by real-time Q-RT-PCR analysis. (<b>C</b>) Protein extracted with T-PER from representative liver homogenates was analyzed by immunoblotting with a monoclonal antibody against human Foxa1 (sc-101058). (<b>D</b>) Total RNA was also purified from NAFL (n = 17) and cholelithiasis (n = 17) patients' liver biopsies and the mRNA level of Foxa1 was quantified by real-time Q-RT-PCR. (<b>E</b>) Adult male OFA rats were randomized into two experimental groups (n = 6 per group) and fed with either MCD or a standard isocaloric diet for 7 weeks <i>ad libitum</i>. Total RNA was purified and the expression level of Foxa1 determined as explained above. Box plots represent the 25^th, the median and the 75^th percentiles, a cross depicts the mean, whiskers indicate the 95^th and 5^th percentile, and dots represent outliers. GAPDH mRNA was used for normalization.</p

Foxa1 repression in NAFL correlates with higher PKC activity/expression.

<p>(<b>A</b>) HepG2 cells were exposed to the PKC activator PMA (phorbol 12-myristate 13-acetate), and the level of Foxa1 mRNA was measured at different time points. Data represent the mean±SD of 3 independent experiments. (<b>B</b>) Foxa1 protein level in HepG2 cells was determined by immunoblotting (sc-101058 antibody) after 4, 20 and 30 h of exposure to PMA (+) or DMSO (−). (<b>C</b>) HepG2 cells were pretreated with PKC inhibitors, Gö-6976 (200 nM) or Ro-318220 (1 µM), for 1 h, and thereafter exposed to 160 nM PMA. Two hours latter cells were collected and Foxa1 mRNA level was determined by Q-RT-PCR. PBGD mRNA was used for normalization. Data represent the percentage of Foxa1 in treated samples (PMA or PMA plus PKC inhibitors) respect to control samples (DMSO or inhibitors alone). (<b>D–E</b>) Protein kinase C epsilon (PKCε) mRNA level was determined by real-time Q-RT-PCR analysis in the human liver samples described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030014#pone-0030014-g008" target="_blank">Fig. 8B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030014#pone-0030014-g008" target="_blank">8D</a>.</p

Foxa1 decreases FA uptake.

<p>(<b>A</b>) Cultured cells were infected with Ad-Foxa1 or Ad-Control for 48h, and the mRNA level of the plasma membrane fatty acid transporter protein 2 (FATP2) was measured by real-time Q-RT-PCR. (<b>B</b>) Human hepatocytes and HepG2 cells were infected with Ad-Foxa1 for 24h and were then exposed to 0.2 mM FA (oleate: palmitate, 2∶1) for an additional period lasting 16 h. The amount of non-consumed (residual) free FA, expressed as the percentage of the initial amount in the culture medium, were determined with a specific kit. (<b>C</b>) The uptake of a fluorescent FA analog (C1-BODIPY 500/510-C12) was measured in HepG2 cells and human cultured hepatocytes with an Olympus ScanR Screening Station. Data represent the mean±SD of 4–5 independent experiments. * p<0.05; ** p<0.01; *** p<0.001 in relation to Ad-Control infected cells.</p

Foxa1 reduces lipid accumulation and ApoB100 secretion in human liver cells.

<p>(<b>A</b>) Cultured human hepatocytes and HepG2 cells were transduced with Ad-Foxa1 or Ad-Control for 24 h and then exposed to FA (oleate: palmitate, 2∶1). Twenty-four hours later, accumulated lipids were quantified by Nile Red fluorescence. Data represent the mean±SD of 4–5 independent experiments. * p<0.05; ** p<0.01; *** p<0.001. (<b>B</b>) <u>Left panels</u>: Representative microphotographs of living HepG2 cells and human cultured hepatocytes showing cytoplasmic neutral lipids stained with BODIPY 493/503 (green), nuclei stained with Hoechst 33342 (blue) and death cell nuclei stained with propidium iodide (pink). <u>Right panels</u>: Quantification of the BODIPY 493/503 fluorescent signal with the Olympus ScanR Screening Station in HepG2 cells (mean±SD, n = 4) and in a representative human hepatocyte culture. (<b>C</b>) Secreted (culture medium) and intracellular ApoB100 protein was analyzed by immunoblotting at 48h after infection with adenoviral vectors. (M, fresh culture medium; Cont, non-infected controls; Ad-C, Ad-Control; Ad-F, Ad-Foxa1).</p

Clinical and functional characteristics of COPD patients at first hospital admission (Spain, 2004–2006).

<p>Abbreviation<i>: mMRC</i>, Modified Medical Research Council; Number (%) is given, unless otherwise indicated.</p><p>*Chronic cough with phlegm;</p>†<p>Scores range from 0 (no health impairment) to 100 (maximum impairment).</p