1. EPS15R, TASP1, and PRPF3 Are Novel Disease Candidate Genes Targeted by HNF4α Splice Variants in Hepatocellular Carcinomas 
Monika Niehof, Jürgen Borlak 
Gastroenterology 
Volume 134, Issue 4, Pages (April 2008)
DOI: /j.gastro
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2. Figure 1
HNF4α expression (all, P1, P2) in Caco-2 cells; in human HCC cell lines; and in human, rat, and mouse liver. (A) HNF4αall Western blotting of 30 μg Caco-2 cell or rat liver nuclear extract. (B) EMSA with 2.5 μg Caco-2 cell or rat liver nuclear extract, probe: A-site of the HNF1α promoter (HNF1pro). For supershift assays, an antibody directed against HNF4αall was added. (C) HNF4αall gene expression in Caco-2, HepG2, and Hep3b cells. Gene expression was measured by real-time qRT-PCR and determined relative to expression of mitATPase, ie, the housekeeping gene. HNF4αP1 and HNF4αP2 gene expression was determined to that of HNF4αall. (D) HNF4αall, P1, and P2 Western blotting of 30 μg Caco-2, Hep3b, or HepG2 nuclear extract. (E and F) EMSA with 2.5 μg Caco-2, Hep3b, or HepG2 cells (E) or 2.5 μg human, rat, or mouse liver (F) nuclear extracts, probe: HNF1pro. For supershift assays, antibodies directed against HNF4αall, P1, or P2 were added.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
HNF4α DNA binding to ChIP clones. (A) Independent ChIP experiments were performed with cultures of Caco-2 cells and an antibody against HNF4αall (IPP HNF4α) or no antibody (noAB). Following DNA purification, samples were subjected to PCR with primers designed to amplify putative HNF4α-binding sites of clones and their promoters. (B and C) EMSA with Caco-2 (B) or HepG2 (C) cell nuclear extract and 32 P-labeled oligonucleotides to probe for DNA binding to putative HNF4α-binding sites. In supershift assays, antibodies directed against HNF4αall, P1, and P2 were added. (D) Gene expression of TASP1, EPS15R, and PRPF3 in cultures of Caco-2 cells was analyzed by RT-PCR. A linear range of amplification cycles is depicted.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Changes in P1 and P2 promoter-driven HNF4α expression in HCCs of transgenic EGF2B mice. (A) HNF4α gene expression (HNF4αall) in nontransgenic control livers (C57/BL6 mice, n = 3), in tumor-free transgenic tissue (n = 3), and in tumors of transgenic EGF2B mice (n = 7, see also Table 1). HNF4αP1 gene expression was calculated relative to HNF4αP2 gene expression (HNF4αP1/HNF4αP2), and HNF4αP1 (HNF4αP1/HNF4αall) and HNF4αP2 (HNF4αP2/HNF4αall) gene expression was computed relative to HNF4αall gene expression. (B) Western blotting of nuclear extract derived from nontransgenic control livers (C57/BL6 mice), from tumor-free transgenic tissue, and from tumors of transgenic EGF2B mice with antibodies directed against HNF4αall, HNF4αP2, and actin. (C and D) EMSA with 2.5 μg nuclear extract isolated from nontransgenic control livers (C57/BL6 mice), tumor-free transgenic tissue, and tumors of transgenic EGF2B mice, probe: HNF1pro. For supershift assays, antibodies directed against HNF4αP2 and HNF4αall were added. (D) Dried gels were analyzed with a Molecular Imager (Bio-Rad) using the Quantity One software. HNF4αP1 and HNF4αP2 DNA binding was calculated in relation to HNF4all DNA binding. Ratios for HNF4α isoforms in liver nuclear extracts of controls (n = 3) were set to 1, and fold changes were calculated for liver nuclear extracts of nontumorous transgenic tissue (n = 3) and tumors (n = 3) of transgenic EGF2B mice, respectively.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Changes in P1 and P2 promoter-driven HNF4α gene expression in human HCCs (group A). (A) P1 and P2 promoter-driven HNF4α gene expression in human control liver (H, healthy, n = 4) and in human HCCs (T, tumor, n = 6). Gene expression was measured by real-time RT-PCR and computed relative to expression of mitATPase, ie, the housekeeping gene. (B) P1 and P2 promoter-driven HNF4α gene expression were calculated in relation to total HNF4α (HNF4αall) gene expression. (C) TASP1, PRPF3, EPS15R, and EPS15 gene expression in 3 human HCCs were determined in relation to HNF4αP1, HNF4αP2, and HNF4αall gene expression. Ratios found in human control liver (n = 4) were set to 1.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Changes in P1 and P2 promoter-driven HNF4α protein expression in human HCCs (group B). (A) HNF4αall, P1, P2, and actin Western blotting of 30 μg healthy human liver or human HCC nuclear extracts. (B) EMSA with 10 μg healthy human liver or human HHC nuclear extracts, probe: HNF1pro. For supershift assays, antibodies directed against HNF4αall, P1, or P2 were added. (C) EMSA were performed with nuclear extracts from patients 2–4. Dried gels were analyzed as indicated in Figure 3D. The ratio of HNF4αP2/HNF4αall DNA binding is given for individual patients (right column) and as mean values (left column). The nonparametric 2-tailed Mann–Whitney U test was used for comparison. The results were considered significant, and the P value was (D) P1 and P2 promoter-driven HNF4α gene expression in human healthy liver and in human HCC (n = 4 patients) were determined in relation to total HNF4α (HNF4αall) gene expression. Data for individual patients are given. (E) TASP1, PRPF3, EPS15R, and EPS15 gene expression in human healthy liver and in human HCC (n = 4 patients) were measured by real-time qRT-PCR and calculated relative to expression of mitATPase, ie, the housekeeping gene.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Changes in disease candidate protein expression in HCCs of transgenic EGF2B mice, in transgenic AT-myc mice, and in human HCCs. (A) EPS15 and tubulin Western blotting of 100 μg and 10 μg total cellular extract, respectively, isolated from nontransgenic control livers (C57/BL6 mice), from tumors of transgenic EGF2B mice, and from tumors of AT-myc mice (B). EPS15, TASP1, and actin Western blotting of 100 μg, 100 μg, and 10 μg total cellular extract, respectively, isolated from healthy human liver and from human HCC (4 patients, group B). Western blotting of patients 1 and 2 was done with TASP1 antibody kindly provided by James Hsieh,13 whereas Western blotting of patients 3 and 4 was done with TASP1 antibody purchased from BioCat Heidelberg, Germany (Abgent No. AP1330a).
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Immunohistochemistry of HNF4αP2 and EPS15 in human HCCs. Tissue sections of 5 patients (group C) were stained with a polyclonal antibody against HNF4αP2 (A, control tissue; B, HCC) or EPS15 (C, control tissue; D, HCC). Patient identification numbers are given individually. Original magnification, ×400.
Gastroenterology  , DOI: ( /j.gastro )
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