β-catenin knockdown decreases <i>RHBG</i> expression in SW480 colon cancer cells.

<p>A) Representative results of immunofluorescence of β-catenin localization in SW480 cells using β-catenin antibody. Nuclei were stained with DAPI. B-G) SW480 cells were reverse transfected with β-catenin or control (scramble) siRNAs. 72 hours after transfection, expression levels of <i>β-catenin</i> mRNA (B), β-catenin protein (C), <i>RHBG</i> mRNA (D), <i>Axin2</i> mRNA (E), <i>Cyclin D1</i> mRNA (F) and <i>RHCG</i> mRNA (G) were determined.</p

Activation of β-catenin signaling increases <i>RHBG</i> expression.

<p>A) HEK293T cells were treated with LiCL (20mM) for 24 hours. β-catenin localization was determined by immunofluorescence using β-catenin antibody. Nuclei were stained with DAPI. B) HEK293T cells were treated with LiCL (10 or 20mM) for 24 hours. β-catenin protein level was determined in total cell lysates by immunoblotting using β-catenin antibody. C) Same culture conditions as in B. The <i>RHBG</i> mRNA level was determined by qRT-PCR.</p

<i>RHBG</i> and <i>GLUL</i> are highly expressed in HepG2 hepatoma cells.

<p>A) The level of mRNA expression of <i>RHBG</i> in HEK293T, HepG2 and Hep3B cells was determined by qRT-PCR and normalized to β-actin. B) The mRNA expression of <i>GLUL</i> in HepG2 and Hep3B was determined by qRT-PCR. C) Western Blot analysis of GS protein from total cell lysates of HepG2 and Hep3B cells. D) The level of mRNA expression of <i>RHCG</i> in HEK293T, HepG2 and Hep3B cells was determined by qRT-PCR and normalized to β-actin. E) Western Blot analysis of β-catenin protein from total cell lysates of HEK293T, HepG2 and Hep3B cells.</p

β-catenin knockdown decreases <i>RHBG</i> expression in HepG2 hepatoma cells.

<p>A-F) HepG2 cells were reverse transfected with β-catenin or control (scramble) siRNAs. 72 hours after transfection, expression levels of <i>β-catenin</i> mRNA (A), β-catenin protein (B), <i>RHBG</i> mRNA (C), <i>Axin2</i> mRNA (D), <i>Cyclin D1</i> mRNA (E) and <i>RHCG</i> mRNA (F) were determined.</p

<i>RHBG</i> expression is dependent on TCF4.

<p>A-B) HepG2 cells were treated with PKF118-310 (0,2 or 0,4 μM) for 24 hours. The <i>RHBG</i> (A) and <i>GLUL</i> (B) mRNA levels were determined by qRT-PCR. C-G) HepG2 cells were reverse transfected with TCF4 or control (scramble) siRNAs. 72 hours after transfection, levels of <i>TCF4</i> mRNA (C), TCF4 protein (D), <i>RHBG</i> mRNA (E), <i>Axin2</i> mRNA (F), and <i>Cylcin D1</i> mRNA (G), were determined.</p

Deletion analysis of <i>RHBG</i> promoter sequence.

<p>HepG2 cells were transfected with the empty plasmid (pGL3) or <i>RHBG</i> promoter (pGL3-RHBG) together with Renilla plasmid. 48 hours after transfection, <i>RHBG</i> promoter activity in total cell lysates was determined by luciferase assay. B) HepG2 cells were transfected with <i>RHBG</i> promoter (pGL3-RHBG) or the indicated construct together with Renilla plasmid. 48 hours after transfection, <i>RHBG</i> promoter activity was determined by measuring luciferase activity in total cell lysates. Data are expressed as mean of triplicate determinations ± S.E.M of the pGL3-RHBG construct relative to pGL3-Basic. C) HepG2 cells were transfected with the indicated construct together with Renilla plasmid. 48 hours after transfection, <i>RHBG</i> promoter activity was determined by measuring luciferase activity in total cell lysates.</p

Promoter region of <i>RHBG</i> gene.

<p>The potential human <i>RHBG</i> promoter sequence was obtained from eukaryotic promoter database (<a href="http://epd.vital-it.ch/" target="_blank">http://epd.vital-it.ch/</a>). Black arrow (↓) indicates the predicted transcription start site (TSS) which is designated nucleotide 0. The GC boxes are shadowed. A selection of potential binding sites (with 0 or less than 5% dissimilarity) of transcription factors identified using PROMO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128683#pone.0128683.ref054" target="_blank">54</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128683#pone.0128683.ref055" target="_blank">55</a>] is underlined. Potential TCF4 binding sites are indicated with empty boxes. Horizontal arrows (→) indicate the starting residue position of each promoter construct analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128683#pone.0128683.g008" target="_blank">Fig 8</a>.</p

TCF4 knockdown decreases <i>RHBG</i> expression in SW480 cells.

<p>A-E) SW480 cells were reverse transfected with TCF4 or control (scramble) siRNAs. 72 hours after transfection, levels of <i>TCF4</i> mRNA (A), TCF4 protein (B) <i>RHBG</i> mRNA (C), <i>Axin2</i> mRNA (D), and <i>Cyclin D1</i> mRNA (E), were determined.</p

TCF4/β-catenin binds to the <i>RHBG</i> promoter.

<p>HepG2 cells were cross-linked with formaldehyde followed by chromatin digestion. Chromatin immunoprecipitations were performed using antibodies targeting either β-catenin, TCF4 or IgG, as a control. Purified DNA was analyzed by qPCR using the indicated primers. The amount of immunoprecipitated DNA with each antibody is represented as signal relative to IgG (equivalent to 1) (n = 2).</p