The lack of helix C in β-catenin does not prevent interaction with ICAT.

<p><b>A</b>. Schematic representation of WT and mutant Δ665 HA-tagged β-catenin-NLS proteins. <b>B</b>. Total cell lysates from Lu1205 cells transfected with WT or mutant β-catenin Δ665 were analyzed by WB or affinity immunoprecipitated with WT ICAT-GST recombinant protein and blotted with anti-HA and anti-ICAT antibodies. Numbers represent mean ± SD of normalized densitometry values from three independent experiments, *p<0.05.</p

ICAT negatively regulates the M-MITF promoter activity by competing with LEF1.

<p><b>A.</b> Mel501 cells were transfected with a <i>M-MITF</i>::<i>luciferase</i> vector in the presence of increasing amounts of <i>CMV</i>::<i>ICAT-WT</i> expression vector. Data are presented as means ± SEM of three independent experiments. <b>B.</b> qRT-PCR analysis of <i>M-MITF</i> mRNA levels in Mel501 cells transfected with empty or <i>ICAT-WT</i> expression vectors. <b>C.</b> WB analysis of MITF and ICAT proteins in Mel501 cells transfected with empty or <i>ICAT-WT</i> expression vectors. <b>β</b>-actin = loading control. <b>D.</b> WB analysis of MITF and p27^Kip1 protein levels in siRNA and ICAT-transfected Mel501 cells. SiMITF treatment and ICAT overexpression induce respectively a 42% and 35% increase of p27 protein amount; Scrb = control scrambled siRNA. <b>E</b>. Mel501 cells were transfected with <i>M-MITF</i>::<i>luciferase</i>, <i>LEF1</i> and <i>ICAT-WT</i> expression vectors. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p< 0.001; ****p<0.0001.</p

List of ICAT and β-catenin mutants created by site directed-mutagenesis.

<p>List of ICAT and β-catenin mutants created by site directed-mutagenesis.</p

Interactions between ICAT and β-catenin mutants, K312E, K435E and R386G in Lu1205 cell extracts: Consequences on <i>NEDD9</i> promoter activity.

<p><b>A</b>. Left: WB analysis of lysates (Input) from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: Pull-down assay of HA-tagged WT and mutant β-catenin (K312E, K435E and R386G) by WT ICAT-GST recombinant protein. <b>B</b>. Left: WB analysis of lysates (Input) from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: Pull-down assay of HA-tagged WT and mutant β-catenin (K312E and K435E) by LEF1-GST recombinant protein. <b>C</b>. Lu1205 cells were transiently transfected with <i>NEDD9</i>::<i>luciferase</i> and either <i>β-catenin-WT</i> or <i>β-catenin</i> mutants <i>(K312E</i>, <i>K435E and R386G)</i> expression vectors. <b>D</b>. Lu1205 cells were transiently transfected with <i>NEDD9</i>::<i>luciferase</i> vector in the presence of <i>CMV</i>::<i>LEF1</i>. Cells were also transfected with <i>β-catenin-WT</i> or <i>β-catenin</i> mutants <i>(K312E</i>, <i>K435E and R386G)</i> expression vectors. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.</p

The characteristics of the first X residue in the consensus peptide of several β-catenin binding proteins regulate their interactions with β-catenin.

<p><b>A</b>. Zoom is made on the first conserved Aspartate residue of the consensus peptide and its adjacent non conserved residues (shown as sticks) in ICAT, LEF1, TCF4, APC and E-cadherin (yellow ribbons) and the facing β-catenin Arm repeats 8 and 9 (purple cylinders). The first X residue of the consensus is encircled because residue numbering diverges between various β-catenin regulators, although they are facing the same β-catenin residues forming a basic patch. Hydrogen bonds between basic β-catenin residues and their counterpart in β-catenin regulators are presented as black dotted lines. In ICAT, V67 does not establish any hydrogen bond, whereas in LEF1 and TCF4/TCF7L2, E20 and E17, respectively make an H-bond with the facing β-catenin K508. In APC and E-cadherin, T1487 and S675 respectively form hydrogen bonds with the facing β-catenin R469. The color scheme of stick residues based on their characteristics is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172603#pone.0172603.g001" target="_blank">Fig 1</a>. PDB codes: ICAT (1luj), LEF1 (3ouw), TCF3/TCF7L1 (1g3j), TCF4/TCF7L2 (1jdh), APC (1t08) and E-cadherin (1i7w).</p

Crystal structure of the β-catenin/ICAT complex.

<p><b>A</b> Crystal structure of ICAT bound to the core domain of β-catenin (PDB code 1LUJ,[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172603#pone.0172603.ref022" target="_blank">22</a>]). ICAT is shown as yellow ribbons and β-catenin as purple cylinders. The secondary structures were calculated using the program STRIDE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172603#pone.0172603.ref037" target="_blank">37</a>]. Residues mutated in this study are shown as hard spheres. ICAT residues are colored according to their characteristics: white for hydrophobic, green for polar, red for acidic and blue for basic residues. β-catenin F660 is in pink and the basic residues facing the C-terminal domain of ICAT are in cyan. <b>B</b>. Sequence alignment of the consensus peptide from several β-catenin binding proteins. The conserved acidic residues are in red and the aromatic residue in green. The first X residues, when they are hydrophilic, are boxed. <b>C</b>. β-catenin/ICAT complex showing the interaction between ICAT consensus peptide of the C-terminal domain (ribbon and sticks) and its facing β-catenin residues (surface). All residues are colored according to their characteristics. Figures were drawn using VMD software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172603#pone.0172603.ref038" target="_blank">38</a>].</p

β-catenin residue F660 is critical for ICAT anchoring to Arm repeat 12 but plays no role in the affinity for LEF1.

<p><b>A</b>. Left: Western blot (WB) analysis of lysates (Input) from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: pull-down assay of β-catenin WT and mutants F660S and F660A by WT ICAT-GST recombinant protein. <b>B</b>. Left: Input from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: pull-down assay of β-catenin WT and mutants F660S and F660A by LEF1-GST recombinant protein. <b>C</b>. Lu1205 cells were transiently transfected with a <i>NEDD9</i>::<i>luciferase</i> vector. These cells were also transfected with either <i>NLS-</i>β<i>-catenin-WT</i> or <i>NLS-</i>β<i>-catenin-F660S</i> expression vectors in the absence (left) or presence (right) of exogenous LEF1. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.</p

Histological analysis of WT and ctnnb1Δex3 lungs at P28.

<p>(A) WT ( = <i>Tyr::Cre</i>/°; +/+) and (B) ctnnb1Δex3 mice. Note the disorganized alveolae of the mutant lung. Nonetheless, the lung cells do not express the ctnnb1Δex3 transgene, suggesting that the effect is cell non-autonomous. Scale bars, (A, C, D)  = 50 µm, (B)  = 20 µm.</p

Melanoblasts are numerous in ctnnb1Δex3 DA.

<p>Ventral view of WT-Dct (A) and ctnnb1Δex3-Dct (B) E18.5 hearts stained with X-gal. Note that ctnnb1Δex3-Dct samples contain numerous β-galactosidase-stained cells (arrow) in the ductus arteriosus (DA). High magnification of the WT-Dct (C) and ctnnb1Δex3-Dct (D) DA regions, including the aorta (Ao) and the pulmonary trunk (PT). Scale bar (A, B)  = 1 mm.</p

Indomethacin treatment and survival of ctnnb1Δex3 mice.

<p>Indomethacin treatment results in the closure of WT and ctnnb1Δex3 ( = <i>Tyr::Cre/°; ctnnb1Δex3</i>/+) DA and allows the survival of ctnnb1Δex3 mice. Mock (A, B) and indomethacin (indo, 10 mg/kg body weight) (C, D) intraperitoneal injections into pregnant <i>Tyr::Cre/Tyr::Cre</i>; +/+; <i>Dct::LacZ/Dct::LacZ</i> females carrying <i>Tyr::Cre/</i>°; +/+; <i>Dct::LacZ/</i>° (A, C) and <i>Tyr::Cre/</i>°; <i>ctnnb1Δex3</i>/+; <i>Dct::LacZ/</i>° (B, D) E18.5 embryos. Four hours later, embryos were isolated, fixed, X-gal stained, transversally sectioned through the DA and counterstained with eosin. We treated three pregnant females and sectioned ten embryonic hearts (five WT and five mutants). The ductus arteriosus was closed in all cases. Note that the numbers of Dct+ cells derived from ctnnb1Δex3-Dct embryos obtained from pregnant mothers injected with indomethacin or mock-injected were similar. (E) Kaplan-Meier curves of WT and ctnnb1Δex3 newborn pups treated or mock-treated with indomethacin (6 mg/kg body weight indomethacin within 12 hours of birth). Ultrasound analysis was performed on treated versus non-treated animals during the second and third months, which associated survival of treated ctnnb1Δex3 to the size of the left atrium (not shown). Indomethacin-treated ctnnb1Δex3 mice survived significantly longer than mock-treated mice (p<0.009). Note similar results were obtained when ctnnb1Δex3 mi mice were treated with indomethacin or mock. Scale bars, (A, B)  = 100 µm, (C, D)  = 50 µm.</p