Cadherins are required for transcriptional activation by Wnt3a and β-catenin.

<p>(A–D) Confocal microscopy of MDCK cells without (control, −Tet) or with (+Tet) induction of E-cadherin shRNA expression by tetracycline, after treatment without or with HGF, stained for β-catenin. Insets in (C) and (D) (C′,D′) are exogenous β-catenin detected by myc tag. For the exogenous β-catenin in the presence of E-cadherin, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023899#pone-0023899-g003" target="_blank">Fig. 3J,K</a>. E-cadherin-depleted cells do not scatter by HGF and the majority of β-catenin remains on the cell membrane, while the minority distributes in the cytoplasm in a punctuated manner. Transfected β-catenin localises at the cell membrane in the absence of HGF (C′), while it distributes in the cytoplasm in the presence of HGF (D′), similar to E-cadherin-positive MDCK cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023899#pone-0023899-g003" target="_blank">Fig. 3J,K</a>). Scale bar, 20 µm. (E) TOPflash reporter assay in MDCK cells (control, −Tet) or MDCK cells depleted of E-cadherin (E-cadherin shRNA, +Tet). Cells transfected with TOPflash reporter were either treated with Wnt3a medium or co-transfected with β-catenin. Treatment of cells with HGF enhances cells' response to Wnt3a or β-catenin, as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023899#pone-0023899-g001" target="_blank">Fig. 1E and 1F</a>. In cells depleted of E-cadherin, HGF fails to enhance the response to Wnt3a and β-catenin. (F) Western blot analysis of membrane (upper two rows) and cytosolic (lower four rows) fractions of MDCK cells without or with induction of E-cadherin shRNA expression, without or with HGF. Some groups of cells were treated with Wnt3a conditioned medium or with 2 µM BIO, a GSK3β inhibitor, or transfected with myc-tagged β-catenin, as indicated at the top. The membrane fraction analysis shows that E-cadherin shRNA completely abolishes E-cadherin protein. The cytosolic fraction was analysed with anti-Active-β-catenin (ABC) and anti-β-catenin (total) antibodies, along with anti-myc antibody to detect exogenous β-catenin and with anti-β-tubulin for loading control. Cell lysate of A431 human carcinoma was used as a positive control for anti-active-β-catenin antibody. In all experimental groups, active-β-catenin is increased in response to HGF when E-cadherin expression is kept intact. In contrast, in the absence of E-cadherin the ability of HGF to increase active-β-catenin is abolished. Note that myc-tagged β-catenin, larger than endogenous one in size, is also detected by ABC antibody, responding to HGF and to the loss of E-cadherin in a manner similar to endogenous ones treated with Wnt3a or BIO. (G) TOPflash reporter assay in MDCK cells (control, −Tet) or MDCK cells depleted of E-cadherin (E-cadherin shRNA, +Tet). Cells were transfected with TOPflash reporter along with β-catenin (β-cat) or the stabilised form in which four serine and threonine residues of GSK3β targets have been mutated to alanine (β4A) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023899#pone.0023899-Yost1" target="_blank">[45]</a>. Similar to β-catenin, β4A construct does not show any significant activation of the reporter in E-cadherin-depleted cells, regardless of presence or absence of HGF.