Here, we review previous work on the role of Wnt/β-catenin signalling in glioma initiation, proliferation, and invasion. Understanding the molecular mechanisms regulating GIC biology and glioma progression may help in identifying novel therapeutic targets for GBM treatment.Glioblastoma multiforme (GBM) is a commonly occurring brain tumor with a poor prognosis. GBM can develop both “de novo” or evolve from a previous astrocytoma and is characterized by high proliferation and infiltration into the surrounding tissue. Following treatment , tumors often reappear. Glioma-initiating cells (GICs) have been identified in GBM and are thought to be responsible for tumors initiation, their continued growth, and recurrence.  Gliomas are the most common primary malignancies in the central nervous system, comprising a heterogeneous group of tumors that display some histological similarities to glia . Astrocytomas account for the majority of gliomas, which can be classified into four different grades according to the World Health Organization (WHO) classification system , 2: gradGBMs can present as primary or secondary. Primary or “de novo” GBMs, representing the majority of GBM cases, arise without any prior evidence of a lower-grade precursor lesion and more commonly affect older patients (mean age of 62 years). Secondary GBMs progress from a lower-grade glioma and typically develop in younger patients (median age of 45 years). Gliomas exhibit a vast array of genetic changes that contribute to the malignant phenotype . These iβ-catenin signalling, a pathway primarily involved in embryogenesis and displaying important functions in adulthood when aberrant Wnt signalling has been linked to disease and cancer [β-catenin signalling regulates gliomagenesis and tumor progression may lead to novel therapeutic interventions in GBM. Firstly, therefore, we discuss the role of glioma-initiating cells (GICs), a type of “cancer stem cells,” in glioma development and evidence suggesting that Wnt/β-catenin signalling regulates GICs biology. We then review progress in the understanding of the involvement of Wnt/β-catenin signalling in the proliferation and invasion of glioma tumor cells.Here we focus our attention on Wnt/d cancer –14. UndeThe existence of brain tumor stem cells was proposed about a decade ago, following advances in the stem cell field and the discovery that neurogenesis persists in the adult brain –18. Glioβ (TGF-β)/bone morphogenetic proteins (BMP) families) maintains the self-renewal capacity of embryonic stem cells and supports cancer stem cell growth [β signalling through the induction of LIF and the JAK-STAT pathway promotes the self-renewal of patient-derived GICs [β inhibitors prevents tumor initiation and recurrence. This work identifies CD44 and Id1 levels as prognosis markers in GBM and shows that TGF-β signalling is key to maintain this GIC population [Signalling by several morphogens and cytokines (including leukemia inhibitory factor (LIF), fibroblast growth factor (FGF) and members of the Wnt, transforming growth factor-l growth . Thus, Tved GICs . Seoane pulation . Aberranpulation –30. Hedgpulation .β-catenin canonical pathway (β) in the so-called destruction complex , Axin and β-catenin) phosphorylates β-catenin, which is then targeted for proteasomal degradation. Upon Wnt binding to Frizzled (Fz) (of which there are 11 family members in humans) and low-density-lipoprotein-related protein (LRP)5/6 receptors, the scaffolding protein Dishevelled (Dvl) and LRP5/6 become phosphorylated by Gsk-3 and Casein-Kinase Iγ. Consequently, the destruction complex components are recruited instead to the receptor complex, leading to β-catenin stabilization [β-catenin because it competes with Axin to bind Gsk-3 and removes it from the destruction complex. Stabilized β-catenin translocates to the nucleus, where it binds to lymphoid enhancer factor-1 Lef-1/T-cell factor (Tcf) transcription factors and regulates expression of Wnt target genes. In the absence of nuclear β-catenin, Tcf/Lef factors suppress the expression of target genes through their binding to members of the Groucho/transducin-like enhancer of split (TLE) family of transcriptional corepressors. β-catenin does not have a DNA binding domain but it has a potent transcription activation domain. Conversely, Lef/Tcf transcription factors do not have a strong transcription activation domain, but they do have a good DNA binding/bending domain [β-catenin binds to a Lef/Tcf protein, a potent transcription regulatory complex is formed. Nuclear translocation of β-catenin converts Tcf proteins into potent transcriptional activators by displacing Groucho/TLE and recruiting an array of coactivator proteins, including CBP, TBP, BRG1, BCL9/PYGO, Legless, Mediator, and Hyrax [Wnt factors are a family of secreted glycoproteins (19 members exist in humans) that regulate embryonic patterning and play different roles throughout development of the nervous system , 33. Wnt pathway . In the lization . The prog domain . Thus, wnd Hyrax   cells and regulating adult stem cells [β-catenin signalling pathway is a hallmark of many tumors [PLAG2 is amplified in GBM and promotes GICs proliferation and gliomagenesis. PLAG2 increases the expression of Wnt-6, Fz-9, and Fz-2, inhibits differentiation, and increases proliferation of neural progenitors [PLAG2 amplification correlates with increased β-catenin levels in GBM samples. These results indicate that PLAG2 imparts stem-cell properties to glioma cells by regulating Wnt signalling. Another gene regulating Wnt signalling in glioma is PEG3 , an imprinted gene with a tumor suppressor activity. Hypermethylation of PEG3 promoter in glioma decreases PEG3 mRNA expression and correlates with high-grade gliomas [β-catenin that promotes the proliferation of CD133+ GICs [β-catenin that promotes β-catenin nuclear localization in tumor cells and maintains GIC self-renewal has been recently described [β-catenin signalling in GICs to decrease GIC proliferation and stop glioma growth, while increasing GICs differentiation.Canonical Wnt/em cells , 41–45. em cells , 47. Intem cells . As abery tumors , 14, thegenitors . It is w gliomas . In turn33+ GICs . Finallyescribed . Novel tMGMT promoter methylation status improves the benefits of chemotherapy. According to Liu eta l., CD133 expression in tumor tissue is higher in recurrent GBM than in newly diagnosed tumors and CD133+ GICs are chemoresistant to temozolomide [Following surgery, chemotherapy, and radiotherapy GBM recurrence is common. Therefore, a particularly relevant feature of the cancer stem cells is their ability to export drugs and develop resistance mechanisms to cytotoxics and irradiation . Currentzolomide . Howeverzolomide . Consequ2) derived from GBM showed increased expression of BCRP1, CD133 and the neural precursor marker Nestin and at the same time maintained Wnt-1 expression [Another mechanism of drug resistance is the expression of the ATP-binding cassette (ABC) transporters by cancer stem cells. BCRP1 and MDR1 ABC transporters allow the exclusion of Hoechst 33342 dye, a feature that defines the pluripotential side population (SP) originally reported in haematopoietic stem cells and now used to identify cancer stem cells. Expression of these ABC transporters accounts for the chemoresistance of some cancers and high drug efflux capacity. CD133+ GICs express higher levels of BCRP1 compared to CD133− cells . Consistpression . Interespression . This suβ-catenin signalling confers radioresistance to mammary progenitors cells through survivin upregulation [β inhibition enhances DNA repair of double-strand breaks following radiation of hippocampal neurons [As regards radioresistance, the CD133+ stem cell fraction is enriched after glioma radiation . Furthergulation . In addi neurons . Taken tβ-catenin signalling plays a role in the proliferation of glioma tumor cells and tumor progression. β-catenin has been proposed as a prognostic marker in GBM, as both mRNA and protein levels increase in high-grade astrocytomas and GBM, thus correlating with malignancy [Cyclin D1 and c-myc) [β-catenin was observed that associates with high-grade astrocytoma and GBM [β-catenin that escapes proteasomal degradation, in addition to the elevated β-catenin mRNA levels reported in GBM. Silencing β-catenin, Wnt-2, and Pygo-2 expression demonstrated the involvement of Wnt/β-catenin signalling in the proliferation of U251 glioma cell line [β-catenin signalling as a mediator of Wnt-induced proliferation of glioma cells. Moreover, expression of noncanonical Wnt-5a is also upregulated in high grade gliomas, in which Wnt-5a stimulates cell proliferation [GICs represent a small percentage of the brain tumor mass, which is thought to contain a heterogeneous mixture of tumor cells with limited proliferation capacity. Molecular analysis on whole tumor samples is expected to mainly represent non-GIC cells. Wnt/lignancy , 61, 62.lignancy , 63, 64 d c-myc) , 62 alsoell line . Togetheferation . Wnt-5a feration , 68. Howβ-catenin exon 3, a hot spot affecting the GSK-3 phosphorylation sites and β-catenin degradation that renders β-catenin active in glioma samples and cell lines [β-catenin-driven colorectal carcinoma, has not been associated with gliomagenesis (with the exception of Turcot syndrome patients) [secreted-Frizzled-related protein (sFRP) promoters is a significant event in primary “de novo” GBM, whereas hypermethylation of the promoter of the LRP antagonist Dickkopf (Dkk) associates with secondary GBM [β-catenin nuclear localization and transcriptional activation (both constitutive and Wnt-induced) that controls Wnt target gene expression and glioma tumorigenesis has been described, which involves the interaction of β-catenin with FoxM1 [In contrast to other cancers, no mutations have been found in ll lines , 70. Truatients) . These oatients) . Also, hdary GBM , 74. In th FoxM1 .β-catenin binds to cadherin, thus regulating cell-cell adhesion. Altering the binding of β-catenin to cadherin or to α-catenin downregulates cell adhesion, while promoting cell migration and epithelial-mesenchymal transition [β-catenin nuclear signalling is not only achieved by Wnt factors in tumor development [β-catenin, resulting in increased migration [α-catenin at serine 641, which correlates with glioma malignancy [α-catenin phosphorylation promotes β-catenin transactivation and glioma cell invasion [β-catenin signalling not only as a mediator of Wnt but also downstream of growth factor signalling in glioma invasion. On the other hand, enhanced expression of the Fz antagonist sFRP2 reduced glioma invasion by decreasing β-catenin tyrosine phosphorylation and downregulating matrix metalloprotease-2 (MMP-2) [β-catenin levels, its cytoplasmic/nuclear distribution, or its serine phosphorylation status [β-catenin tyrosine phosphorylation requires further investigation.As a component of the cell adhesion complex, ansition . Howeverelopment . Growth igration , 77–79. igration . EGF/EGFlignancy . Interesinvasion . These r (MMP-2) . Howevern status . How sFRβ-catenin independent pathways , enhancpathways . Moreovepathways , 80. Thepathways , 81, 82.invasion , 80, 83.We are accumulating knowledge on the signalling pathways responsible for the maintenance of GICs, sustaining the proliferation of bulk tumor cells and dictating the invasive properties of glioma cells. Current and future research should offer novel opportunities for anticancer drug discovery. Undoubtedly, the cancer stem cell hypothesis has provided a promising framework for investigations into an incurable disease. Future combined therapies including cytotoxics, tumor-targeted drugs, and agents that target GICs should be expected to reduce glioma growth and recurrence, raising hopes for glioma patients.


Since NF-κB concurrently induces Wnt/β-catenin signaling implicated in cell proliferation, cell survival, and apoptosis evasion, we examined antiproliferative, apoptosis-inducing and Wnt/β-catenin signaling-modulatory mechanisms of PE during DENA rat hepatocarcinogenesis. PE (1 or 10 g/kg) was administered 4 weeks before and 18 weeks following DENA exposure. There was a significant increase in hepatic proliferation (proliferating cell nuclear antigen) and alteration in cell cycle progression (cyclin D1) due to DENA treatment, and PE dose dependently reversed these effects. PE substantially induced apoptosis by upregulating proapoptotic protein Bax and downregulating antiapoptotic protein Bcl-2. PE dose dependently reduced hepatic β-catenin and augmented glycogen synthase kinase-3β expression. Our study provides evidence that pomegranate phytochemicals exert chemoprevention of hepatic cancer through antiproliferative and proapoptotic mechanisms by modulating Wnt/β-catenin signaling. PE, thus, targets two interconnected molecular circuits  to exert chemoprevention of HCC.Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, and chemoprevention represents a viable approach in lowering the mortality of this disease. Pomegranate fruit, an abundant source of anti-inflammatory phytochemicals, is gaining tremendous attention for its wide-spectrum health benefits. We previously reported that a characterized pomegranate emulsion (PE) prevents diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis though inhibition of nuclear factor-kappaB (NF- Hepatocellular carcinoma (HCC), the major primary malignant tumor of the liver, is one of the most life-threatening human cancers in the world, resulting in almost one million deaths every year . There hβ-catenin signaling cascade which plays a decisive role in cell fate, favoring cell proliferation over apoptosis. Accumulating evidence has shown that the canonical Wnt/β-catenin pathway is frequently activated in HCC and responsible for initiation and progression of the disease .κB and members of the Bcl-2 family consisting of both proapoptotic and antiapoptotic proteins contribute to the regulation of apoptosis in hepatocytes . Acc in rats  and DENA in rats . Our immβ mRNA has been observed in human hepatic cancer linked to HCV infection , veriewed in ]. β-catenin signaling in hepatocarcinogen-exposed animals. The interference of Wnt/β-catenin signaling may be achieved, at least in part, through degradation of β-catenin protein, the cardinal effector of Wnt pathway, by up-regulation of GSK-3β. This is further supported by the diminished hepatic expression of cyclin D1, a β-catenin-dependent target gene, and an indicator of activation of Wnt/β-catenin pathway. Thus, our study provides encouraging preclinical evidence and novel mechanism of targeting activated Wnt/β-catenin signaling by pomegranate products to achieve chemoprevention of hepatocellular cancer. Previously, we have shown modulation of the NF-κB pathway by pomegranate constituents in DENA hepatocarcinogenesis in rats [κB and Wnt/β-catenin signaling pathways has emerged as a pivotal mechanism for the regulation of a diverse array of genes involved in tumorigenesis, including HCC [κB and Wnt/β-catenin pathways, to achieve prevention of hepatocellular cancer (summarized in The results of our present study undoubtedly establish that pomegranate bioactive constituents suppress cell proliferation (PCNA), regulate cell cycle progression (cyclin D1), and induce programmed cell death (apoptosis) during DENA-initiated and PB-promoted hepatic tumorigenesis in Sprague-Dawley rats. Pomegranate-mediated apoptogenic signal during experimental hepatocarcinogenesis may be transmitted through up-relegation of proapoptotic protein Bax and downmodulation of antiapoptotic protein Bcl-2. We also present data that show, for the first time, that pomegranate phytochemicals diminish cell proliferation and survival possibly through blockade of activated canonical Wnt/ in rats . As funcding HCC –40, resuding HCC  indisput


The deprivation or abnormality of the molecular function of Wnt/β-catenin triggers the genesis and development of AD, and β-catenin is an important positive mediator in the Wnt/β-catenin signaling pathway. In our previous study, we found that Curcumin could inhibit the expression of β-catenin and prevent AD, but the mechanisms were not fully understood. E-cadherin is a negative mediator in the Wnt/β-catenin signaling pathway, and interacts with β-catenin and PS1 form a trimeric complex, so, we hypothesized that Curcumin prevented AD through increasing PS1/E-cadherin/β-catenin complex by overexpression of E-cadherin.TM2000. The cells were treated with Curcumin at 0, 1.25, 5.0, 20.0 μmol/L for 24 h, or with Curcumin at 5.0 μmol/L for 0, and 12, 24 and 48 h for time course assay. Cell lysates were collected for RT-PCR, Western blot assay for detecting the effect of Curcumin on the expression of E-cadherin, β-catenin and PS1. And immunofluorescent staining was carried out for detecting the effect of Curcumin on the expression of PS1/E-cadherin/β-catenin complex. ELISA was carried out to detect the generation of Aβ.Plasmid APPswe and BACE1-mychis were transiently co-transfected into SHSY5Y cells by Liposfectamin40/42. RT-PCR and Western blot results showed that the expression of PS1 and β-catenin at mRNA and protein levels were significantly decreased in the transfected cells treated after treatment; however, the protein expression of E-cadherin was increased (P <0.05). Furthermore, all the changes were in a dose and time-dependent manner (P <0.05). Immunofluorescent staining results not only confirmed the above changes, but also showed that the PS1/E-cadherin/β-catenin complex was increased.ELISA results showed that Curcumin reduced markedly the production of AβCurcumin exerts its preventive effects on AD through increasing PS1/E-cadherin/β-catenin complex by overexpression of E-cadherin.


The large conductance calcium-activated potassium channel alpha-subunit (Slo) is widely distributed throughout the body and plays an important role in a number of diseases. Prior work has shown that Slo, through its S10 region, interacts with β-catenin, a key component of the cytoskeleton framework and the Wnt signaling pathway. However, the physiological significance of this interaction was not clear.domesticus leghorn white) hair cells treated with siRNA to β-catenin. HSlo reciprocally co-immunoprecipitates with β-catenin, indicating a stable binding between these two proteins, with the S10 deletion mutant having reduced binding with β-catenin. We also observed that mutations of the two putative GSK phosphorylation sites within the S10 region affect both the surface expression of Slo and the channel's voltage and calcium sensitivities. Interestingly, expression of exogenous Slo in HEK cells inhibits β-catenin-dependent canonical Wnt signaling.Using a combination of proteomic and cell biology tools we show the existence of additional multiple binding sites in Slo, and explore in detail β-catenin interactions with the S10 region. We demonstrate that deletion of this region reduces Slo surface expression in HEK cells, which indicates that interaction with beta-catenin is important for Slo surface expression. This is confirmed by reduced expression of Slo in HEK cells and chicken (Gallus gallus These studies identify for the first time a central role for β-catenin in mediating Slo surface expression. Additionally we show that Slo overexpression can lead to downregulation of Wnt signaling. We demonstrate that the interaction between Slo and β-catenin is important for Slo surface expression in HEK-293 cells and chick hair cells in culture. Both deletion of the proposed S10 interacting region on HSlo and siRNA knockdown of β-catenin reduces Slo surface expression in HEK cells. Similarly, siRNA mediated knockdown of β-catenin resulted in decreased Slo on the surface of chick hair cells. Mutations of two putative GSK phosphorylation sites within the S10 region alter the surface expression of Slo in HEK cells, while also affecting the biophysical properties of the expressed channels. Since β-catenin is a major component in the Wnt signaling pathway, we also explored the effect of channel expression on Wnt signaling. Interestingly, expression of exogenous HSlo inhibits canonical Wnt signaling in HEK cells. This shows that Slo may modulate the Wnt signaling pathway presumably through its binding and immobilization of β-catenin.Iberiotoxin-LC-Biotin was a kind gift from Dr. John-Paul Bingham (University of Hawaii). N-dodecyl beta-D-maltoside (DDM) was obtained from CalBiochem.Ca (Alamone Labs); mouse β-catenin (BD Transduction Laboratories); mouse GSK3β (BD Transduction Laboratories); rabbit GSK3β ; mouse anti-vinculin (Chemicon). bSloC was an affinity-purified rabbit antibody to the last C-terminal 21 amino acids of the bovine Slo channel.Antibody suppliers were as follows: mouse anti-Flag M2 and rabbit anti-Flag (Sigma); anti-BKHEK293 cells and H28 (NCI-H28) cells were from American Type Culture Collection (ATCC). 293T was a specially maintained cell line, which is responsive to stimulation of Wnt signaling 5′ctgtacctcacgcagcccaatgacaatatcctcaccctgatacgtac gctggtgacc 3′, backward primer 5′ggtcaccagcgtacgtatcagggtgaggatattgtcattgggctgc gtgaggtacag 3′. For S918A and S922A mutant the forward primer was 5′ gcatttgccgtcgctgtcctggacgctctcatgagcgctacgtacttca atg 3′, and the backward primer 5′cattgaagtacgtagcgctcatgagagcgtccaggacagcgacg gcaaatgc 3′.For S918D and S922D the forward primer was 5′ ggacagcatttgccgtcgacgtcctggacgatctcatgagcgcgacg 3′ and the backward primer 5′ cgtcgcgctcatgagatcgtccaggacgtcgacggcaaatgctgtcc 3′. The mutant inserts were sequenced in their entirety after mutagenesis. β-catenin-EGFP was subcloned from a LZRS-GFP-bCAT vector  into pcDNA3.1_hygro(+) vector using BamHI/NotI restriction sites.A pcDNA3 vector encoding a His6-Flag-epitope tagged version of the human BK channel (HSlo) originally cloned by Wallner et al. (1995) was kindly supplied by Dr. Andrew Tinker, University College London Surface labeling of HSlo/mutant in transfected HEK293 cells with Ibtx-LC-biotin was done as described previously Slo and β-catenin staining in hair cells was done as described previously 2+ free PBS, and HSlo surface expression detected as described above. FACS experiments were done as previously described on a FACS Caliber machine (BD BioSciences) using FlowJo software For FACS analysis cells were detached from culture dishes using CaHSlo was purified from a stable HSlo HEK cell membrane by affinity chromatography using an M2 anti-Flag/agarose (Sigma) column. Cells were lysed in 16 mM DDM, 250 mM KCl, protease inhibitor cocktail (Sigma). After washing with 20 column volumes of 16 mM DDM, 250 mM KCl, HSlo was eluted off the column with 100 µM Flag peptide (Sigma) in 16 mM DDM, 250 mM KCl. The eluate was separated on SDS-PAGE using 4–15% ReadyGel (BioRad), and protein detected with Gel-Blue staining solution (Pierce). For western blots, protein was wet transferred to PVDF membrane, blocked with PBS, 5% non-fat milk, 0.05% Tween-20 followed by incubation with the appropriate primary antibody in blocking solution. Blots were washed extensively in PBS, 0.05% Tween-20 before incubation with the appropriate secondary antibody. Secondary antibodies were matched with the corresponding primary antibody. These included bovine anti-goat IgG-HRP (mouse/human cross-adsorbed), donkey anti-rabbit IgG-HRP (mouse/human crossadsorbed), and chicken anti-mouse IgG-HRP (human cross-adsorbed) from Santa Cruz. Blots were again washed extensively in PBS, 0.05% Tween-20 before enhanced chemilluminescence (ECL) detection. WestDureUltra (Pierce) substrates were used for ECL detection.5′r(UGCUUGGUUCACCAGUGGAUU)3′and r(GGUGUAGAACACUAAUUAA)d(TT); siRNA sequences to HSlo (sense) are 5′r(GGGAUGUUACGUCAACCAU)D(TT)3′ AND 5′R(GGCGGAUGGCACUCUCAAA)d(TT)3′; siRNA sequences to β4 (sense) are 5′r(UUUCUGCUUGUACGUGACAUU)3′. In these experiments, cells were transfected using 40 nM of each siRNA, or 80 nM for β4.In experiments using HEK cells siRNAs were transfected using Oligofectamine or RNAiMax (Invitrogen) at a final concentration of ∼80 nM total siRNAs. Paired plates of cells were transfected with siRNAs or buffer, then either stained live with anti-Flag/488 for surface protein or stained after fixation and permeabilization for total HSlo protein. Assays were done 48–72 hrs after transfection. Unless indicated otherwise, a combination of siRNAs against the same gene were used. siRNAs to HSlo and β-catenin were purchased from Qiagen and IDT. siRNA sequences to β-catenin (sense) are: CAACCAAGCAGGAGGGAAUUU (sense); CTNNB1-2 CAACAAGACAAAUGUGAAAUU (sense); CTNNB1-3 CCAUGGAGCCAGACAGAAAUU (sense). Here siRNA was transfected at a final concentration of 33.3 nm each . In these experiments we used the x-TREMEGene SiRNA Transfection Reagent  as previously described In experiments on hair cells we used three siRNAs to chick β-catenin that had the following sequence: CTNNB1-1 HEK 293T cells grown in 12-well culture plates were transiently transfected with TOP-FLASH TCF reporter plasmid (0.2 µg), β-galactosidase expression vector (0.2 µg) and various expression plasmids as indicated for individual experiments. The total quantity of DNA (1.6 µg) added to each well for each of the transfection protocols was held constant by adding mock DNA (pcDNA3.1) where necessary. Cells were maintained in serum replete medium and then harvested 24–30 h post-transfection in 300 µl cell lysis buffer (Promega). Luciferase activity was determined using a luciferase assay system (Promega) and luminometer according to the manufacturer's specifications. β-galactosidase activity was measured at 420 nm using a spectrophotometer and was used to normalize for transfection efficiency. An aliquot from the cell lysates of each well were saved and used to determine protein expression of the transfected plasmids by Western blotting.I. Only records where the estimated maximum series resistance (sR) voltage error sIR was less than 10 mV were used for analysis.Macro-patch currents were obtained in the inside-out patch-clamp configuration 2 (pH 7.2). The bath solutions contained (in mM): 140 KCl, 20 KOH, 10 Hepes, 5 mM EGTA (for 0 Ca2+) or 5 mM HEGTA (for 10 µM Ca2+), and added CaCl2 to reach 10 µM free Ca2+ concentration (pH 7.2). The amount of CaCl2 needed to obtain 10 µM Ca2+ was calculated using Max Chelator (http://www.stanford.edu/~cpatton/downloads.htm). The desired free Ca2+ concentration was confirmed with a Ca2+-sensitive electrode . Following initial recording in 0 µM Ca2+, currents were then recorded in 10 µM Ca2+ after fast perfusion exchange using a QMM perfusion tip (ALA Scientific Instruments). Activation time constants were determined by single exponential fitting of the traces following a 150 µs delay.The pipette solution contained (in mM): 140 KCl, 20 KOH, 10 Hepes and 2 MgClHSlo peptide arrays were made by JPT Peptide Technologies . Glass slides were spotted with individual peptides. Human and mouse IgG spots were used as controls and served to normalize fluorescence intensity between arrays. Purified GST-β-catenin in PBS was incubated with each triplicate array at a concentration of 1 µg/ml for 1 hour, followed by 3 washes in PBS. Bound GST- β-catenin was detected by 1∶1000 rabbit anti GST antibody followed by 3 washes in PBS, 0.05% Tween-20 and incubating with Alexa 546 conjugated goat anti-rabbit antibody (1∶5000). The slides were washed ×3 with PBS, 0.05% Tween-20 dried and fluorescence detected on a Genepix 4000B Microarray Scanner . Fluorescence intensity was normalized to mouse IgG controls. A second triplicate set of arrays probed with GST alone and processed in an identical manner served to identify non-specific binding. Mean fluorescence data from each spot was averaged between the triplicate arrays. Mean fluorescence intensity for each peptide from arrays probed with GST alone was subtracted from corresponding data from arrays probed with GST- β-catenin to obtain an index of binding of β-catenin to each peptide. Fluorescence intensity was expressed in arbitrary units. We noted a range of values from 0–60,000 A.U. We considered a peptide to have true binding when it along with the two adjacent peptides had fluorescence intensity above 10,000 A.U.Cell lysates were made as in immuno-precipitation experiments. Slo peptides used for competitive binding were made by Biomatik (Canada). Peptide AchE is a 15mer fragment from acetylcholinesterase, used as a negative control. All peptides were dissolved to make 1 mg/ml stock solution, and used at a final working concentration of 100 µg/ml in peptide competitive binding assays. Briefly, 50 µl glutathione/agarose beads (from Pierce) were incubated with 1.5 µg of purified GST-tagged β-catenin  in 0.5 ml wash buffer (same as IP wash buffer) for 30 min at 4°C. After washing, 20 µg of each peptide together with 0.2 ml HSlo HEK cell lysate (with fresh protease inhibitors added) was mixed with the β-catenin-bound beads, and incubated at 4°C over night. After extensive washing with wash buffer the bound HSlo- β-catenin was eluted by incubating with 50 µl 4× SDS sample buffer at room temperature for 30 min and collected by centrifugation. Western blotting, chemilluminescence detection, and densitometry measurement protocols were as described as in co-IP experiments.To study β-catenin-HSlo interactions we used an HSlo-HEK stable cell line, and ascertained the distribution of HSlo on the surface of these cells. We labeled Slo in these cells with a biotin-derivative of the highly specific BK channel blocker Iberiotoxin (Ibtx), which was in turn labeled with streptavidin-Alexa488 conjugate We then sought to determine if β-catenin colocalize with HSlo in these cells. Staining for the cell surface population of HSlo channels revealed sharp co-localization of these channels with β-catenin .Drosophila through humans suggesting that this region is critical to the normal functioning of this channel . We therefore sought to determine how β-catenin knockdown would affect Slo expression in hair cells. In these experiments we used chick hair cells in culture and knocked down β-catenin using siRNA transfection. As shown in The initial rationale that led to the detection of interactions between Slo and β-catenin was to ascertain mechanisms that led to Slo localization at the basolateral surface of hair cells  leading them to conclude that this interaction was indirect. We tested the binding between β-catenin and HSlo by purifying HSlo tagged with the FLAG epitope. We column-purified HSlo from HEK cell lysates using an anti-FLAG antibody. β-catenin was detected by western blotting in purified HSlo sample . ConversWe also tested for co-immunoprecipitation with the deletion mutant HSloΔS10. β-catenin immunoprecipitates from stable cells expressing the deletion construct showed reduced levels of HSlo . ConsistOur co-IP data indicates that the mechanism of the interaction of HSlo with β-catenin through the S10 region and its significance in HSlo surface expression may lie in a simple binding between these two proteins. However, other factors may modulate this interaction or its physiological consequences. For instance, there are two potential GSK phosphorylation sites in the S10 region that contain the consensus GSK phosphorylation motif (SXXXS). A recent work attempting to identify putative phosphorylation sites in Slo determined these sites to have a high probability of phosphorylation based on two phospho-prediction algorithms The surface expression of the S10AA mutant is markedly reduced compared with the S10DD construct, which shows an increased level of surface expression of HSlo . In perm2+ (0 and 10 µM). In contrast, the phosphodeletion form of HSlo (S10AA) activated more slowly . Conversely, we do not observe differences in Wnt signaling between HSlo and the S10 deletional form, even though immunoprecipitations suggest a clear difference in interaction strength. To be sure any number of reasons could explain these seemingly discordant results. These include differences in threshold between the functional assays and immunoprecipitation assays, allosteric effects on β-catenin's transcriptional activity mediated by regions of HSlo outside the S10 region, functional effects between the two proteins additionally mediated by a third protein etc. Irrespective of the exact mechanisms that underlie these phenomena that must await future exploration, these data point to a complex and dynamic interaction between HSlo and β-catenin.It is widely recognized that β-catenin has a dual cellular function and involved in cell adhesion and, separately, is integral to the developmentally important Wnt signaling pathway. Using a transcriptional assay we find a dose-dependent inhibition of Wnt signaling by HSlo expression. Wnt signaling induced either by expression of the Wnt3a ligand or by overexpression of β-catenin is suppressed as the expression of HSlo is increased. This effect is likely due to HSlo interactions with β-catenin, which may in turn reduce the size of the free pool of β-catenin available for activating Wnt signaling. It is unlikely that HSlo expression led to increased degradation of β-catenin, since the levels of total β-catenin were invariant irrespective of the mutants of HSlo transfected .Drosophila Eag channel to bind and activate Ca2+-calmodulin-dependent protein kinase II (CaMKII), independent of calmodulin and autophosphorylation in vivo. This is particularly the case since Wnt signaling affects planer cell polarity of hair cells, and since Slo is restricted to the basolateral pole of hair cells.The observation that HSlo affects Wnt signaling has substantial implications. It is one of the first instances where the expression of an ion channel has been shown to affect signaling possibly independent of its effects on ionic flux. One instance where this has been previously shown is the ability of the 2+-binding states of the channel may also affect this interaction, since mutations on the putative phosphorylation sites in the S10 region alters channel activation kinetics and steady state voltage and Ca2+ sensitivity. In addition to the effect of β-catenin on Slo channel surface expression, our data show that HSlo overexpression also affects canonical β-catenin-dependent Wnt signaling pathway. The expression of HSlo can, in a dose dependent manner, inhibit Wnt signaling in TOP-Flash assays. This is among the first in a growing literature demonstrating direct effects on Wnt signaling by ion channels.In conclusion, we extend previous work showing interactions between the S10 region of Slo and β-catenin. In particular we elucidate the physiological significance of this interaction. We show that this interaction is important for surface expression of HSlo, and may be also important for the stability of Slo channel. The binding of these two proteins extends to regions outside the S10 region, as HSlo reversibly precipitates β-catenin in co-IP experiments, while S10 deletion mutant channels have reduced, but not absent, binding to β-catenin. This possibility was confirmed by β-catenin binding to multiple Hslo peptides in a peptide array. Possible phosphorylation by GSK3 on S10 region of HSlo, and the CaFigure S1ΔS10 mutants have more cellular fragments compared with wt HSlo. Shown is a western blot of several cell lines expressing HSlo (left lane) and HSlo lacking the S10 region . Cell lysates were separated on SDS-PAGE and western blotted. The blot was probed with an anti-FLAG antibody in turn detected by HRP conjugated anti-mouse antibody using enhanced chemiluminescence (ECL). Extra fragments between 40 kDa to 75 kDa, likely representing breakdown products containing the N-Terminus of the protein, were detected in the three cell lines lacking the S10 region. We interpret these data to suggest that interaction with β-catenin protects HSlo from proteolytic degradation.(TIFF)Click here for additional data file.Figure S2β-catenin binds to multiple regions of the Slo C-terminus. Shown is a schematic of the HSlo C-terminus with its recently determined secondary structure superimposed. Alpha helices (blocks) and β-strands (arrows) are indicated beneath the primary sequence. Secondary structure from the RCK1 and RCK2 regions are shown in pink and blue respectively. Twelve contiguous overlapping peptide sequences that bound to β-catenin on peptide arrays are indicated in green. The S10 region that was previously identified as binding to β-catenin is underlined. Note that the actual binding regions are likely to be smaller than indicated since the peptide arrays involved overlapping peptides.(TIFF)Click here for additional data file.Figure S3Multiple regions of the C-terminus of HSlo binds to β-catenin. Shown in graph form is fluorescence intensity of β-catenin bound to an HSlo peptide array. Triplicate arrays were incubated with GST- β-catenin and probed with fluorescent-tagged antibodies to GST. GST alone was used to probe a separate set of triplicate arrays and these fluorescence values subtracted from the corresponding values of the arrays probed with GST- β-catenin to obtain a profile of true β-catenin binding. Binding to twelve separate peptide regions that reached an arbitrary cutoff of 10,000 fluorescence units is indicated. The three peptides used for binding correspond to the peaks 2 (D410DNV…), 5 (E562DT..) and 11 (D1014RC…). The S10 region corresponds to the 10th fluorescence peak. While the peptide with the highest binding (peak 5 E562DT) also inhibited interactions between Slo and β-catenin, we are unable to assert a relationship between intensity of fluorescence in the peptide array with strength of protein-protein interactions. In part this is because such an assertion would require the non-trivial assumption that all peptides were synthesized with equal efficacy. The S10 region for instance is resistant to synthesis (and purification) to allow testing in a non-competitive binding assay.(TIFF)Click here for additional data file.


Xenopus embryos to explore functional differences between two α-catenin family members, α-E- and α-N-catenin, and their interactions with the different classical cadherins that appear as tissues of the embryo become segregated from each other. We show that they play both cadherin-specific and context-specific roles in the emerging tissues of the embryo. α-E-catenin interacts with both C- and E-cadherin. It is specifically required for junctional localization of C-cadherin, but not of E-cadherin or N-cadherin at the neurula stage. α-N-cadherin interacts only with, and is specifically required for junctional localization of, N-cadherin. In addition, α -E-catenin is essential for normal tissue size control in the non-neural ectoderm, but not in the neural ectoderm or the blastula. We also show context specificity in cadherin/ α-catenin interactions. E-cadherin requires α-E-catenin for junctional localization in some tissues, but not in others, during early development. These specific functional cadherin/alpha-catenin interactions may explain the basis of cadherin specificity of actin assembly and morphogenetic movements seen previously in the neural and non-neural ectoderm.Alpha (α)-E-catenin is a component of the cadherin complex, and has long been thought to provide a link between cell surface cadherins and the actin skeleton. More recently, it has also been implicated in mechano-sensing, and in the control of tissue size. Here we use the early  The assembly of filamentous actin at the cell cortex has long been known to be essential for cell shape and motility. In embryos, actin-based changes in cell shape and motility are responsible for creating form, both of the whole embryo, and of its constituent organs. Since cells can migrate as individuals or can undergo complex morphogenetic migrations as cell masses, the relationship between cell-cell adhesion and cell migration must be tightly regulated. Cadherins mediate cell-cell adhesion and are also known to be the sites of membrane attachment of the actin skeleton The cadherin complex consists of the transmembrane glycoprotein cadherin, and cytoplasmic catenins that bind to the cadherin intracellular C-terminal tail. p120 catenin (p120ctn) is an armadillo repeat protein which binds to the juxta-membrane domain (JMD), close to the cell membrane, whilst two related proteins, β-catenin and plakoglobin, bind in a mutually exclusive fashion to the more C-terminal catenin-binding domain (CBD). β-catenin binds in turn to the vinculin homolog α-catenin, which has long been considered to link the cadherin/catenin complex to the actin skeleton through its C-terminal actin-binding domain. However, the precise relationship between α-catenin and actin at the cell membrane requires further examination, given evidence that α-catenin binding to β-catenin and actin are mutually exclusive in vitro Xenopus embryo to study the role of cadherins in cortical actin assembly. At the blastula stage, all of the components of the cadherin complex, as well as the actin assembly proteins so far identified, are synthesized on mRNAs transcribed and stored in the oocyte, later to be replaced by zygotic mRNAs during gastrulation. Depletion of the stored maternal mRNA from the oocyte by microinjection of antisense oligodeoxynucleotides (AS oligos) into cultured oocytes, results in a corresponding depletion of the cognate protein until the zygotic mRNA is switched on We have previously used the early These data indicate that cadherins do not simply link adhesion sites to a pre-assembled cortical actin cytoskeleton, but rather play a critical role in directing tissue-specific assembly of actin filaments at sites of cell-cell contact. This makes it extremely important to understand the mechanisms that regulate the amount and position of cadherin at the cell surface during morphogenesis, and the mechanism of actin assembly on cell surface cadherin.Xenopus embryo. α-E-catenin (αEC) is required both for junctional localization and the adhesive function of C-cadherin, both in the pre-gastrula embryo, and in the neural and non-neural ectoderm after gastrulation. In the post-gastrula ectoderm, α-N-catenin (αNC) is switched on in the neural ectoderm, whilst αEC continues to be expressed in both the neural and non-neural ectoderms. As these changes occur, the first signs of specificity appear in alpha-catenin-cadherin interactions. N-cadherin interacts only with αNC, and requires αNC, but not αEC for junctional localization and actin assembly. C-cadherin interacts with αEC but not αNC, and requires αNC for junctional localization and actin assembly at all stages and tissues. E-cadherin is more complex. At the neurula stage, it’s expression on the junctions in the non-neural ectoderm is αEC-independent. However, at the late blastula stage, it shows a context-dependent requirement for αEC. In the polarized outer cells of the blastula it’s cell surface expression is αEC-independent, whereas in the non-polarized inner cells, the opposite is the case. These data help explain the mechanism of the cadherin-based specificity of actin assembly and morphogenetic movements shown previously for the neural and non-neural ectoderms.In this paper, we show that the α-catenins play crucial, and cadherin-specific, roles in the expression of cadherins on the cell surface in the developing ), it was not associated with αEC , and was non-functional in cell adhesion. The result is shown more clearly in The function of αEC at the late blastula stage was investigated first by comparing the effects of replacing C-cadherin, the major cadherin at this early stage, with mutant forms either lacking the CBD domain (C-cad ΔCBD), and thus unable to bind αEC, or with the CBD domain replaced by the C-terminus of αEC. This was achieved by depleting the endogenous C-cadherin mRNA using an antisense oligo as described previously We then carried out a similar experiment in which C-cadherin was replaced by a construct in which the CBD domain of mouse E-cadherin was replaced by the C-terminal half of αEC (nEαC).In contrast to the C-cad ΔCBD protein, nEαC was efficiently expressed on the cell surface and rescued cell adhesion Fig. 2A.Xenopus laevis αEC. To test this further, we depleted early embryonic αEC directly, using the antisense oligo described previously Thus the two approaches; replacement of C-cadherin with a mutant that lacked the αEC binding domain, or reducing directly the αEC protein levels, both gave the same result; that αEC is required for cell adhesion because it controls the expression levels of C-cadherin in the adherens junctions of the blastula cells.Xenopus whole mounts confirmed the previously reported patterns of expression of these two alpha catenins in other species .The ectoderm was used as a model in this study, since its cells can be directly visualized on the outside of the embryo, and it differentiates into two components, the neural and non-neural ectoderm, each with a distinct expression pattern of cadherins and α-catenins. The non-neural ectoderm expresses C- and E-cadherins, whilst the neural ectoderm expresses C- and N-cadherins, each with characteristic sub-cellular distributions en face view of the non-neural ectoderm stained for C-cadherin (red) and αEC (green). In clones of cells depleted of αEC  or non-neural  ectoderm respectively. The efficiency of the αEC-MO is shown in d of αEC Fig. 5C,Next we depleted the α-catenins in the neural ectoderm. Clones of cells depleted of αEC, αNC, or both, were generated on one side of the neural ectoderm by injection of MO, together with a lineage tracer, into one dorsal animal cell at the 8-cell stage. As it did in the non-neural ectoderm, depletion of αEC caused a dramatic decrease in junctional localization of C-cadherin Fig. 6A.In summary, αEC is required for C-cadherin expression on the cell surface in both the neural and non-neural ectoderm, but is not required for E-cadherin expression in the non-neural ectoderm, or N-cadherin expression in the neural ectoderm. αNC is essential for N-cadherin cell membrane expression in the neural ectoderm, but not required for C-cadherin expression. These data indicate that there is considerable specificity of function of the α-catenins in the post-gastrula embryo.Movie S1). Depletion of αNC in both sides of the neural plate, by injection of both dorsal animal cells at the 8-cell stage, caused an effect identical to that caused by the depletion of N-cadherin, the failure of the neural plate to invaginate  was found to be distributed throughout the baso-lateral cell surfaces, but concentrated in a sub-apical belt of junctions. αEC in the same fields of view , and that either E-cadherin-containing junctions do not bind αEC, or do not require binding (because αEC depletion did not cause a loss of E-cadherin from cell junctions). To test these possibilities, high resolution confocal imaging was carried out on cleared ventral ectoderm tissues co-stained for E-cadherin and αEC Fig. 10Aen face under the confocal microscope. The results are shown in  A, B, C), although the E-cadherin protein was translated and was present at high levels in these embryos ( F). However, when the superficial layer of cells in the animal caps was examined, in samples where all the deeper (blastocelic roof) cells had fallen off, E-cadherin was seen to be expressed at cell-cell contact sites and rescued the dissociation of αEC- depleted cells . Imaging the animal caps from the outside also showed the same result (data not shown). These results suggest that E-cadherin expression and function in cell adhesion is context-dependent. In the deep cells of the animal cap, E-cadherin required αEC, whereas in the superficial cells it did not.These data suggest that the dependence on αEC binding for E-cadherin junctional expression may be context-dependent in the non-neural ectoderm. In some junctions the two proteins overlapped. In others they did not. Depletion of αEC showed that E-cadherin expression at the cell surface is not αEC–dependent, although we cannot rule out the possibility that some of it has gone after αEC depletion. Ectopic expression of E-cadherin in the blastula offered the opportunity to test this context dependence. We showed previously that E-cadherin can rescue the depletion of C-cadherin in the blastula Xenopus embryo is a useful model system for this kind of study. It is an in vivo model in which the distribution and concentration of target proteins can be directly correlated with the phenotypic effects in vivo. The large size of its cells allows the visualization of cadherins and the actin skeleton at relatively low magnification. The ability to deplete individual target proteins, and replace them with either mutant forms, or related proteins, allows functional analysis of the differences between structurally related proteins, and of specific domains. We have used this model previously to show that the cadherins play a much larger role in morphogenesis than simply anchoring F-actin at the cell surface to stabilize cell contacts. They are also essential for F-actin assembly in the cell cortex of the early embryo The Previous work showed that depletion of αEC caused a reduction in cell adhesion at the blastula stage Xenopus.The exact role played by αEC in this process is still unknown. It is possible that it may be involved in either targeting cadherin/catenin complexes to the cell surface or stabilizing cadherins in junctions by preventing endocytosis. αEC was recently shown to bind dynamitin and to regulate dynactin-based trafficking of lysosomes Xenopus, are expressed on the cell surface, and assemble F-actin, in pluripotent blastula cells lacking the endogenously expressed C-cadherin. However, by the neurula stage, in the embryonic ectoderm, they lose their ability to substitute for one another. The mechanism of this specialization is not known, although recent work suggested that the cadherin extracellular domain plays a role in regulating distribution through interactions with other cell adhesion molecules such as nectin We have shown previously that all three major classical cadherins , in One of the most interesting questions that arises from this work is how cadherin-α-catenin interactions can be specific, when α-catenins bind, not directly to cadherins, but through β-catenin in each case. The answer to this will only come from a fuller understanding of how each component in the complex affects the binding of others. A number of proteins, in addition to actin and β-catenin, are known to bind α-catenin, including vinculin, α-actinin, ZO-1, afadin, formin, and Rho Xenopus). In addition, as has been shown previously in the epidermis In conclusion, it is becoming increasingly obvious from this and other studies that the cadherin-α-catenin interaction is not a simple linear link from cell surface cadherins to the actin cytoskeleton. α-catenins are responsible for maintaining cell surface expression of the cadherins, but in both a cadherin-specific, and context-dependent manner. They are also required for adhesive function of C- and N-cadherins, but probably not E-cadherin . Host transfer experiments were done following the protocol described previously All Xenopus expression vector pCS107, using EcoRI and NotI sites by Rebecca Daugherty (Northwestern University). Capped mRNA for microinjecting was synthesized by linearizing the constructs with Ascl and using the SP6 mMESSAGE mMACHINE Kit. The morpholino resistant α-N-catenin-HA construct was made by PCR amplifying the full length Xenopus laevis α-N-catenin , using the following primer pair: Left 5′-TCGAGCGGCCGCGCCACCATGTCTTCTGCC ACATCACCCATCATTCTCAAATGG-3′ ; Right 5′-TCGAAGGCCTTTAGA GGCTAGCATAATCAGGAACATCATACGGATAGAAT GAATCCATAGCTTT AAATTC-3′ and sub cloned into the NotI and StuI sites of the pCS107 expression vector. Capped mRNA was synthasized by linearizing the template with AscI and the SP6 mMESSAGE mMACHINE Kit. Xenopus tropicalis N-cadherin–Myc, and Xenopus laevis E-cadherin-HA mRNA were synthasized as previously described C-cadherin mRNA used in this study were synthesized using the Sp6 mMESSAGE mMACHINE Kit (Ambion) using the same construct as previously described Anti-sense oligos (designated as AS throughout the text) used in this study are the following:5′-ATGTTTCCTGTATTGAGAGTCATGC-3′, α-N-catenin morpholino: 5′-GGCAGAAGACATGTTCCTCTATTG -3′ . A dose of 40 ng of α-E-catenin morpholino was injected into oocytes for maternal depletion experiments, and doses of 10–20 ng of either alpha catenin morpholino (per cell) was used for 2–8 cell stage embryonic injections.C-cadherin AS, 5′-C*C*T*CTCCAGCTCCCT*A*C*G-3′ in-situ protocol using double Dig labeled custom LNA mRNA detection probes (Exiqon). For α-N-catenin: 5DigN/ATGCACAACTGACACTACAAT/3DigN; for α-E-catenin: 5DigN/TACGCTCACCAGAAACCAGT/3DigN probes were used. Probes were resuspended in hybridization buffer to make 20 µM (40X) stock solutions, and were then hybridized at a 0.5 µM final concentration at 53°C. Samples were developed using BM Purple detection reagent (Roche) at 4°C. After the stainings were complete, samples were washed, and fixed in Bouin’s fixative overnight and bleached using a standard bleaching solution , and a fluorescent light source to remove pigments.Wholemount In-situ’s for α-E-catenin, and α-N-catenin, were carried out using a standard Xenopus C-, and E-, cadherin at a 1∶200 dilution as previously described Late blastula Stage 9,  animal cE, F, G, H and E, F, G, H were created using the Nikon image analyser volume function. The 3D projections in S4, were created using Bitplane’s Imaris software.A Zeis LSM 510 inverted confocal microscope was used for imaging immunostained samples in this study. A LD C-Apochromat 40X/1.1 W korr UV-vis IR objective was used for visualizing both F-actin and C-cadherin staining under high magnification. Same imaging settings including the laser power level, gain, and pinhole size were used between all groups compared in an single experiment. Imaging conditions were set so that more than 90% of the imaged pixels were in the linear range in the control group of an experiment. All immunostaining pictures represent maximum intensity projections of Z-stacks. High resolution imaging of cleared samples and sections  and equal volumes of supernatants were removed and diluted in equal volumes of 4X sample buffer. Samples were then boiled for 5 minutes and 2 embryos equaling lysate  was loaded in to a single lane of a 8% SDS PAGE gel and run for 2 ½ hours at 100 volts. Protein bands were then transferred into nitrocellulose membranes using the wet-transfer method and blocked in 5% non-fat milk. For C-cadherin staining, the 6B6 monoclonal antibody, and for E-cadherin, the 5D3 monoclonal antibody, were used at 1∶1000 and incubated at 4°C overnight. For α-tubulin, the DM 1A monoclonal antibody  was used at 1∶12000 dilution and incubated for no more than 3 hours in room temperature. For α-E-catenin staining, the rabbit ployclonal antibody against human α-E-catenin  was used at 1∶1000 and incubated at 4°C overnight. For β-catenin staining, a mouse monoclonal antibody aginst human β-catenin (E5) (Santa Cruz antibodies Cat. # Sc-7963) was used at 1∶2500 dilution. For HA staining, the rat anti-HA monoclonal antibody 3F10 (Roche) was used at 1∶5000 dilution. For Myc tag staining, a rabbit polyclonal anti-Myc antibody  was used at 1∶5000 dilution. After washing the membranes for at least 1hour in PBSTw, HRP conjugated goat anti mouse, or anti rabbit, secondary antibodies (Jackson Immuno Research) were used at 1∶5000 dilutions and incubated for 1 hour at room temperature. ECL western blot developing solutions (Amersham) were used to detect the secondary antibodies after a 1 hour PBS wash step. Membranes were then exposed to X-ray films accordingly to obtain unsaturated bands and the films developed using a standard western blot-developing machine. For stripping and reprobing membranes, Onemiute westernblot stripping buffer (GM Biosciences) was used as directed.5 embryos or oocytes for each experimental group were frozen in dry ice and lysed using 50 µl of ice-cold PBSTx (PBS+1% Triton X-100) containing 1 mM PMSF and 0.01% PIC . Samples were then cleared by a low speed centrifugation step  was used.For each immunoprecipitation (IP), at least 50 embryos were used per sample. All immunoprecipitation experiments were carried out using st. 19 embryos and protein-G conjugated agarose beads (Roche), as previously described Movie S1Cyst formation in α-E-catenin depleted embryos. 10 ng of αEC morpholino was injected to one cell at the two cell stage. Embryos were cultured in1XMMR following gastrulation. Photographs for the timelpase were taken at 5 minute intervals.(MOV)Click here for additional data file.Movie S2Blockade and rescue of neurulation movements in α-N-catenin-depleted embryos. Embryo on the left is an uninjected control. Middle embryo was injected with 20 ng of αNC morpholino in to each dorsal animal cell of the 8 cell stage, the embryo on right was injected with a morpholino-resistant αNC mRNA following the injection of the morpholino.(MOV)Click here for additional data file.Movie S33D-image projection of a cleared, E-cadherin and α-E-catenin stained non-neural ectoderm, aquired by high resolution confocal microscopy. E-cadherin is stained green and αEC is stained red.(MOV)Click here for additional data file.Movie S43D-image projection of a cleared, C-cadherin and α-E-catenin stained non-neural ectoderm, aquired by high resolution confocal microscopy. C-cadherin is stained green and αEC is stained red.(MOV)Click here for additional data file.


However, the relationship between these two pathways is not understood. We targeted the E-cadherin JMD to mitochondria (WT-JMD) to isolate this domain from the plasma membrane and internalization, and to examine protein modifications and degradation. WT-JMD localized to mitochondria, but did not accumulate there except when proteasome activity was inhibited. We found WT-JMD was ubiquitinated, and arginine substitution of lysines at position 5 (K5R) and 83 (K83R) resulted in the stable accumulation of mutant JMD at mitochondria. p120-Catenin did not localize, or bind to WT-JMD even upon proteasome inhibition, whereas the K5,83R-JMD mutant bound and localized p120-catenin to mitochondria. Mutation of the p120-catenin binding site in combination with these lysine mutations inhibited p120-catenin binding, but did not decrease JMD stability or its accumulation at mitochondria. Thus, increased stability of JMD lysine mutants was due to inhibition of ubiquitination and not to p120-catenin binding. Finally, mutation of these critical lysines in full length E-cadherin had similar effects on protein stability as WT-JMD. Our results indicate that ubiquitination of the JMD inhibits p120-catenin binding, and targets E-cadherin for degradation. For example, Epithelial Growth Factor Receptor (EGFR) is ubiquitinated and internalized leading to the recycling of EGFR and/or the degradation of both receptor and its ligand  was transiently expressed in WT-JMD stable cells. Total proteins were extracted under normal conditions in both mock and Ub-HA transfections, upon proteasome inhibition, or NEM treatment (to inhibit de-ubiquitinating enzymes). Sequential immunoblotting were performed using mouse monoclonal antibodies for tubulin, and then HA. A protein smear was present throughout the lane in whole cell lysates of WT-JMD cells transfected with Ub-HA only after immunoblotting for HA , lane 12n) in four different experiments as either a single band  did not appear to change when Src-GFP was over-expressed , and wasExpression of Hakai-FLAG in cells stably expressing WT-JMD affected the level of WT-JMD ubiquitination. Hakai-FLAG expression in WT-JMD cells decreased the level of WT-JMD compared to mock transfections , lane 12Western blots of p120-catenin showed a reduction in the amount of p120-catenin bound to WT-JMD upon proteasome inhibition in Hakai-FLAG expressing cells compared to the small amount present in mock transfections under similar conditions . The fasAs an independent test that WT-JMD instability was due to ubiquitin-dependent proteasomal degradation, we examined the JMD sequence for lysine residues that could be targets for ubiquitination. Two lysines are present within the JMD domain, and they were mutated to arginine : lysine The level of K5R-JMD appeared greater than WT-JMD, but less than ActA , while tp120-Catenin binds to the E-cadherin JMD These results indicate that p120-catenin was not recruited to the E-cadherin JMD. Surprisingly, p120-catenin was also not recruited to the E-cadherin JMD when proteasomal degradation was inhibited. This result suggests that p120-catenin binding to WT-JMD is inhibited.In light of the fact that we could not find p120-catenin co-localized with, or bound to WT-JMD, we asked whether increased levels of the JMD lysine mutants were due to the lack of ubiquitination or p120-catenin binding. Significantly, p120-catenin clearly co-localized with K5R-JMD  and K5,8RFP immunoprecipitation was used to quantify the levels of the JMD lysine mutants and examine p120-catenin binding. Transiently transfected MDCK cells were extracted in the presence or absence of proteasome inhibition. The level of WT-JMD increased 4-fold upon inhibition of the proteasome, as expected -graph. TAlthough the data thus far indicate a correlation between increased stability of JMD and increased p120-catenin binding to the JMD, it is also possible that increased JMD stability is due to the lack of ubiquitination. To test whether the increased level of K5,83R-JMD was due to loss of ubiquitination or increased binding of p120-catenin, we mutated the binding site for p120-catenin These results indicate that K5R-JMD contains a mutation in the primary ubiquitination site, and that mutation of K5 within WT-JMD allows the recruitment of p120-catenin to the JMD.To test directly if p120-catenin affects WT-JMD levels, we depleted p120-catenin by ∼95% using specific siRNAs compared to p120-catenin levels in WT-JMD and ActA stable cells treated with scrambled siRNAs  or contained lysine mutations ; the RFP fusion proteins were isolated by RFP immunoprecipitation from whole cell lystates . The levPlasma membrane protein levels are regulated by post-translational modifications such as ubiquitination, and phosphorylation in vivo that were dependent upon JMD recruitment of p120-catenin, Hakai and Src to the mitochondria. Importantly, we found little or no affect of expression of mitochondrial targeted WT-JMD on endogenous E-cadherin, β-catenin and α-catenin levels, nor the organization or migration of an epithelial sheet. Therefore, this approach did not appear to affect the cell generally, and thus could be used as an in vivo assay for JMD binding to p120-catenin compared to JMD ubiquitination, and the consequences on JMD degradation.p120-Catenin binds the JMD in the cytoplasmic domain of E-cadherin, and is required for E-cadherin stability at the plasma membrane Although p120-catenin binds to a minimal octapeptide sequence within E-cadherin JMD When we examined WT-JMD for ubiquitination in the presence of NEM or Ub-HA, we detected a small amount of ubiquitinated protein (JMD-Ub), which was sensitive to the deubiquitination enzyme Usp2. The bulk of WT-JMD under any of these conditions did not appear to be ubiquitinated but still did not bind p120-catenin. We suggest that WT-JMD was ubiquitinated in cells, but deubiquitinating enzymes were released upon cell lysis and deubiquitinated most of the WT-JMD, even in the presence of NEM. We cannot rule out other modifications of JMD such as tyrosine phosphorylation that might have additional effects on sterically inhibiting JMD/p120-catenin binding.Significantly, p120-catenin was recruited to the JMD that had mutations of specific lysine residues . These mutations blocked JMD degradation and resulted in JMD accumulation. We showed that K5R-JMD was sufficient to inhibit proteasomal degradation of JMD, and result in p120-catenin binding to either mitochondrial targeted JMD or full length E-cadherin K5R mutants. An additional mutant AAA-K5,83R deleted the p120-catenin binding site in combination with both lysine mutations and also resulted in accumulation of JMD, but, as expected, did not recruit p120-catenin. Therefore, stabilization and accumulation of JMD was conferred by the lysine mutations that resulted in inhibition of JMD degradation, and not binding of p120-catenin.in vitro studies of JMD and p120-catenin binding will be needed to fully test this possibility.The K5R mutation is 17 amino acids upstream of the p120-catenin binding site and increased the stability of the JMD to a greater extent than K83R, which is closer to the β-catenin binding domain of the E-cadherin cytoplasmic tail. We suggest that p120-catenin binding to JMD might, therefore, depend on the state of K5 ubiquitination; the lack of ubiquitination in K5R mutants allows p120-catenin binding, whereas the addition of an ubiquitin to wild-type K5 might sterically inhibit p120-catenin binding. Further Previous studies expressed a truncated E-cadherin containing the extracellular domain and only the first 15 amino acids of the JMD, which included K5. This mutant E-cadherin did not accumulate within the plasma membrane, but instead colocalized with endocytic markers It has been proposed that the E3 ligase Hakai ubiquitinates E-cadherin in a Src-dependent manner and p120-catenin is not bound during this process in vitro binding studies will be needed to test this hypothesis directly.These data, combined with p120-catenin binding to mutant K5R-JMD, further indicate that once ubiquitination of K5 has occurred p120-catenin cannot bind to E-cadherin and thereby confer stability to E-cadherin. On the other hand, binding of p120-catenin to the JMD might mask the primary ubiquitination site in E-cadherin (K5) thereby preventing ubiquitination, internalization and degradation. Since Hakai binds E-cadherin JMD With the close proximity of p120-catenin and ubiquitination sites in the JMD it is difficult to determine exactly how p120-catenin confers stability on E-cadherin. The E-cadherin-JMD/p120-catenin binding interface includes a static region of protein-protein interaction that contains the highly conserved octapeptide sequence, and a more dynamic region upstream containing K5 Our results support the hypothesis that competitive binding between p120-catenin and modifications of E-cadherin by Hakai (ubiquitination) or Src (tyrosine phosphorylation) at the JMD regulate E-cadherin degradation. While it is possible that p120-catenin binding to the JMD may inhibit Src or Hakai binding and hence ubiquitination and degradation, our results show that ubiquitination on at least K5 inhibits p120-catenin binding, and results in proteasomal degradation of E-cadherin. In a broader context, ubiquitination of E-cadherin JMD may block or even displace p120-catenin, resulting in E-cadherin internalization from the plasma membrane and rapid proteasomal degradation.ActA-RFP Fluorescence Activated Cell Sorting (FACS) to increase the number of positive RFP cells, and then selected with 0.3 mg/ml of G418 for 10–14 days. Cells positive for RFP were plated at single cell density, and single cell clones were isolated using cloning rings, expanded and replicates were frozen and stored in liquid nitrogen. The use of DNA and generation of cell lines is approved by Stanford APLAC .Cell lines were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS (Cell Generations). MDCK cells MDCK cells were transfected with plasmids  using LipofectAMINE 2000 (Invitrogen) according to manufacturer’s instructions. Cells were plated for 40 hours prior to experiments. All transiently transfected cell lines were transfected on the same day and time for direct comparison between all cell lines. Ub-HA, Hakai-FLAG and Src-GFP were transfected into WT-JMD cell line. Mock transfections were performed with transfection reagents only.MDCK cells were incubated with siRNAs using LipofectAMINE 2000 (Invitrogen). Scrambled siRNA was siGENOME non-targeting siRNA #3 , p120-Catenin siRNA utilized the sequence CAGUCAAGAAAGUG-GUGAAUU (Dharmacon siRNA Technologies). 100 ng of siRNA was transfected with 10 ul of lipofectAMINE in a final volume of 200 ul in reduced serum media. Medium was replaced 24 hours post-transfection with DMEM, 10% FBS without antibiotics.Stable cell lines were plated on collagen I-coated coverslips and grown for 40 hours. Transiently transfected cells were plated on collagen I-coated coverslips 6 hours post-transfection and grown for 40 hours. Coverslips were rinsed with PBS-ME and fixed in 3% paraformaldehyde in PBS-ME for 13 minutes at room temperature (RT). After washing, cells were incubated in 0.5% saponin in PBS-ME for 15 minutes, followed by blocking buffer  for 1 hr at RT. Cells were incubated with primary antibodies in blocking buffer overnight at 4°C, washed in PBS-ME, incubated with the secondary antibody for 30 min at RT, and washed in PBS-ME. The following antibodies were used: rabbit RFP ; p120-catenin ; mouse E-cadherin antibody raised to extracellular domain ; mouse ZO-1 ; mouse vinculin ; mouse tubulin ; mouse β-catenin ; mouse α-catenin . Secondary antibodies used were FITC-conjugated goat anti-mouse antibody  and Rhodamine-conjugated goat anti-rabbit antibody . Cells were mounted in Vectashield containing DAPI. Images of antibody-stained cells were taken using a Zeiss Axiovert 200 microscope and Axiovision software . All images within an experiment were taken using the same exposure on the same day. Images were adjusted identically within the same experiment using Adobe Photoshop to optimize image quality.2) with 0.1 ug/ml Pefabloc. RFP containing protein chimeras were immunoprecipitated using a rabbit polyclonal RFP antibody at 1∶100 dilution . E-cadherin immunoprecipitations used E2 antibody raised against the cytoplasmic domain of E-cadherin All extractions were performed at 4°C. Cells were washed twice in TBS. Cells were extracted with 0.1% Triton X-100 in CSK buffer  for 1 hr at RT. Blots were incubated with antibodies overnight at 4°C. The following antibodies were used: rabbit RFP , p120-catenin , mouse tubulin , mouse β-catenin , mouse HA.11 , mouse GFP . Secondary antibodies used were goat anti mouse 800CW  and goat anti-rabbit Alexa Fluor . Membranes were scanned using an Odyssey imager and software (LI-COR Biosciences) and bands were quantified using Image J. Resulting images of entire blots were adjusted using Adobe Photoshop and cropped only after all adjustments were performed. In all cases an n of 1 is equivalent to analysis from 1 gel and subsequent blot.2+ in DMEM) and plated to confluency on collagen I-coated 35 mm glass bottom dishes (MatTek) for 75 minutes. Medium was replaced with DMEM containing 1.8 mM Ca2+ and incubated for 3hours. A single scratch was made along the length of the dish using a 1 ml pipette tip and the media was replaced with imaging media . Cells were imaged at 10X magnification every 15 min for 15 hours at 37°C on an inverted microscope (Axiovert 200 M) controlled with Slidebook software (Intelligent Imaging Innovations); 5 sites were imaged along the wound edge. The total wound area covered was quantified using Image J software. Time zero was 15 min post-scratch.Cells were trypsinized and resuspended in low calcium media  Immunofluorescence images of MDCK stably expressing ActA or WT-JMD. Cells were fixed and labeled for E-cadherin, ZO-1, vinculin, tubulin, phalloidin (green) and RFP (red). All images were from the same experiment and processed identically between cell lines. Scale bar is 25 µm in 100X images. (B) Still images of MDCK cells and MDCK cells stably expressing either ActA or WT-JMD at 0 and 15 hours of wound healing assays. Scale bar is 100 µm in 10X images (C) Quantification of total area covered during 15 hours of a wound healing assay of MDCK cells, and MDCK cells stably expressing either ActA or WT-JMD.(TIF)Click here for additional data file.Figure S2ActA and WT-JMD localize at mitochondria. (A) Endogenous RFP fluorescence of MDCK cells stably expressing ActA or WT-JMD under normal conditions and upon proteasome inhibition. Mitotracker was used to identify mitochondria. Images were taken on the same day at the same exposure, and brightness and contrast were adjusted identically between cell lines to demonstrate locazlization of RFP with mitochondria. Scale bar is 25 µm in 100X images.(TIF)Click here for additional data file.Figure S3- and α-Catenin do not localize at mitochondria. (A-B) β-catenin (A-green) or α-catenin (B-green) and RFP (red) immunofluorescence images of MDCK cells stably expressing ActA or WT-JMD under normal conditions and upon proteasome inhibition. Images were taken and processed under identical conditions. Scale bar is 25 µm in 100X images.β(TIF)Click here for additional data file.Figure S4Lysine Mutations Differentially Affect p120-catenin localization. (A-D) MDCK cells were transiently transfected with either K5R-JMD (A), K83R-JMD (B), K5,83R-JMD (C) or AAA-K5,83R-JMD (D). Cells were fixed and processed for immunofluorescence for RFP and p120-catenin. Insets are cropped area denoted in 100X images . All images were from the same experiment and processed identically. Scale bar is 25 µm in 100X images and 5 µm in insets.(TIF)Click here for additional data file.


Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy. The canonical Wnt/β-catenin signaling pathway plays a key role in embryogenesis and cellular homeostasis and regulates cell fate, differentiation, proliferation and self-renewal of stem cells and progenitor cells. The activity of the central signaling molecule β-catenin is mainly determined by regulation of proteolysis by the β-catenin destruction complex including the principal constituents casein kinase 1α (CK1α), glycogen synthase kinase 3 (GSK3), adenomatous polyposis coli (APC) and axin Several studies have shown that the Wnt/β-catenin signaling pathway regulates formation of neural-crest derived melanocytes and by this influences melanocyte development We recently described that melanoma cells developed an efficient new mechanism to activate the β-catenin signaling pathway by suppression of CK1α expression, defining CK1α as a novel tumor suppressor in melanoma We observed that RNA expression of the β-catenin target genes fibronectin (FN1) and axin (Axn2) were upregulated in primary melanomas and melanoma metastases compared to benign nevi . Furthermore, expression of either lentivirally delivered shRNA against β-catenin or doxycycline-inducible knockdown of β-catenin using shRNA efficiently reduced the amount of β-catenin protein . Inhibition of β-catenin expression and signaling in metastatic melanoma cell lines  using PKF115–584 treatment resulted in a dose-dependent reduction of viability . Furthermore, knock down of β-catenin expression in metastatic melanoma cell lines using doxycycline-inducible shRNA significantly inhibited proliferation of the metastatic melanoma cells lines SKMEL28 and 451LU (50) are summarized in To evaluate the role of β-catenin during melanoma progression we downregulated β-catenin in several well-defined human melanoma cell lines derived either from primary melanomas or from melanoma metastases , Figure S5). Similarly, the inducible shRNA against β-catenin strongly reduced melanoma cell migration and showed a reduced invasiveness after shRNA induction compared to the non-induced cells . Furthermore, inhibition of β-catenin induced mitochondrial swelling and the mitochondria showed degraded cristae indicating strong mitochondrial damage and cytochrome C release .To reveal the mechanism of cell death induced by β-catenin downregulation in metastatic melanoma cell lines we analyzed whether apoptosis is induced by β-catenin downregulation using shRNAs or PKF115–584 treatment. Cell cycle analysis showed that β-catenin downregulation leads to a reduced G2/M cell population and the emergence of a subG1 fraction indicative for apoptosis induction , whereas proliferation and migration of benign melanocytes are not affected. Several other studies showed that the loss of β-catenin in a number of differentiated cell types is compatible with normal cell viability and tissue structure. Posthaus et al. showed that β-catenin signaling is dispensable during proliferation and early terminal differentiation of epidermal keratinocytes in vitro and in vivo their biological behaviour of the original tumor cells and are therefore representative members of melanoma cells in the different progression stages Human metastatic melanoma cell lines were cultured in RPMI 1640 medium with 10% fetal bovine serum , penicillin and streptomycin. The non-metastatic melanoma cell lines were maintained in “2% Tumor” medium composed of MCDB153∶L15 (4∶1) supplemented with 2% FBS and 5 µg/ml insulin, unless otherwise stated. The isolation and culturing of these cell lines have been described elsewhere All cell lines used for expression analyses were cultured 24 hours before RNA extraction in the same medium (RPMI/10%FCS). DNA-free total RNA was extracted with the NucleoSpin RNA II kit  and 1 µg RNA reverse transcribed with Superscript II using hexamer primers into first strand cDNA. The cDNA was used with the corresponding primers  into a SYBR green real-time PCR and analyzed in a LightCycler480  by the ΔΔCt-method. The following primer sequences were used:Gene sense antisensettgttacaggaagtcccttgcc atgctatcacctcccctgtgtgACTINB gaataagctgtaccatcgcaaa tgtaaccaccagtctcatgtggFN1 atgattccatgtccatgacg cttcacactgcgatgcatttAXN2 5 cells per well) were seeded in 6well plate cavities and grown for 24 h. Cells were transfected using Lipofectamin 2000  with 2.0 µg Super8xTOPFlash and transferred into 96 well plates. 36 hours after transfection cells luciferase activity was measured in lysates using the Dual Luciferase Reporter Assay (Promega) in a TriStar luminometer . RLU firefly values were background subtracted and normalized to the protein concentration of the lysate.Melanoma cells  in melanoma cell growth medium. After gelation, 1 ml of growth medium was applied on top and inhibitors were added in the indicated concentrations into the feeding 1 ml. Every treatment regimen was set up in triplicates. After 14 days, the formed colonies were counted in every well and photomicrographs were taken.Migration and invasion assays were performed in Boyden chambers coated with or without a Matrigel basement membrane matrix  as described previously 2. Readings were taken every 15 min for 24 h and plotted curves represent the averages from three independent wells/measurements.The kinetics of cell invasion was assayed using the xCELLigence Real-Time Cell Analyzer . CIM-plate 16 wells were pre-coated with 30 µl of matrigel diluted 1∶10 in DMEM for 1 h at 37°C, then 10,000cells were plated in each well in fibroblast conditioned serum-free DMEM. The lower medium chamber contained fibroblast conditioned DMEM with 10% FCS. Cells were allowed to settle for 30 min at room temperature before being placed in the RTCA DP in a humidified incubator at 37°C with 5% COin vivo tumor growth assays, 8×105 451LU cells stably expressing tetracycline-inducible shRNA against β-catenin were subcutaneously injected into SCID mice. 3 weeks post injection small tumor nodules had developed in all the mice. One group was subsequently fed with 200 µg/ml doxycycline in the drinking water for one week. Tumor size was monitored for six weeks post injection by measuring tumor length, width and height. The tumor volume was calculated using the formula for ellipsoids (V = 4/3π*a*b*c). In case of subcutaneous injection of the radial growth phase melanoma cell line SbCl2, 2×106 cells were injected in 100 ul PBS with 10% matrigel into the flanks of the mice. Subcutaneous tumor nodules were prepared four weeks post injection and the tumor size was determined. All animal experiments were approved by the Regierungspräsidium Tübingen (AZ HT6/06).For the Lentiviral particles were produced in HEK 293T cells  using the previously described transfer plasmids of Skokowa et al. Cells were washed, centrifuged and the resulting pellets were fixed for 24 hours in Karnovsky's fixative , 4% paraformaldehyde (Merck), 0.2 M Na-cacodylate (Merck). After centrifugation, the sediment was embedded in 3% agarose at 37°C and then cooled on ice. Post-fixation was based on 1.0% osmium tetroxide containing 1.5% K-ferrocyanide in Aquadest for 2 hours. After embedding in glycide ether the blocks containing cells were cut using an ultra microtome  and ultra-thin sections (30 nm) were examined using a Zeiss Libra120 transmission electron microscope  operating at 120 kV.3 cells were seeded into 96-well plates and cultured for the indicated periods of time. After washing, 4-methylumbelliferyl heptanoate in PBS (100 µg/ml) was added and incubated for 1 h at 37°C. Microplates were measured in a fluorescence microplate reader  with Ex355/Em460 nm in quintuplicates. In case of lentiviral shRNA transfer, the increase of DsRed fluorescence which is expressed only in transduced cells was measured from day 2 to day 6 using a microplate fluorescence reader . This fluorescence intensity correlates in a linear way to cell number.For the analysis of proliferation and survival of melanoma cells, 2.5×105 cells were permeabilized in ice-cold 70% ethanol for at least four hours and resuspended in 500 µl PBS with 50 U RNAseA  and 50 µg/ml of propidium iodide . After 30 min cells were analyzed in a LSRII FACS .2×10the following antibodies were used as recommended by the manufacturer: anti-cytochrome c ; anti-COX IV .5 cells were resuspended in 50 µl binding buffer  containing 5 µl of AnnexinV-FITC  and stained for 15 min. Then 10 µg/ml of propidium iodide  was added for 5 min and the cells were analyzed in a LSRII FACS.After the indicated time of culture, 1×104 cells were seeded into 96 well plates in quintuplicates, treated as indicated and lysed in 50 µl caspase lysis buffer . 150 µl reaction buffer containing 50 mM HEPES pH 7.4, 100 mM NaCl, 2 mM CaCl2, 5 mM DTT, 10% sucrose, 0.1% CHAPS and 70 µM DEVD-AMC  were added to each well and fluorescence was measured every 15 min for one hour in a Tristar microplate reader  at Ex488 nm/Em512 nm.2×104 cells per well) were seeded in 24well plates and grown for 24 h. Cells were transfected using Lipofectamin 2000  with 0.4 µg Super8xTOPFlash. 36 hours after transfection cells luciferase activity was measured using the Dual Luciferase Reporter Assay (Promega) in a TriStar luminometer . RLU firefly values were background subtracted and normalised to protein content.Melanoma cells (8×10http://rsb.info.nih.gov/nih-image/) by using the actin signal as a reference.30 µg of lysate were subjected to SDS PAGE and blotted onto PVDF membrane . The following antibodies were used: anti-Mcl-1, anti-Bcl-xl , anti-Bcl-2, anti- β-catenin, anti-Cyclin D1, anti-C-myc , anti- β-Actin, anti-CK1α and anti-LaminB . For preparation of nuclear protein extracts the cells were trypsinised and lysed in a hypotone buffer containing 0.5% NP-40. The detergent insoluble fraction containing nuclei was further treated with a hypertone buffer. Semiquantification of western blots was done with the freeware software tool scion image  Pharmacological β-catenin inhibition reduces β-catenin signaling and protein level in melanoma cells. SkMel28 melanoma cells were treated with 1 µM of the inhibitor PKF115–584 and nuclear β-catenin expression was analyzed by confocal immunofluorescence microscopy over time  (left panel). In addition, β-catenin-TCF/LEF signaling was determined by a Super8xTOPflash reporter assay. PKF115–584 inhibits the signaling in a concentration dependent manner (right panel). Asteriks denote significant differences as measured by t-test with one asteriks being p<0.05, two asteriks being p<0.01 and three asterisk being p<0.005 (t-test). (B) PKF115–584 treatment reduces protein levels of β-catenin, Cyclin D1, Mcl-1 and Bcl-xl. Shown are western blot analyses of melanocytes (NHM), WM 35, WM115, WM793, 451LU, 1205LU, Mewo and SKMel28 melanoma cells treated for 24 hours with increasing concentrations of PKF115–584 (0–2 µM). Lysates were analyzed for expression of β-catenin, cyclin D1, Mcl-1, Bcl-xl and actin. The colorectal carcinoma cell line HCT116 served as a reference cell line.(TIF)Click here for additional data file.Figure S2(A) Inhibition of β-catenin expression by shRNA reduces β-catenin protein level in melanoma cells. SkMel28 and 451LU melanoma cells were lentivirally transduced with shRNA against β-catenin or luciferase as a control. Total protein lysates were produced at the indicated time points and 30 µg of lysate were subjected to a β-catenin western blot (two top panels). On the right semiquantification of the western blots  is shown. (B) Inhibition of β-catenin expression by inducible shRNA reduces nuclear β-catenin protein level. Two clones stably expressing a Docycycline-inducible shRNA against β-catenin were generated from the parental cell lines SKMel28 and 451LU with pcDNA6/TR and pTER-β-catenin. Both cell lines were induced with 1 µg/ml doxycycline for the indicated time points. Protein isolates of the induced clones revealed a β-catenin downregulation in nuclear protein fractions after 4 days (shown is the 451LU clone). As shown in the right diagram β-catenin RNA expression measured by real-time PCR is reduced after doxycycline treatment in the clones expressing shRNA against β-catenin.(TIF)Click here for additional data file.Figure S3PKF115–584 treatment induces apoptosis in metastatic melanoma cells. (A) AnnexinV-FITC/PI staining: SKMel28 melanoma cells were treated for 24–72 hours with increasing concentrations of PKF115–584 (0–2 µM) and stained with AnnexinV plus PI to detect apoptotic and necrotic cells in a flow cytometry analysis. In a time and concentration dependent manner the melanoma cells got AnnexinV positive (apoptotic) before ending as AnnexinV and PI double positive dead cells. Only a small amount of cells were necrotic as indicated by the early appearance of AnnexinV/PI +/+ cells. (B) Caspase 3 activity: 451LU and SKMel28 melanoma cells were treated with 1 µM PKF115–584 for 24 h or 48 h and cell lysates were used to measure the DEVD-AMC cleavage activity as indicator for activated caspase 3. The treatment induced a significant (p<0.01) higher caspase activity of 10–30-fold, normalized to untreated controls. A staurosporine treatment for 4 h served as a positive control.(TIF)Click here for additional data file.Figure S4shRNA against β-catenin reduces melanoma cell growth of metastatic melanoma cell lines (raw data to ). Different melanoma cell lines were lentivirally transduced with shRNA against β-catenin or a non-silencing control (both with RFP as a marker). The proliferation of the metastatic melanoma cell lines 451LU, SKMel28, 1205LU and Mewo ceased. Shown are the measured fluorescence values of RFP expression of transduced cells over time, normalized to the first day of measurement (day 2 after transduction). Increased RFP values are indicative for an increase in cell number/cell proliferation.(TIF)Click here for additional data file.Figure S5Ablation of β-catenin activity in melanoma cells inhibits migration, invasion (raw data to). Melanocytes (NHM) and non-metastatic melanoma cell lines or metastatic melanoma cell lines were used for Boyden-chamber experiments. Matrigel coated transwells were used to perform invasion assays and non-coated transwells to assay the cell migration capability after treatment with 0.5 µM PKF115–584 for 12 hours or after the induced expression of shRNA against β-catenin for 24 hours. Cell viability was not affected as seen by trypan blue staining. Shown is the number of counted cells per field. All reductions in migration and invasion were significant except for NHM (p<0.05).(TIF)Click here for additional data file.