CTNNB1 gene, leading to a deregulation of the WTN/β-catenin pathway, is a common feature of desmoid tumors (DTs). Many β-catenin inhibitors have recently been tested in clinical studies; however, BC2059 , a selective inhibitor of nuclear β-catenin that works through binding TBL-1, is the only one being evaluated in a clinical study, specifically for treatment of desmoid tumor patients. Preclinical studies on BC2059 have shown activity in multiple myeloma, acute myeloid leukemia and osteosarcoma. Our preclinical studies provide data on the efficacy of BC2059 in desmoid cell lines, which could help provide insight regarding antitumor activity of this therapy in desmoid tumor patients. In vitro activity of BC2059 was evaluated using desmoid tumor cell lines. Ex vivo activity of BC2059 was assessed using an explant tissue culture model. Pharmacological inhibition of the nuclear β-catenin activity using BC2059 markedly inhibited cell viability, migration and invasion of mutated DT cells, but with lower effect on wild-type DTs. The decrease in cell viability of mutated DT cells caused by BC2059 was due to apoptosis. Treatment with BC2059 led to a reduction of β-catenin-associated TBL1 in all mutated DT cells, resulting in a reduction of nuclear β-catenin. mRNA and protein levels of AXIN2, a β-catenin target gene, were also found to be downregulated after BC2059 treatment. Taken together, our results demonstrate that nuclear β-catenin inhibition using BC2059 may be a novel therapeutic strategy for desmoid tumor treatment, especially in patients with CTNNB1 mutation.Mutation in the  CTNNB1 gene (encoding for β‐catenin) which results in nuclear accumulation of β-catenin [Desmoid tumors (DTs) are rare fibroblastic lesions that can occur anywhere in the body. Although considered benign due to their lack of metastatic potential, DTs have a high risk of local recurrence and can be very locally invasive, thereby significantly decreasing patient quality of life. The estimated incidence in the general population is two to four per million population per year . Desmoid-catenin . Mutatio-catenin –8, exempWhile many therapeutic options are available, the standard treatment for desmoids remains uncertain, due to their rarity, there is a lack of randomized clinical trials to inform therapeutic comparisons . When feCTNNB1-mutated desmoid cell lines. We have also confirmed BC2059-related decreased nuclear β-catenin levels and the decreased expression of Axin2, a downstream target of β-catenin after BC2059 treatment. In addition, BC2059 decreased cell viability and β-catenin activity in an ex vivo desmoid tumor model, thus providing possible predictive insights regarding antitumor activity of this therapy in desmoid tumor patients.Giving the importance of Wnt/β-catenin pathway in many malignancies, several drugs to inhibit the β-catenin gene have been developed. However, there are significant challenges in targeting the Wnt/β-catenin pathway, including identifying effective compounds that do not affect β-catenin-mediated normal functions in cellular repair and tissue homeostasis –20 SeverCTNNB1 by Sanger sequencing to confirm that the cell lines were truly desmoid tumor cells. Iterion Therapeutics  provided BC2059.All DT cells included in this study were created in the Sarcoma Research Lab at the Ohio State University and the MD Anderson Cancer Center. This study was conducted with approval from both the Ohio State University and the MD Anderson Cancer Center institutional review board (IRB) with written informed consent of patients. All cell lines and corresponding tumors were genotyped for CTNNB1 wild-type cells. The IC50 values were determined using GraphPad Prism Version 6.05 software. The capacity to migrate and invade was evaluated as previously reported [CellTiter-Glo Luminescent Cell Viability Assay (Promega) assessed cell viability after six days of exposure to BC2059 for the mutated-DT cells and after thirty days for the reported . BrieflyCell cycle progression and apoptosis induction using Annexin V-PI staining (BD Biosciences) were measured as previously described . CaspaseThe collected cells were lysed in 1x lysis buffer  containing protease inhibitor. The immunoprecipitation was performed using the PureProteome™ Magnetic Bead according to the manufacturer’s instructions. Briefly, the protein lysate was incubated with 2μg of the β-catenin antibody  overnight at 4°C. PureProteome™ Magnetic Bead (Millipore) was added to a 1.5 mL microcentrifuge tube and washed with binding buffer using the PureProteome™ Magnetic Stand (Millipore). The antibody-antigen sample was added to the beads, and the mixture was incubated at room temperature for 30 minutes with continuous mixing. Finally, 40 μl of loading buffer was added to the beads and boiled for 10 minutes. Elutions were collected using the PureProteome™ Magnetic Stand and loaded into the SDS-PAGE gel for protein analysis. TBL-1 (Santa Cruz) antibody was used for the immunoprecipitation assay.Western blotting analysis were performed as previously described . BrieflyAxin2 probe (Hs00610344) (ThermoFisher). Relative expression levels were normalized against β-actin and GAPDH RNA expression.Total RNA was extracted using RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. cDNA was generated using TaqMan® Reverse Transcription Reagents (ThermoFisher) and analyzed by quantitative real-time PCR using StepOnePlus™ Real-Time PCR System, using Explant culture was performed as previously described . Brieflyt test analysis assessed statistical significance between experimental groups. For cell viability analysis, Two-way ANOVA test followed by Tukey post-test were performed. P < 0.05 was considered statistically significant.Unpaired two-tailed Student This study was conducted with approval from both the Ohio State University and the MD Anderson Cancer Center institutional review boards (IRB).CTNNB1 mutations found in desmoid patients . BC2059 exhibited potent cytotoxicity, particularly against cell lines bearing CTNNB1 mutations, with IC50 values in the low nM range . We next evaluated the impact of BC2059 on desmoid tumors migration and invasion. After BC2059 treatment, CTNNB1 wild-type desmoid cells showed markedly less decreases in migration and invasion as compared to mutated DT cells (P<0.001)  .CTNNB1-mutated subset of DT cells that underwent apoptosis as per the previous flow analysis  , resultiCTNNB1-mutated desmoid cells; however, no significant effect was observed in CTNNB1 wild-type cells Click here for additional data file.S2 FigTransducin β-like protein 1 (TBL1) recruits β-catenin to the Wnt target gene promoter. BC2059 disrupts the binding of β-catenin to TBL1, facilitating β-catenin destruction, resulting in the inhibition of the Wnt/β-catenin pathway.(TIF)Click here for additional data file.S1 Raw images(ZIP)Click here for additional data file.


APC (Adenomatous Polyposis Coli) are reflected in β-Catenin hyperactivation and loss of proliferation control. Certain intestinal bacteria metabolites have shown the ability to limit CRC cell proliferation and CRC pathogenesis. Here, we investigated the molecular mechanism underlying the anti-proliferative activity of butyrate, a microbiota-derived short chain fatty acid, in two CRC cell lines, namely HCT116 and SW620, which bear a mutation in β-Catenin and APC, respectively. In particular, we focused on autophagy, a lysosome-dependent degradation pathway, which was shown to control intestinal tissue homeostasis. Butyrate reduced CRC cell proliferation, as witnessed by the downregulation of proliferation markers. TCGA bioinformatic transcriptomic analysis of CTNNB1 (β-Catenin) gene correlation in CRC patients showed that β-Catenin negatively correlates with the autophagy gene ATG4D. In CRC cells, regardless of the mutational state of APC or β-Catenin genes, butyrate caused the autophagy-mediated degradation of β-Catenin; thus, preventing its transcriptional activity. Autophagy gene silencing restored β-Catenin levels, allowing it to translocate into the nucleus to promote the expression of downstream genes associated with cancer cell proliferation. CRC-affected patients show driver mutations in the WNT pathway; thus, targeting its crucial effector may be a promising therapeutic strategy in CRC treatment; for instance, by using ad hoc probiotics that stimulate autophagy.Colorectal cancer (CRC) pathogenesis is mainly driven by alterations in WNT signaling, which results in altered transcriptional activity of β-Catenin. Mutations in  Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer-related death globally, with one million new cases diagnosed every year and 600,000 deaths per year, the incidence being higher in developed countries .APC) [CRC is a heterogeneous disease at genetic level, due to the many different mutations occurring in the epithelium of the large intestine , and an APC) .APC [APC mutation has been linked to intestinal microbiota dysbiosis [Interestingly, the canonical WNT/β-Catenin pathway has been found to be constitutively activated in 80% of colorectal tumors , and theAPC . Consistysbiosis . The staysbiosis . When thysbiosis ,10. Mutaysbiosis . A similysbiosis . These mysbiosis . HoweverAPC, along with β-Catenin mutated in the phosphorylation domain, and a truncated APC mutant, along with wild-type β-Catenin, respectively [Here, we addressed these issues, employing two CRC cell lines, namely HCT116 and SW620, that express a wild-type ectively ,15,16,17We investigated the fate of β-Catenin in these cells exposed to butyrate, the microbiota metabolite that has been shown to prevent CRC through inhibition of the Wnt signaling . We hypoAutophagy (herein meaning macroautophagy) is a catabolic lysosome-driven process that is crucial for the maintenance of cellular homeostasis and found to be deregulated in cancer . AutophaCTNNB1) gene negatively correlated with the autophagy gene ATG4D. Notably, butyrate promotes the interaction of β-Catenin with LC3, indicating its sequestration within the autophagosome. The genetic silencing of the autophagy gene ATG7 restores the level of β-Catenin and abrogates the cell cycle arrest in CRC cells exposed to butyrate, confirming that, in CRC cells bearing mutations in the machinery for the degradation of β-Catenin, this probiotic metabolite still elicits anti-proliferative activity, by activating an alternative degradation of the downstream effector of the WNT pathway. The present findings may translate to clinics through the designing of probiotic supplements that enhance autophagy in intestinal cancer cells as pre- and post-operative adjuvant therapies.Here we show that butyrate counteracts CRC cell proliferation through induction of autophagy. Accordingly, interrogation of the cancer genome atlas (TCGA) transcriptome in CRC patients revealed that the β-Catenin (v/v% air: 5 v/v% CO2) in Dulbecco’s Modified Eagle Medium  and RPMI-1640 Medium , respectively, supplemented with 10% heated-inactivated fetal bovine serum , 1% glutamine  and 1% penicillin/streptomycin . Treatments included 50 ng/mL interleukin-6 , 1 mM and 2 mM sodium butyrate , and 30 μM chloroquine . All three reagents were dissolved in sterile water.Human colorectal cancer HCT116 (CCL-247™) and SW620 (CCL-227™) cell lines, with different genetic background  and tumo2. The day after, cells were counted (time 0) and then treated with 2 mM sodium butyrate for 24, 48, and 72 h. At the end of each experimental time point, cells were trypsinised and collected, and cell suspension was diluted 1:1 with trypan blue , a dye that stains dead cells. Cell counting was performed in triplicate for each experimental condition. For cell cycle analysis, cells were fixed in 70% ice-cold ethanol and stored at −20 °C. Before starting the cytofluorimetric analysis, RNAse (50 μg/mL) was added to cells for 30 min at 37 °C. Cells were stained with propidium iodide  and acquired using a FacScan flow cytometer . A fraction of 5000 events was analyzed for each sample. The processing of the data obtained was performed using the free tool Flowing Software 2.5.1 .Cells were plated in p35 petri dishes at the density of 40,000–50,000 cells/cmThe following primary antibodies (at the dilution indicated) were used for either immunofluorescence or Western blotting: rabbit anti-Ki67 ; mouse anti-p21 ; mouse anti-Histone H3 ; mouse anti-LC3 ; rabbit anti-LC3 ; mouse anti-LAMP1 ; rabbit anti-β-Catenin ; rabbit-anti ATG7 ; mouse-anti ATG7 ; mouse anti-β-tubulin ; rabbit anti-GAPDH ; mouse anti-β-actin .2 and treated as indicated when confluence reached approximately 80%. Cell homogenates were prepared by freeze–thawing and ultrasonication in Ripa lysis buffer containing protease inhibitors. Equal amounts of proteins (25 μg) from total lysates were separated on SDS-PAGE and then transferred onto PVDF membranes . The membranes were blocked with 5% non-fat dry milk  +0.2% Tween for 1 h at room temperature. Then, membranes were incubated with specific primary antibodies overnight at 4 °C, followed by incubation with secondary HRP-conjugated antibodies  for 1 h at room temperature. The bands were detected using enhanced Chemiluminescence reagents  and developed with a ChemiDoc XRS instrument . Intensity of the bands was estimated by densitometry using Image Lab 6.0 . Western blotting data were reproduced at least three times, separately.Cells were seeded in p35 petri dishes at the density of 40,000–50,000 cells/cm2, left to adhere, and treated as reported. At the end of the experiment, the coverslips were fixed within ice-cold methanol, permeabilized with 0.2% Triton-PBS, and incubated overnight at 4 °C with specific primary antibodies dissolved in 0.1% Triton-PBS + 10% FBS. The following day, coverslips were incubated for 1 h at room temperature with secondary antibodies (diluted in 0.1% Triton-PBS + 10% FBS), and either AlexaFluor488-conjugated goat-anti rabbit IgG  or AlexaFluor555-conjugated goat-anti mouse IgG , as appropriate. Nuclei were stained with UV fluorescent dye DAPI . Coverslips were mounted onto glass using SlowFade reagent  and imaged with a fluorescence microscope .Cells were seeded onto sterile coverslips at the density of 25,000–30,000 cells/cmPoly(2-hydroxyethylmethacrylate) or polyhema  was dissolved in 95% ethanol and left under rotation overnight at 50 °C. The stock solution was diluted in ethanol to obtain a working solution of 120 mg/mL. P35 petri dishes were coated with this solution, left under a biological hood to completely dry, and then stored at room temperature until used. Cells were seeded at the density of 500,000 cells/p35 and treated according to the experimental conditions. The growth of spheroids was monitored by taking pictures with a phase-contrast microscope at each time point. The quantification of the dimensions of the spheroids was analyzed with ImageJ software 1.51 .Three-dimensional immunofluorescence was performed on spheroids cytospotted on glass slides at day 5. Spheroids were fixed in methanol, permeabilized for 30 min in 0.5% Triton-PBS, and processed as described above .To assess cell proliferation in 3D spheroids, we employed plasma membrane staining with a vital fluorescent dye that undergoes dilution with cell division. Cell lines were stained with 1μM DiD  in serum-free medium at 37 °C for 30 min, then dissolved in complete medium, plated as described above for 3D spheroids, and treated as indicated. At day 0 and day 5 the spheroids were cytospotted on glass slides. The intensity of Did retention was immediately acquired with a fluorescence microscope  and then quantified with the software ImageJ 1.51 .This assay has previously been described in detail . EssentiATG7 was performed with small interference RNA (siRNA) technology in cells cultivated on coverslips (for immunofluorescence purpose) or in petri dishes (for Western blotting purpose). Cells were plated on coverslips at the density of 20,000–30,000 cells/cm2 and on petri dishes at the density of 50,000–60,000 cells/cm2 and left to adhere for 24 h before transfection. Cells were transfected with siATG7  using Lipofectamine 3000 Reagent , as described previously [Post-transcriptional silencing of eviously . The siRhttps://cancergenome.nih.gov, accessed on 2 October 2021). The mRNA gene expressions were downloaded from cBioportal.org [RNA-seq and clinical data of 526 colorectal adenocarcinoma  patients were downloaded from the TCGA data repository . In the current study, CTNNB1 gene expression was correlated with 20,040 other groups of genes using Spearman’s correlation coefficient analysis and p-values were obtained. TBtools  was used to identify the differentially expressed genes (DEGs) in correlation with CTNNB1, which are represented in the form of Volcano plots. To identify the DEGs, cut-off criteria were set based on Spearman’s correlation value  or lower than −0.25 (negatively correlated) and p-value < 0.0001  threshold was fixed above 5.0) [https://david.ncifcrf.gov/summary.jsp, accessed on 10 October 2021) was used to analyze gene ontology (GO) biological processes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were obtained with the help of positive and negative-differentially expressed genes. Data are presented in bar graphs showing the number of transcripts belonging to each positively and negatively associated biological process [RNA-seq data of colorectal adenocarcinoma samples were downloaded from the TCGA database (ove 5.0) . The DAV process .p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.All data refer to at least three separate experiments performed by different operators. Data in histograms are shown as average ± S.D. GraphPad Prism was employed (GraphPad Software Inc.) for statistical analysis. The statistical significance of the data was given by t student test and one-way ANOVA analysis of variance followed by Bonferroni’s test. Differences were considered statistically significant for * = APC, β-Catenin (CTNNB1) and GSK3β in 526 colorectal adenocarcinoma patient samples . The oncoprint  is altered in 7% of the cases, and GSK3β is altered in just 2.5% of cases. Moreover, the comparison of available CRC cell lines shows the presence of APC or β-Catenin (CTNNB1) mutations, confirming that these mutations are mutually exclusive. Additionally, genetic mutations of GSK3β are absent or undetectable [APC or β-Catenin (CTNNB1) genetic alterations, respectively, as representative of the majority of CRC diagnosed.We searched the TCGA database for the presence of altered ncoprint  shows thtectable . TherefoThe cell counting of viable (trypan blue-excluding) HCT116 and SW620 colorectal cancer (CRC) cells exposed to 2 mM sodium butyrate (NaB) showed a large decrease in cell population, compared to untreated culture counterparts, from 48 to 72 h A. We perWe moved to a 3D spheroid model that better mimics the in vivo tumor growth . FurtherWe monitored the ability of colon cancer cells to form spheroids for up to 5 days. Butyrate impaired the growth of CRC spheroids, as indicated by the fact that the diameter remained the same at day 0. IL-6 increased the dimension of CRC spheroids starting from day 2, and this effect was significantly counteracted by NaB  gene expression in CRC growth, we performed an in-silico transcriptomic analysis in a dataset of colorectal adenocarcinoma patients. We retrieved the RNA-Seq data (mRNA expression profile) from the TCGA database (PanCancer Atlas dataset) and performed a co-expression analysis to identify the genes that are positively and negatively correlated with CTNNB1. Up to 1000 differentially expressed genes (DEGs) were identified, out of which 501 genes were positively correlated (up-regulated) and 499 genes negatively correlated (down-regulated) with CTNNB1. We selected the most significant 250 DEGs, which are represented in the Volcano plot reported in CTNNB1-positively and CTNNB1- negatively regulated DEGs. As shown in CTNNB1-positively correlated genes are significantly enriched for the regulation of the mitotic cell cycle, cell proliferation, angiogenesis, cell-to-cell communications, response to growth factor signals, cell migration, and epithelial to mesenchymal transition. In more detail, these genes are associated to PI3K (phosphatidylinositol 3-kinase), MAPK (mitogen-activated protein kinase), and Wnt signaling pathways. In contrast, the CTNNB1-negatively correlated genes are significantly enriched for the regulation of apoptotic, proteolytic, and autophagy-lysosomal pathways. Of relevance to our initial hypothesis, high expression of CTNNB1 correlated with low expression of ATG4D gene, which codes for the autophagy protein ATG4 that contributes to autophagosome building by processing MAP-LC3 [To obtain an insight into the functional role of  MAP-LC3 .In line with our hypothesis and based on the gene correlation analysis, we then investigated whether butyrate exerted its anti-proliferative action on CRC cells by inducing β-Catenin degradation and whether this was carried out via autophagy. As shown in Next, we investigated whether and how autophagy is modulated by butyrate in CRC cells. The treatment with butyrate induced the conversion of the cytosolic LC3-I isoform into the autophagosome-associated LC3-II isoform, suggestive of autophagy . An incrWe then investigated whether autophagy was responsible for the reduced level of cellular β-Catenin in butyrate-treated CRC cells. We first checked by immunofluorescence whether β-Catenin was co-localized with autophagy-lysosomal organelles, using LC3 and LAMP1 as markers for autophagosomes and lysosomes, respectively. Butyrate increased the number of LC3-positive organelles and promoted their fusion with LAMP1-positive organelles , confirming the induction of autophagy and neo-genesis of autophagosomes A. This sTo definitively confirm that β-Catenin was sequestered  within the autophagy-lysosomal vacuoles, we performed a co-immunoprecipitation assay to check whether this protein indeed interacted with LC3 upon butyrate challenge. Data in ATG7, which codes for one of the proteins involved in the post-translational lipidation of LC3-I mandatory for autophagosome formation. Effective inhibition of autophagy was indicated by the reduced level of ATG7 protein and accumulation of unprocessed LC3-I in siRNA-transfected cells  was greatly reduced, and this was paralleled by the decreased co-localization with β-Catenin (yellow puncta) and increased signal of the latter (green puncta) C. These To verify whether the restoration of β-Catenin following autophagy disruption rescued cell proliferation, we assayed in the autophagy knocked-down cells the expression of Ki67, marker of cell proliferation, and of p21, marker of cell cycle block. To this end, we performed the immunofluorescences double-staining Ki67/LC3 and LC3/p21. Images show that the siATG7-transfected cells where formation of autophagosomes was efficiently prevented (as indicated by low LC3 puncta) also expressed a low level of p21 and increased level of Ki67, indicative of cycling cells D. These Taken together, these data support the view that the knock-down of autophagy abrogates the butyrate-induced degradation of β-Catenin and results in the rescue of cell proliferation.The gut microbiota has been implicated in CRC development ,37. MicrImportantly, genetic alterations of the WNT/β–Catenin pathway play a pivotal role in the stepwise development of colorectal cancers . In thesTo address these issues, in the present study we employed two CRC cell lines defective in the proteasomal degradation of β-Catenin. In particular, the HCT116 cell line carries the S45del mutation of β-Catenin, resulting in the deletion of an amino acid at a GSK3β phosphorylation site, conferring a gain of function to β-Catenin ,15, and CTNNB1 (β-Catenin) expression performed on a database of CRC-affected patients showed that it is positively correlated to several genes involved in biological processes, which include mitotic cell cycle process, cell migration, epithelial-to-mesenchymal transition (EMT), regulation of gene expression; in contrast, CTNNB1 expression negatively correlated with genes exerting a tumor suppressive role and genes involved in the autophagy-lysosomal pathway, particularly ATG4D. Starting from this correlation analysis and having proven that NaB can induce autophagy, we investigated the link between Wnt pathway and autophagy.Given the critical role of the Wnt pathway in colorectal carcinogenesis , here weAPC and β-Catenin genes, and that this effect is mechanistically linked with the autophagy-mediated degradation of β-Catenin. We demonstrate that butyrate promoted the translocation of β-Catenin into autophagosomes and autolysosomes. In a co-immunoprecipitation assay, β-Catenin was not decreased with LC3 in control cells, indicating that, in this condition, it was free to translocate into the nucleus for the transcription of genes promoting cell proliferation. By contrast, in butyrate-treated cells, β-Catenin was precipitated along with LC3, indicative of autophagosome sequestration. Our finding agrees with the notion that β-Catenin contains an LC3 interacting domain (W/YXXI/L motif), which directs the target substrates for selective autophagy degradation [We tested the hypothesis that autophagy could mediate the beneficial effects of microbiota metabolites, using butyrate as representative of the latter. Here, we provide evidence that butyrate induces the growth arrest of cell proliferation in CRC cells bearing dysfunctional mutations of the radation . AutophaOf potential clinical relevance, butyrate could also elicit its beneficial effect in 3D spheroid cultures, which better resemble the in vivo growth of cancers, and even in the presence of IL-6, an inflammatory cytokine that is abundantly present in the tumor microenvironment and that has been reported to inhibit autophagy ,45. IL-6In summary, here, we demonstrated the functional role of autophagy in the protective and anticancer activities of the microbiota metabolite butyrate. This may open the way for novel approaches in the management of CRC. Currently, drug repositioning to inhibit both the Wnt/β-Catenin and autophagy pathways represents a new frontier of targeted cancer therapies .Innovative therapeutic approaches, having reduced side effects and enhanced effectiveness for increasing the survival rate in colorectal cancer-affected patients, are urgently needed. Since diet affects the composition and the function of commensal bacteria, one adjuvant approach could be the use of dietary supplements that promote a “non-carcinogenic” microbiota and increase the probiotic strains producing butyrate.


We evaluated the mechanistic link between circadian rhythms and gut barrier permeability. Mice were subjected to either constant 24-h light (LL) or 12-h light/dark cycles (LD). Mice housed in LL experienced a significant increase in gut barrier permeability that was associated with dysregulated ß-catenin expression and altered expression of tight junction (TJ) proteins. Silencing of ß-catenin resulted in disruption of barrier function in SW480 cells, with ß-catenin appearing to be an upstream regulator of the core circadian components, such as Bmal1, Clock, and Per1/2. In addition, ß-catenin silencing downregulated ZO-1 and occludin TJ proteins with only limited or no changes at their mRNA levels, suggesting post transcriptional regulation. Indeed, silencing of ß-catenin significantly upregulated expression of matrix metallopeptidase (MMP)-2 and MMP-9, and blocking MMP-2/9 activity attenuated epithelial disruption induced by ß-catenin silencing. These results indicate the regulatory role of circadian disruption on gut barrier integrity and the associations between TJ proteins and circadian rhythms, while demonstrating the regulatory role of ß-catenin in this process. With rising disruption of light–dark cycles in human populations due to night-shift work, excessive use of artificial light, and/or travel across different time zones, there is increasing concern for the effects of circadian rhythm misalignment on susceptibility to disease. Circadian rhythms act as ~ 24 h pacemaker for the human body and its individual cells. The “master clock”, so named for its direct synchronization to light and its influence over all other peripheral clocks throughout the body, is located in the suprachiasmatic nucleus (SCN) of the hypothalamus . AlthougThe core members of the mammalian molecular clock are transcription factors Bmal1 and Clock that bind to the E-box promoter and activate the transcription and translation of Per and Cry genes –6. The pWhen circadian rhythms are disrupted by chemical or environmental interference, processes regulating homeostasis are altered, contributing to the development of various diseases, such as gastrointestinal diseases, sleep disorders, metabolic syndromes, and cancer formation . MechaniThe gut barrier is a multilayer system composed of tight junction (TJ) proteins such as zonula occludens (ZO)-1, tricellulin, and occludin that regulate its functions . TJ protThe exact mechanistic interrelationships between the circadian rhythm and gut barrier integrity are not fully understood. Therefore, the aim of the present study is to examine the link between circadian rhythm disruption and alterations of gut barrier permeability. Our data indicate that loss of barrier function as the result of circadian disruption is mediated, at least in part, by ß-catenin-induced modulation of circadian clock gene and protein expression as well as upregulation of matrix metallopeptidase (MMP) 2/9 leading to a reduction in TJ protein expression.Mice were subjected to constant 24 h light (LL) for 4 weeks, with the control group maintained under 12-h light/dark cycles (LD). Voluntary wheel running was measured to evaluate behavioral circadian rhythms affected by exposure to constant light. All mice showed regular running rhythms at baseline and in normal light (LD) Fig. . The phaIn addition to wheel running, body temperature was measured as a second method to evaluate circadian rhythmicity. Following two weeks of normal light, mice showed a regular pattern of body temperature, elevated during the night/active time (18:00 [ZT12] until 06:00 [ZT0]) and decreased during the day/resting time. Mice exposed to circadian disruption displayed an arrhythmic pattern in body temperature throughout the day  experienced a significant increase in intestinal permeability as compared to LD controls both in the ileum Fig. A and thep = 0.015, p = 0.023, and p = 0.007, respectively)  Fig. A. The loIn order to investigate a potential correlation between β-catenin and clock gene expression, we returned to in vitro studies based on human colon SW480 cells. β-catenin was silenced using specific siRNA, followed by analysis of clock genes mRNA and protein expression Fig. . SilenciTo evaluate the impact of clock genes on epithelial barrier function, β-catenin, Clock, or Bmal1 expression was silenced in SW480 cells with specific siRNAs, followed by measuring permeability for FITC-dextran 20 kDa in the Transwell system. Permeability across SW480 monolayers was significantly increased by 24% and 27% upon silencing with ß-catenin siRNA at 20 and 100 nM, respectively Fig. A. In addTaking into consideration the substantial impact of β-catenin on the regulation of epithelial barrier function and the role of β-catenin in circadian rhythm regulation, we next analyzed the consequence of β-catenin knockdown on TJ protein expression. Silencing of β-catenin with 20 nM of specific siRNA did not alter ZO-1 mRNA levels; however, it decreased its protein levels Fig. C. Similap < 0.01)  Fig. C, suggesCircadian rhythms are critically important to maintain normal gut function, to provide intestinal barrier function, to engage bacterial dissemination, and to regulate inflammation, which when disrupted can have several pathophysiological consequences –54. The The gut barrier is vital for maintaining homeostasis and performing functions such as providing a selective barrier, degrading pathogens and antigens, preventing bacterial adhesion and colonization, and initiating an immune response , 56. CirIn the present study, circadian rhythm disruption through light cycle manipulation led to dysregulation of ß-catenin and circadian rhythm molecules Bmal1 and Clock as well as disrupted TJ protein expression of occludin and ZO-1 at both the mRNA and protein levels Figs.  and 4. TΔ19/Δ19) treated group − [CT of target gene − CT of housekeeping gene]untreated control group. For the treated samples, evaluation of The total RNA was isolated and purified using RNeasy Mini Kit (Qiagen) according to the protocol of the manufacturer. Then, 1 µg of total RNA was reverse transcribed at 25 °C for 15 min, 42 °C for 45 min and 99 °C for 5 min in 20 µl of 5 mM MgCl earlier . The dat2, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 1 × EDTA-free protease inhibitor cocktail [Roche Applied Science], and 1 mM phenylmethylsulfonyl fluoride) for the total cell extract. Protein concentration was determined using BCA protein assay kit . Then, 10 μg protein of cell lysates was electrophoresed on SDS–polyacrylamide gels, transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with 3% BSA in PBS-T (0.1% Tween-20) solution and incubated with the primary antibodies overnight at 4 °C. After incubation with the secondary antibody for 2 h, immunoblots were visualized using the ECL detection system (Amersham Biosciences). GAPDH or actin was determined as the loading control. The band density was measured using Image J software (NIH).Protein expression levels of β-catenin, Bmal1 and Clock, Per1 and Per2, Cry1 and Cry2, ZO-1, occludin and tricellulin were assessed in the intestinal epithelial cell-enriched fractions of the isolated colon or in SW480 cells by immunoblotting. All primary antibodies were purchased from ThermoFisher, and HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology . Briefly, gut homogenates or treated cells were lysed with RIPA lysis buffer (1.0% Nonidet P-40, 0.5% deoxycholic acid, 0.2% SDS, 40 mM Tris–HCl [pH 7.6], 1 mM EDTA, 1 mM EGTA, 10 mM MgClp < 0.05 was considered statistically significant. The results are expressed as means ± S.D. All experiments were repeated at least 3 times.The data were statistically analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. Statistical probability of


Wnt/β-catenin signaling, also referred as canonical Wnt signaling, is a highly conserved cellular pathway that regulates multiple biological processes involved in normal physiological and disease processes, including cell proliferation and differentiation, tissue regeneration, migration, autophagy, and apoptosis. The main human disorder associated with dysfunctional Wnt/β-catenin signaling is cancer . Indeed,This Research Topic entitled “Targeting the Wnt/β-catenin Signaling Pathway in Cancer” represents a review of the current knowledge of the intrinsic link between Wnt/β-catenin signaling and cancer. The published original research and review articles are briefly described below:Zhou et al. investigated the molecular mechanisms by which uterine leiomyoma (UL) cells proliferate and unveiled that the specific long noncoding RNA Alu-mediated p21 transcriptional regulator (APTR) was overexpressed in UL human tissues and promoted cell proliferation by increasing the expression of the proteins of the Wnt/β-catenin signaling pathway and by directly interacting with the estrogen receptor alpha (ERα).Raoul et al. reported the clinical outcome and molecular profile of two cases of metastatic pancreatoblastomas, a rare pancreatic tumor typically found in young children. In this report, they demonstrated that both patients benefited from systemic chemotherapy (FOLFIRINOX regimen). Furthermore, by performing high-throughput sequencing of the tumors analyzed, they found specific mutations of members of the Wnt/β-catenin pathway.Behrooz and Syahir provides an overview of the state-of-the-art of the interplay between CD133, Wnt/β-catenin and TERT signaling pathways in glioblastoma multiforme (GBM) to demonstrate that this axis may represent a possible therapeutic target against this aggressive cancer type.She et al. demonstrated that the Fas apoptotic inhibitory molecule 2 (FAIM2) is highly expressed in non-small cell lung cancer (NSCLC) tissues and NSCLC tissues with bone metastasis, and promotes cell proliferation, migration, and invasion, and inhibits cell apoptosis. Interestingly, the authors unveiled that FAIM2 exerts these effects by regulating the Wnt/β-catenin signaling pathway.Ding et al. identified high expression levels of tumor endothelial marker 8 (TEM8) in samples and cells of lung adenocarcinoma. Such increased levels were associated with tumor size of primary tumor. Mechanistic investigations performed both in vitro and in vivo showed that TEM8 promoted lung cancer cell proliferation and invasion by activating Wnt/β-catenin signaling.Ke et al. found that exposure to polycyclic aromatic hydrocarbons  suppressed the microRNA (miR), 377-3p, and downregulated the expression of early growth response 1 (EGR1) to facilitate malignant transformation and tumor growth of lung cells by regulating the Wnt/β-catenin pathway.Zheng et al. demonstrated that the protein A-kinase interacting protein 1 (AKIP1) was elevated in diverse thyroid carcinoma cells and regulated different aspects of tumorigenesis  by controlling the activity of PI3K/AKT and β-catenin pathways.Shah et al. explored the current knowledge of the phosphorylation-dependent regulations of β-catenin and Wnt signaling in cancer providing a detailed overview of the main kinases which regulate the Wnt/β-catenin pathway.Wang et al. performed a study aimed to investigate the significance of Wnt in the prognosis and tumor immunity in NSCLC and found that WNT2B and WNT7A might have prognostic value in lung adenocarcinoma and in squamous cell lung cancer. Authors performed this investigation by using a publicly available tumor database and validated their findings in NSCLC pathological tissues and cell lines.The Wnt/β-catenin signaling pathway was discovered over 30 years ago and has been extensively studied as a key cellular pathway involved in both physiological and pathological processes. While it has been widely reported that Wnt/β-catenin signaling plays a critical role in multiple aspects of tumorigenesis from early stages of malignant transformation to invasion and metastasis, there are no approved anticancer drugs that target this pathway for either the treatment or prevention of cancer. Nonetheless, multiple lines of evidence suggests that targeting the Wnt/β-catenin pathway with small molecule inhibitors can selectively inhibit proliferation and induce apoptosis of cancer cells to inhibit tumor growth. Despite all these aspects, the exact function of Wnt/β-catenin signaling, all of its molecular components, and other intracellular signaling pathways  that may intersect with Wnt/β-catenin signaling, within the context of normal physiology and cancer biology remain only partially understood. A more complete understanding of these questions holds promise for providing insight to novel molecular targets and the development of efficacious anticancer drugs for a wide range of malignancies that are driven by Wnt/β-catenin signaling.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.SP was supported by Fondazione Umberto Veronesi and by A-ROSE. DB was supported by the American Board of Obstetsrics & Gynecology, the Norma Livingston Foundation, and the Foundation for Womens Cancer. GP was supported, in part, by the National Cancer Institute of the National Institutes of Health under awards R01CA197147, R01CA254197, and R01CA238514.We thank all the authors who contributed to this Research Topic as well as various reviewers/editors of the respective manuscripts, for their efforts, timely responses and enthusiasm. We also thank the Frontiers Editorial Office for their assistance and support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


While this adhesive showed good lap-shearstrength and shear resistance under static load and temperature, thecuring process was slow. In order to speed up the curing process,a thiol (trimethylolpropane tris(3-mercapto propionate)) was addedand its impact on the curing process as well as on the adhesive propertieswas evaluated. The trifunctional thiol additive allowed for fastercuring in the presence of the 1,1,3,3-tetramethylguanidine basic catalyst.Moreover, a combination of NH2–PHU–NH2 and the thiol as curing agents for the epoxy resin resultedin adhesives with superior toughness, without any deterioration ofthe ultimate lap-shear strength or shear resistance under load andtemperature, making these adhesives suitable for high-demand applicationsin the automotive industry.The polyadditionbetween dicyclic carbonates and diamines leadingto poly(hydroxy urethane)s (PHUs) has emerged as the preferred methodfor the synthesis of green, non-isocyanate polyurethanes. However,when proposed for use as structural adhesives, the long times forcompletion of aminolysis of the 5-membered cyclic carbonates underambient conditions force the use of complementary chemistries to acceleratethe curing process. In this work, a system that combines an amino-terminatedPHU (NH Some technologieshave decreased cycle times from hours to minutes, especially when(high) temperatures can be applied, making adhesives more cost-effective.It has to be noted that the automotive part manufacturers’process is not adapted to the adhesive, but rather it is the adhesivethat is adapted to the needs of the automotive production lines toget the best performance.Polymer-based adhesives are widely employedin the automotive industryas they allow for a reduction in the weight of the final product andalso increase the safety in terms of energy absorption when comparedto spot welding, riveting, or clinching.3 Their excellent performance, flexibility, and ability to cure underambient conditions in short times make PU-based adhesives an appealingoption in this field. Nonetheless, the hazardous monomers, particularlyisocyanates, which are demonstrated to provoke different illnesses6 as well as the chemicals related to their synthesis, such as phosgene,7 are at odds with current global sustainabilitytrends. Thus, the hunt for harmless and greener alternatives to isocyanatesrepresents one of the biggest challenges in PU industry nowadays.This automotive and transportationindustry constitutes the largestconsumer/end-user application of polyurethane (PU) adhesives, forwhich the market size is projected to grow to USD 9.1 billion by 2024.2 insertioninto epoxy precursors, and the limited moisture sensitivity of thesemonomers.10 However, the slow aminolysis of 5-membered cycliccarbonates at room temperature forces researchers to employ high temperaturesto prepare PHUs that compete with isocyanate-based PU adhesives.11 To overcome this limitation, hybrid systemsthat combine PHUs with other chemistries such as epoxy, sol–gel,or composites have been reported.12 Thecreation of hybrid systems through the combination with epoxy chemistryis particularly popular and has been shown to lead to PHUs with betteradhesion performance19 and also to improve their properties for other applications, suchas coatings.24Poly(hydroxy urethane)s (PHUs) prepared through the polyadditionreaction between dicyclic carbonates and diamines is one of the mostpopular routes to prepare green non-isocyanate PUs (NIPUs) due tothe 100% atom economy of the process, the facile preparation of 5-memberedcyclic carbonates through chemical [3 + 2] CO26 (i) the one-stepterpolymerizationof epoxy and cyclic carbonates with amines and (ii) the chain extensionof amino-terminated PHU (NH2–PHU–NH2) prepolymer with the epoxy resin. The amino-terminated prepolymerstrategy is preferable as the reaction between amines and epoxidesis much faster than the one between amines and cyclic carbonates  takes place by a simple nucleophilicattack of the thiolate to the epoxy groups.32 The protonation of the alcoholate anions via either the base catalystor the thiol gives rise to the final β-hydroxyl S-alkylatedstructure. A remarkable feature of thiol-epoxy condensation is thatit can contribute to the acceleration of the initial hardening ofthe adhesive and decreasing the time required to reach handle strength.Meanwhile, NH2–PHU–NH2 will reactmore gradually and will contribute to the final mechanical propertiesof the adhesive.In our search for systems that could compete toisocyanate-basedPUs and that displays enough reactivity at room temperature, we hypothesizedthat the curing process may be sped up by adding a new component tothe formulation, a thiol compound to promote thiol-epoxy “click”chemistry 1. Thiol-In this work, PHU-thiol-epoxy hybrid adhesivesare prepared andcured at room temperature using an epoxy resin with a combinationof amino-terminated PHU and a thiol compound. First, the influenceof the curing agent ratio, that is, PHU and thiol, on the curing stepis explored through rheological measurements. Moreover, the impactof catalyst on this step is investigated by integrating a strong base,TMG, into the formulation. After assessing the curing parameters,the influence of curing agent ratios on ultimate adhesion performancewas addressed by lap-shear and shear adhesion failure temperature(SAFT) measurements, while development of the lap-shear strength atdifferent times was established through lap-shear tests. Additionally,physical characterization of the materials was performed by dynamicmechanical thermal analysis (DMTA), water uptake, and gel contentmeasurements to evaluate the feasibility of the developed materialsas structural adhesives.Mn ∼ 640 g mol–1) (PPGDGE),resorcinol diglycidyl ether, tetrabutylammonium iodide (98%), trimethylolpropanetris(3-mercaptopropionate) (≥95.0%) (TMPTMP), 1,12-diaminododecane(98%) , and TMG (99%) were purchased from Sigma-Aldrich.1,3-bis(2-hydroxyhexafluoroisopropyl)benzene (97%)was purchased from Fluorochem. Viscous epoxy resin based on bisphenolA (trade name: EPIKOTE Resin 828) and stainless steel AISI 316 werekindly supplied by Oribay Group. Deuterated dimethylsulfoxide (DMSO-d6) and methanol (MeOH-d4) were purchased from Sigma-Aldrich and Eurisotop, respectively.All reagents were used without further purification. The cyclic carbonatesemployed for this work were synthesized by CO2 couplingwith commercial precursors as reported elsewhere.33Poly(propylene glycol) diglycidyl ether  of PPGdiCCand RdiCC were calculated according to mC5 isthe mass of cyclic carbonate introduced into the nuclear magneticresonance (NMR) tube; nfunction of carbonate is the molar amount of function carbonate in cyclic carbonate; Ia, Ib, and Ic are integrations of characteristics peaks a, b, and c of carbonate,respectively; ntoluene is the molar amountof toluene introduced in standard solution; and ICH3 is the integration of peak CH3 of toluene.The CEW values for each cyclic carbonate were obtained in triplicatedeterminations and are presented in Table S1Titration of cyclic carbonates was performedas described elsewhere.–3 equiv),RdiCC , and 1,12-DAD were added toa 100 mL round-bottom flask equipped with a half-moon Teflon helixstirrer and put into an oil bath at 80 °C. Mechanical stirringat 100 rpm was kept for 5 h. Then, the stirring was stopped, the reactionwas cooled down, and amino-terminated PHU oligomers were obtainedand used for the further preparation of H-NIPU materials without purification.PPGdiCC. The number of active hydrogensof amine was fixed at 1 for one amine function reacting with one cycliccarbonate. The active hydrogen equivalent weight (AHEW) was calculatedfrom eq d4. The characteristic signal at2.65 ppm of the methylene group next to the free amine was chosen.The AEW is calculated using 3 of toluene and of the two methylene protons of NH2–PHU–NH2, respectively. Measurements werereplicated three timesThe amine equivalent weight (AEW)is the amount of resin (in grams) containing 1 g equivalent of aminefunctions. It was determined following the procedure used for thedetermination of the CEW. Toluene standard solution was prepared inMeOH-The amine hydrogen equivalent weight(AHEW) for primary amines was calculated from –3 equiv), TMPTMP , and EPIKOTE 828  were weighed in a 26mL vial. The reactants were stirred for 2 min at 50 °C, followedby 1 min at room temperature to obtain a homogeneous mixture. Then,the viscous oligomeric solution was transferred into a plastic 6 mLsyringe for applying onto the substrates for the preparation of lap-shear(∼80 mg) or SAFT (∼200 mg) joints.The synthesis of a 70/30 compositionis described as a typical procedurefor the preparation of hybrid materials. Amino-terminated PHU oligomer was first mixed with TMG  and then, TMPTMP  was added. Mixing was done as previously explained.The homogeneous mixture was applied onto substrates and second substrateswere placed after 20 min, allowing the adhesive to get enough viscosityto avoid spreading out from the bonding line. The addition of TMGwas necessary as epoxy and thiol compounds do not react without thepresence of a catalyst.For the preparationof 0/100 composition, EPIKOTE 828  spectra were obtained using an FT-IR spectrophotometer equipped with attenuatedtotal reflection (ATR) with a diamond crystal. Spectra were recordedbetween 4000 and 525 cmG″)and storage modulus (G′) (gel time). The experimentswere carried out using 15 mm disposable parallel plate geometry.Time sweepexperiments were performedin a stress-controlled Anton Paar Physica MCR101 rheometer at roomtemperature at a frequency of 1 Hz and a strain of 1% in order todetermine the crossover between loss modulus .38DMTA experimentswere performed using a rectangular sample of the crosslinked materials(2 × 3.5 × 1 mm) using a Triton 2000 DMA from Triton Technologyin single cantilever bending deformation mode. Tests were performedat 1 Hz at a heating rate of 4 °C minms is theweight of the swollen sample and mi isthe initial weight of the sample.The swelling index was measured afterSoxhlet extractions in refluxing THF for 24 h, wiping samples to removeresidual THF before weighing. Values were calculated withmf is thefinal weight of the dried sample and mi is the initial weight of the sample.Thegel content was measured by Soxhletextraction in refluxing THF for 24 h. Afterward, samples were driedin oven at 70 °C for 24 h. Values were calculated withms is theweight of the swollen sample and mi isthe initial weight of the sample.Three samples (∼30mg) of each formulation were immersed separately into 10 mL of deionizedwater for 96 h. The equilibrium water content (EWC) was calculatedby34 For lap-shear test, a parallel force to theadhesive bond with a displacement rate of 1 mm min–1 was applied. The adhesive joints were allowed to cure at ambienttemperature for 24 h prior testing. Addressing the evolution of thelap-shear strength over time, samples were allowed to cure the correspondingtime .Lap-shear and SAFT tests wereperformed following the procedure as reported elsewhere.As mentioned in the introduction, in the automotive sector, theadhesive must be designed by considering the requirements of the automotiveproduction lines. In particular, liquid structural adhesives thatare used in production plants must harden as fast as possible to achieveenough strength in order to hold the parts of the ensemble together(handle strength). Afterward, the adhesive will remain there untilit reaches the ultimate strength of the adhesive joint related tothe final application properties 2.25 in all cases, the process is still relatively slow and long timesare required to obtain a minimum handle strength. We envisioned thatthe curing process may be sped up by adding a new component to theformulation. Therefore, in the first part of the article, we haveprepared some NH2–PHU–NH2 oligomersthat are further combined with an epoxy resin and a multifunctionalthiol. The effect of these components on the curing process as wellas on the adhesion properties is evaluated.When designing NIPU-type materials for this type of application,it is clear that the reaction between 5-membered cyclic carbonatesand diamines is not fast enough to meet the industrial specificationsand that the maximal strength values are not comparable to the onesof isocyanate-based PU adhesives. The enhancement in the ultimatestrength values has been solved by the use of hybrid chemistries suchas epoxy chemistry. However, while several different chemistries havebeen used for curing NIPUs at room temperature,36 a mixture of balanced soft to hard segment monomers,that is, PPGdiCC/RdiCC in 60:40 molar ratio, was reacted with 2 equivof 1,12-DAD at 80 °C for 5 h under continuous mechanical stirringfor the preparation of an amino-terminated PHU. FT-IR–ATR wasused for tracking this reaction by following the disappearance ofthe carbonyl stretching vibration band of the cyclic carbonates. After5 h, these bands  had totally disappeared and a new band appearedat 1701 cm–1, corresponding to the carbonyl of theurethane group, was clearly observed . Additionally, vibration bands at 3304 ,1593 , 1536 (δ N–H), 1492 , 1463 (C–O), 1254 , and 1101cm–1  overlapped withC–O stretching vibration bands of primary and secondary alcoholsand at 772 and 721 cm–1  confirmedthe structure of the prepolymer. 1H NMR spectroscopy ofNH2–PHU–NH2  showed the splitting of the signal at 2.65 ppm(NH2–CH2−)and the appearance of a new signal at 3.11 ppm (CH2–NH–C(O)–O), confirming the functionalizationof the NH2–PHU–NH2. The AEW wasdetermined by 1H NMR titration  in a standard solution of toluene in CD3OD asdescribed in the experimental section. NH2–PHU–NH2 was characterized with an AEW of 520.4 ± 5.5 g equiv–1. Taking into account that amines possess two activehydrogens when reacting with epoxy resins, an AHEW of 265.2 ±2.2 g equiv–1 was calculated from Based onour previous work,2–PHU–NH2 and TMPTMP. Thus, the sum of activehydrogen equivalents of NH2–PHU–NH2 and thiol equivalents of TMPTMP matched the equivalent number ofepoxy group for all compositions. Five different formulations wereprepared varying the NH2––HU-NH2/TMPTMP ratio . Due to viscosityissues, in all cases, the reagents were mixed at 50 °C for 2min, followed by 1 min at room temperature, to give a homogeneousviscous mixture.To synthesizePHU hybrid materials, EPIKOTE 828 resin (epoxy resin) was reactedwith different equiv ratios of curing agents, that is, NHG′ = G″) of the variousreactive formulations. Catalyst-free compositions were characterizedwith gel times longer than 2 h when NH2–PHU–NH2 was employed, independent of the ratio of curing agents  and C–O–C of the epoxy (914 cm–1) remained unaffected after 2 h .This result suggests that NH2–PHU–NH2 acts as a catalyst for the thiol-epoxy reaction. Ending ofthe reaction after 24 h was confirmed by FT-IR–ATR throughthe disappearance of the stretching vibration band of the thiol at2569 cm–1  and the decreased intensity of the asymmetric C–O–Cstretching vibration band of the epoxy at 914 cm–1 . A shoulder in the zoneof the C–O stretching vibration of the carbonyl group was observedfor compositions in which NH2–PHU–NH2 and TMPTMP were employed due to the carbamate and the estergroup, respectively.The ability of the materials to cure at roomtemperature was checked by rheology by determination of the gel time is dueto vitrification of the system,which causes a dramatic lowering in the mobility of the polymer chains,and therefore, conversion of the system is limited.39 Nonetheless, the formation of a three-dimensional networkwas confirmed by DMTA experiments, in which G′was over G″ at high temperatures  as a catalyst within the reactive formulations.The curing process was found to be highly catalyst- and formulation-dependent.The thermoset material based on only thiol-epoxy chemistry was curedin less than 30 min, while in the absence of the catalyst, no curingwas observed  when the catalyst was employed .The ultimate adhesiveproperties of the hybrid PHUs were addressed by a lap-shear test 5. The NH35Besides shear strength under dynamic deformations (lap-shear tests),the evaluation of structural adhesives under the effect of staticload and temperature is also important. These measurements give informationabout resistance of the material to creep and give an idea of viableservice temperatures of the adhesives. To get this information, SAFTmeasurements were carried out 2. In all40 may be takenas a reference value for handling strength) is mandatory to be competitivein the industrial manufacturing process since this is the principalmethod for improving production speed and reducing costs. Therefore,lap-shear measurements were carried out at different times to evaluatethe development of lap-shear strength for the 70/30 and 30/70 NH2–PHU–NH2/TMPTMP equivalent ratiocompositions  was added to the composition to boost the rate ofthe thiol-epoxy “click” reaction. Despite rheologicalmeasurements demonstrating faster curing when just 0.875 mol % ofTMG was added to the 30/70 composition, resulting in a gelation in76 min, the adhesive could not be applied due to the low pot lifetime. Strategies to add TMG at the time of application are under developmentto accelerate the curing times without altering the pot life of theadhesive.Inthis work, PHU-epoxy hybrid adhesives able to cure at room temperaturewere prepared by mixing an amino-terminated PHU with a viscous epoxy resin, EPIKOTE 828, and a trifunctional thiol(TMPTMP). NH


Cells of the HL-60 myeloid leukemia cell line can be differentiated into neutrophil-like cells by treatment with dimethyl sulfoxide (DMSO). The molecular mechanisms involved in this differentiation process, however, remain unclear. This review focuses on the differentiation of HL-60 cells. Although the Ras proteins, a group of small GTP-binding proteins, are ubiquitously expressed and highly homologous, each has specific molecular functions. Kras was shown to be essential for normal mouse development, whereas Hras and Nras are not. Kras knockout mice develop profound hematopoietic defects, indicating that Kras is required for hematopoiesis in adults. The Wnt/β-catenin signaling pathway plays a crucial role in regulating the homeostasis of hematopoietic cells. The protein β-catenin is a key player in the Wnt/β-catenin signaling pathway. A great deal of evidence shows that the Wnt/β-catenin signaling pathway is deregulated in malignant tumors, including hematological malignancies. Wild-type Kras acts as a tumor suppressor during DMSO-induced differentiation of HL-60 cells. Upon DMSO treatment, Kras translocates to the plasma membrane, and its activity is enhanced. Inhibition of Kras attenuates CD11b expression. DMSO also elevates levels of GSK3β phosphorylation, resulting in the release of unphosphorylated β-catenin from the β-catenin destruction complex and its accumulation in the cytoplasm. The accumulated β-catenin subsequently translocates into the nucleus. Inhibition of Kras attenuates Lef/Tcf-sensitive transcription activity. Thus, upon treatment of HL-60 cells with DMSO, wild-type Kras reacts with the Wnt/β-catenin pathway, thereby regulating the granulocytic differentiation of HL-60 cells. Wild-type Kras and the Wnt/β-catenin signaling pathway are activated sequentially, increasing the levels of expression of C/EBPα, C/EBPε, and granulocyte colony-stimulating factor (G-CSF) receptor. Ras genes has been observed during the differentiation of HL-60 cells with DMSO, all-trans-retinoic acid (ATRA), and 12-O-tetradecanoylphorbol-13-acetate (TPA) [HL-60 cells were originally derived from a patient with acute promyelocytic leukemia  . These nte (TPA) . Ras gene family, Nras and Kras are frequently mutated in AML and related myelodysplastic syndromes [Ras genes result in the expression of activated Ras proteins, which transduce signals for cell proliferation and cell survival [Kras have been found to play essential roles during malignant transformation in human cancers [Ras genes has shown that wild-type Kras (WT-Kras) is involved in normal mouse development, whereas Hras and Nras are redundant [Two members of the yndromes . The Rassurvival ,7. Oncog cancers ,9. In coedundant . Importaedundant . Furtheredundant , suggestedundant ,14,15. HThe Wnt signaling cascade is involved in many cellular events, including development, proliferation, differentiation, and migration ,17,18,19Our previous studies revealed that WT-Kras engages with Wnt/β-catenin signaling to regulate the DMSO-induced differentiation of the HL-60 human AML cell line . At presRas genes are involved in many cellular processes, such as cell proliferation, differentiation, and apoptosis [Ras mutations are the most common molecular alterations in human cancers. Wild-type Ras proteins have intrinsic GTPase activity, hydrolyzing bound guanine triphosphate (GTP) to guanine diphosphate (GDP) and thereby inactivating Ras growth-promoting signaling [The poptosis ,32,33,34ignaling ,34. In cignaling ,33,34,35ignaling . Ras gene family, Hras, Kras, and Nras, are activated by mutations in many types of human cancers [Kras is lethal for mouse embryogenesis, whereas Hras and Nras are not required for normal development [Hras (-/-)/Nras (-/-) mice, expressing only Kras genes, were found to grow normally without any obvious mutant phenotype [Nras and Kras are frequently observed in AML and related myelodysplastic syndromes [Ras oncogenes in leukemogenesis remain unclear. The most well-established Ras effector pathways are the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) signaling pathways [The three members of the  cancers ,34. Thes cancers ,36. Deleelopment . Hras  or lymphoid enhancer factor (Lef); the planar cell polarity (PCP) pathway; and the Wnt-Ca pathway ,18,21,51The Wnt/β-catenin signaling pathway is indispensable in embryonic development and homeostasis, including cell proliferation, differentiation, and apoptosis ,19,52,53In the absence of Wnt, free cytoplasmic β-catenin is rapidly targeted for degradation by the destruction complex. The protein phosphatase 2A is also associated with this complex. CK1 initially phosphorylates β-catenin at Ser45, priming the GSK3β catalyzed phosphorylation of β-catenin at Thr41, which in turn sequentially phosphorylates β-catenin at two other sites, Ser37 and Ser33. These sequential phosphorylations of β-catenin generate a recognition site for the E3-ubiquitin ligase β-TrCP, with ubiquitinated β-catenin subsequently degraded by proteasomes  pathway . However, the difference between these two pathways is not clear-cut and rigorous, because some of the proteins linked to non-canonical Wnt signaling are also involved in canonical signaling. Two non-canonical pathways have been identified to date: the planar cell polarity pathway (Wnt/PCP) and the Wnt-calcium , receptor-like tyrosine kinase (RYK), receptor tyrosine kinase-like orphan receptor (ROR1/2), and Flamingo. These complexes associate with cytoplasmic adaptors, such as Dvl and Dvl-associated activator of morphogenesis 1 (Daam1). Signals are transduced Rac, Rho small GTPases, and Jun-N-terminal kinase (JNK), resulting in actin polymerization and cytoskeleton modification. Rho activates Rho-associated kinase (Rock), a major regulator of the cytoskeleton , Ca2+/Calmodulin-dependent protein kinase II (CaMKII), and Calcineurin, and consequently activate the NFAT transcription factor. Activated genes via the Wnt/Ca2+ pathway regulate cell fate and cell migration [This pathway accompanies an increase in the intracellular Caigration .Csf3 gene, which encodes the G-CSF receptor, as well as by attenuating STAT3 phosphorylation of the G-CSF receptor, thereby impairing the G-CSF receptor-mediated differentiation [Wnt signaling plays an important role in normal hematopoiesis. The involvement of Wnt/β-catenin signaling in leukemia stem cell development remains unclear. The β-catenin/Tcf4 complex directly regulates the level of granulocyte colony stimulation factor (G-CSF) receptor and controls the process by which myeloid progenitor cells differentiate into granulocytes during both steady-state and emergency granulopoiesis . Dominanntiation . These fntiation , with β-ntiation . Aberrant Wnt signaling is involved in the pathogenesis of various cancers, including AML. The Notch, Wnt, and Hedgehog signaling pathways are particularly important in AML-associated stem cells . MoleculApc indicate that Wnt/β-catenin signaling regulates hematopoiesis in a dose-dependent manner [The protein β-catenin is at the core of the Wnt/β-catenin signaling pathway. Fold changes in β-catenin levels, rather than absolute levels, are critical for target gene activation ,73. Elegt manner . Only a t manner . Wnt actt manner ,76,77, wt manner . Thus, pt manner . The comt manner . DetermiVal12, an oncogenic mutant of Kras, enhanced the stability of β-catenin via PI3K and GSK3β and the formation of nuclear β-catenin/Tcf4 complexes [Aberrant activation of Wnt signaling results in malignancy and blocks differentiation. Cross-talk between oncogenic Kras and Wnt/β-catenin signaling has been reported in colorectal cancers, with activity mediated through GSK3β or LRP6 ,79. Krasomplexes . To dateAlthough the Wnt/β-catenin signaling pathway was found to be involved in AML, the involvement of non-canonical pathways in AML is much less frequent. Wnt5a, an almost purely non-canonical Wnt ligand, was found to suppress cyclin D1 expression and negatively regulates B cell proliferation, suggesting that Wnt5a suppresses hematopoietic malignancies . Lethal-Cirmtuzumab, a monoclonal antibody targeting the Wnt receptor ROR1, has shown excellent results in models of chronic lymphocytic leukemia (CLL) and is now in phase I clinical trial . Other trials testing Cirmtuzumab in combination with either the BTK inhibitor Ibrutinib , or the BCL2 inhibitor Venetoclax  are currently ongoing.2+ signaling pathway involving the elevation of intracellular calcium level and phosphorylation of CaMKII and PKC. However, 6-BT regulates expression through both canonical and non-canonical Wnt signaling pathways, indicating that the 6-BT and ATRA-induced leukemic cell differentiation does not specifically involve the non-canonical pathway [Treatment with 6-benzylthioinosine (6-BT) and ATRA induced HL-60 differentiation through activation of the non-canonical Wnt/Ca pathway . GSK3β is a negative regulator of the Wnt/β-catenin signaling pathway and regulates many other cellular processes , includiThe differentiation agent ATRA has been used to treat patients with acute promyelocytic leukemia (APL), a subtype of AML ,95. The The finding that GSK3β regulates programmed death ligand-1 (PD-L1) suggests that GSK3β inhibition may be effective in immunotherapy against cancers . PD-L1 iTaken together, these findings indicate that GSK3β inhibitors are positive regulators of the immune response against cancers, as well as negative regulators of the immune response in autoimmune conditions . GSK3β, The CCAAT enhancer-binding proteins (C/EBPs) are modular proteins, each containing a carboxy-terminal leucine zipper dimerization domain, a DNA binding domain, and an N-terminal activation domain ,106,107.C/EBPα is highly expressed in myeloblastoma, and C/EBPα-deficient mice fail to undergo myeloid differentiation , suggestC/EBPβ-deficient mice show defective macrophage function , as wellC/CEBPε is expressed only in hematopoietic tissues and participates in neutrophilic but not monocytic differentiation . C/EBPε Another critical regulator of granulopoiesis is G-CSF, a cytokine that regulates the differentiation and proliferation of myeloid cells ,130. TheG-CSF mediates signals by binding to a single homodimeric receptor, G-CSF receptor (G-CSFR). G-CSFR does not have intrinsic tyrosine kinase activity. Upon ligand binding, G-CSFR undergoes conformational changes, leading to the activation of downstream signaling pathways, including the JAK/STAT, PI3K/AKT, and MAPK/ERK pathways ,141,142.G-CSF has been found to regulate myeloid differentiation via C/EBPε . G-CSFR 2+-dependent protein kinases are thought to be involved in the early stages of regulation of the phosphorylation/dephosphorylation status in cells [DMSO-induced differentiation of HL-60 cells has been regarded as a model for the differentiation of myeloid cell lines. Because DMSO can affect the fluidity of plasma membranes, it can activate several biological functions of cells by removing surface water from phospholipid bilayers ,151. AMPin cells ,153. SurThe amount of WT-Kras on the membrane and Kras activity is significantly enhanced after DMSO treatment for 1 day and is sustained for up to 5 days. These results indicate that the activation of Kras is the first event during the DMSO-induced differentiation of HL-60 cells. WT-Kras levels on the plasma membrane correlate with Kras activity . GSK3β pConsistent with findings showing that β-catenin levels are low in HL-60 cells , basal lCEBPs are active during hematopoietic differentiation. Upregulation of C/EBPα was initially observed after β-catenin accumulation in the nucleus, with the levels of C/EBPε upregulated following the elevation of C/EBPα . The expGSK3β phosphorylation and CD11b expression are attenuated by treatment with Kras siRNA or Kras inhibitor . FurtherIncreased levels of p-GSK3β stabilize β-catenin, which in turn acts as a nuclear transcriptional co-activator. Interactions with β-catenin and members of the Lef/Tcf family stimulated the transcription of various target genes, such as those encoding C/EBPα, C/EBPε, and G-CSFR, in DHL-60 cells. The basal level of β-catenin was found to be much lower in HL-60 cells than in other cancer cell lines , with leβ-Catenin is also involved in apoptosis, as it acts as a molecular hub for multiple signals and regulates various cell functions. The proapoptotic activity of β-catenin has not been fully determined in hematopoietic cells . InteresRas genes are frequently mutated in cancers, with mutations in Kras being associated with the three most lethal types of cancer: lung, colorectal, and pancreatic cancers. Studies have sought to identify effective inhibitors of Ras, with these genes long thought to be “undruggable” targets. Most previous studies have focused on pancreatic and lung cancers, with direct inhibition of Ras considered a desirable approach for the treatment of tumors induced by mutant Ras genes. The identification of a novel allosteric binding pocket beneath the switch-II region in the Kras G12C protein [The  protein  enabled  protein ,166. In  protein ,95, suggOncogenic Kras 4A possesses leukemogenic activity, and palmitoylation regulates its activity . The palDysregulation of the Wnt/β-catenin signaling pathway has been observed in various malignancies, including AML. Clinical trials of agents targeting the Wnt/β-catenin signaling pathway in AML are shown in FLT3 gene have been detected in approximately 20~30% of patients with AML. FLT3 inhibitors can inhibit the phosphorylation of FLT3 and induce apoptosis [FLT3, an upstream molecule of Ras, is also associated with the Wnt signaling pathway in myeloid transformations. Mutations in the poptosis . FLT3-ITpoptosis . S-palmipoptosis . Cyclooxpoptosis .β-catenin mRNA is not deregulated, and its level does not correlate well with the level of β-catenin protein [Although mutations in Wnt signaling components, such as APC, Axin, or β-catenin, are common in solid cancers such as colorectal, breast, and liver cancers, these mutations have rarely been reported in patients with AML and other hematological malignancies . In AML, protein ,179,180.The proposed pathways of DMSO-induced differentiation of neutrophilic HL-60 cells are schematically described in Upon DMSO treatment, WT-Kras is recruited to the plasma membrane (For full biological activity, WT-Kras proteins must be anchored to the inner leaflet of the plasma membrane. At present, the mechanism by which DMSO stimulates Kras translocation to the plasma membrane has not been determined. The stability of β-catenin is regulated by the β-catenin destruction complex (The WT-Kras signal is transduced by PI3K/AKT signaling, downstream of WT-Kras. AKT is a GSK3β kinase , with AKIn the absence of DMSO, phosphorylated β-catenin in HL-60 cells is degraded by a destruction complex of proteins, including GSK3β, APC, CK1, protein phosphatase 2A, and the E3-ubiquitin ligase β-TrCP (The expression of CD11b was shown to be attenuated by either Kras knockdown or a Kras inhibitor, as well as by PI3K/AKT inhibitors and small molecule inhibitors of β-catenin. In contrast, CD11b expression was enhanced by the GSK3β inhibitor. Thus, elevated β-catenin positively regulates the differentiation of HL-60 cells, with the level of β-catenin correlating with the differentiation status of DHL-60 cells. Moreover, Lef/Tcf-sensitive transcription activity is inhibited by treatment with siRNA against Kras, indicating that WT-Kras is linked to the Wnt/β-catenin signaling pathway. Thus, WT-Kras can enhance the stability of β-catenin, thereby increasing the nuclear level of β-catenin/Tcf4 complexes. In this review, we describe the pathway in which WT-Kras engages with the Wnt/β-catenin signaling pathway to enhance the differentiation of HL-60 cells by DMSO treatment. However, the differentiation of hematopoietic cells is regulated by many signaling pathways. The pathway we have presented here is one of the mechanisms. More detailed analysis is required to fully understand the mechanisms of HL-60 cell differentiation since HL-60 cells can be differentiated into several different cell types depending on inducers.The Wnt/β-catenin signaling pathway is an evolutionarily conserved signal transduction cascade involved in normal human development and diseases. Additional studies are required to fully understand the roles of β-catenin in the hematopoiesis of HSC and myeloid lineage cells. Hyper-activation of Wnt signaling by mutations in genes encoding components of the β-catenin destruction complex, such as APC, Axin, and β-catenin, contributes to tumorigenesis. Mutations in these molecules in solid tumors resulted in the constitutive activation of this pathway. In contrast, such mutations are rarely reported in AML and other hematological malignancies . HoweverFollowing the discovery of Kras G12C inhibitors, Ras becomes a druggable target. Although Kras G12C inhibitors showed great success in solid tumors, therapies targeting Kras in AML are still under development. The fact that activation of WT-Kras can enhance the differentiation of AML cells may give some hope to patients with AML. Agents promoting terminal differentiation have markedly enhanced cure rates in patients with certain AML subtypes, such as ATRA treatment of patients with APL ,95. ATRA


LRP6 are associated with NTDs in humans. Either gain-of-function (GOF) or loss-of-function (LOF) mutations of Lrp6 can cause NTDs in mice. NTDs in Lrp6-GOF mutants may be attributed to altered β-catenin-independent noncanonical Wnt signaling. However, the mechanisms underlying NTDs in Lrp6-LOF mutants and the role of Lrp6-mediated canonical Wnt/β-catenin signaling in neural tube closure remain unresolved. We previously demonstrated that β-catenin signaling is required for posterior neuropore (PNP) closure. In the current study, conditional ablation of Lrp6 in dorsal PNP caused spinal NTDs with diminished activities of Wnt/β-catenin signaling and its downstream target gene Pax3, which is required for PNP closure. β-catenin-GOF rescued NTDs in Lrp6-LOF mutants. Moreover, maternal supplementation of a Wnt/β-catenin signaling agonist reduced the frequency and severity of spinal NTDs in Lrp6-LOF mutants by restoring Pax3 expression. Together, these results demonstrate the essential role of Lrp6-mediated Wnt/β-catenin signaling in PNP closure, which could also provide a therapeutic target for NTD intervention through manipulation of canonical Wnt/β-catenin signaling activities.Neural tube defects (NTDs) are among the common and severe birth defects with poorly understood etiology. Mutations in the Wnt co-receptor  Summary: Conditional ablation of Lrp6 in dorsal neural folds causes spinal neural tube defects that can be rescued by genetic activation of β-catenin or maternal supplementation of Wnt signaling agonists. The neural tube is the precursor structure of the brain and spinal cord. Failure of proper neural tube closure at different anatomical regions along the anterior–posterior body axis results in various types of severe neural tube defects (NTDs), such as spina bifida  and exencephaly , the former of which is the most common type of NTD in humans . AlthougMutant mice have been widely used for NTD mechanistic and preventative studies . More thPtk7 and the Vangl, Celsr, Dvl and Fzd families  and β-catenin-independent  pathways. The noncanonical Wnt/planar cell polarity (PCP) signaling pathway regulates cytoskeleton dynamics and collective tissue movements, such as convergent extension, which may drive neural tube closure . The rarfamilies . Severalfamilies .LRP6 gene from patients with NTDs    are caushic Lrp6 , and Lrpnal NTDs . It has  mutants . HoweverPax3, which encodes a transcription factor critical to neural tube closure, resulting in an open spinal NTD phenotype 8.5 mouse embryos  mice exhibit fully penetrant tail truncations and spinal bifida, as shown by the consistently open PNP at E12.5 and open caudal spinal cord at E18.5   , Tbx6 (CT) Ctnnb1 mice were used, in which the conditional removal of the floxed exon 3 (that encodes a phosphorylation site for β-catenin protein degradation) will stabilize β-catenin, leading to constitutive activation of canonical Wnt signaling  at E10.5  of β-catenin in ignaling . The resat E10.5 . HoweverLrp6-cKOs through maternal supplementation of lithium ion, a commonly used psychotherapeutic medication and well-known Wnt signaling agonist acting through inhibition of intracellular β-catenin degradation  . These results demonstrate effective intervention of spinal NTDs in Lrp6-cKOs by lithium treatment. Lesions of variable lengths at the sacrococcygeal levels were also observed in both the LiCl-treated and placebo groups of Lrp6-cKOs, suggesting that environmental or other factors may affect canonical Wnt signaling activity and NTD severity. After lithium treatment, Pax3 expression in the neural folds of Lrp6-cKOs was restored  A-C. Convrestored D.Fig. 8Together, these results elucidate the essential roles of the Lrp6-mediated canonical Wnt/β-catenin signaling pathway in murine caudal neural tube closure, which may provide significant clues to both the etiology and prevention of human NTDs.Pax3Cre;Lrp6-cKO mutants, which is phenotypically consistent with Pax3Cre;β-catenin-cKO mutants, as demonstrated in our previous study  of Lrp6-mediated Wnt/β-catenin signaling may affect noncanonical Wnt/PCP signaling directly or indirectly. Our results showed that -catenin , it is a-catenin . Interesrulation . The autrulation . Neverthin vitro . These a mutants . Howevercrooked tailLrp6 homozygous embryos exhibit cranial NTDs with defective RhoA signaling and apical–basal cell polarity, suggesting a defective noncanonical Wnt signaling mechanism  LOF by genetic or pharmacological approaches can rescue NTDs in Pax3 mutants, suggesting a role of apoptosis in the development of NTDs  and Bok  can reduce the frequency and severity of spinal NTDs, with ∼18% of tivities  to stabitivities . Thus, ttivities E. There tivities , includitivities . It is itivities . Regardl mutants . Intrigu of NTDs . We did  and Bok , demonstLrp6 in dorsal neural folds resulted in spinal NTDs due to diminished canonical Wnt/β-catenin signaling and downstream target genes. This could be rescued either by genetic activation or pharmacological stabilization of β-catenin in vivo Ctnnb1 mice    were usein situ hybridization using digoxigenin-labeled antisense RNA probes as previously described . At least three mutants and three littermate control (Cre-lacking or double heterozygous) embryos were used for each in situ experiment, which showed consistent results.Mouse embryos were fixed in 1% paraformaldehyde (PFA) for ∼30 min on ice and processed for X-gal staining as previously described . Embryosescribed . AntisenIn vivo stimulation of the Wnt/β-catenin signaling pathway by LiCl was carried out as previously described with minor modifications  to allow for relative comparisons among different experimental groups using the ΔΔCt method.Total RNAs were isolated from the caudal neural folds and pooled from five E9.5 embryos of the mutant or control groups. Heterozygous ications . The mRNAcute BrdU labeling was performed by intraperitoneal injection of BrdU at 100 mg/kg body weight to the pregnant mice 1 h prior to sampling. Immunohistochemistry was carried out on paraffin or frozen sections using primary antibodies against BrdU  and Alexa Fluor-conjugated secondary antibodies . TUNEL assays were performed using the Dead End Fluorometric TUNEL System (Promega) as detailed by the manufacturer. The total numbers of BrdU- or TUNEL-positive cells were counted in the dorsal PNP above the dorsolateral hinge points on each confocal micrograph  then divPax3Cre;Lrp6-cKO embryos. Primary antibodies against Dvl2  and β-actin  were used.Immunoblotting was carried out according to the standard protocol . Proteint-test or pairwise comparison (one-way ANOVA) when appropriate. In all cases, P≤0.05 was considered statistically significant.At least three littermate controls and three mutant embryos were used for each statistical evaluation. Significances were assessed by unpaired, two-tailed Student's