We present an unusual case of subcutaneous granulomas that also highlights the importance of assessing possible associations between exposure and symptoms early in the diagnostic approach to prevent further adverse health effects. Granulomas of the skin are seen in association with several diseases and after foreign body penetration of soft tissue, but have not been described after contact with epoxy. Epoxy resins are commonly used in paints and other protective coatings, including flooring materials.We report a case of granulomatous inflammation in a 58-year-old man after accidental intradermal deposition of unhardened epoxy. Multiple subcutaneous nodules were present on his right forearm, from hand to elbow, for a period of 6 months after the incident. Biopsies and histological analysis showed a granulomatous inflammation without necrosis. Microscopic analysis of the biopsies did not show mycobacterium tuberculosis, other bacteria, or fungal elements. Standard patch testing was negative. The nodules disappeared gradually, but intense pruritus remained. The patient returned to exposure and developed severe work related rhinitis.This case report describes an unusual case of multiple subcutaneous granulomas after a small injury with an epoxy-contaminated tool. Initially no association between the granulomas and exposure was established and the patient returned to work and epoxy exposure. He subsequently developed severe work related rhinitis. The case highlights the challenges of establishing an association between exposure and dermal reactions and that exposure should be reduced or avoided when sensitisation to allergens may have occurred. Granulomas of the skin are seen in association with several diseases and after foreign body penetration of soft tissue, but have not been described following contact with epoxy. The granulomatous response is chronic inflammation characterized by focal collections of macrophages, epithelioid cells, and multinucleated giant cells, and may be accompanied by necrosis and fibrosis . GranuloEpoxy has a wide range of industrial applications, including metal coatings, use in electronic and electrical components, high-tension electrical insulators, fiber-reinforcing materials, and structural adhesives. Epoxy resins, also known as polyepoxides, are reactive prepolymers and polymers containing epoxide groups. Epoxy resins may self-react through catalytic homopolymerization, or combine with a wide range of coreactants including polyfunctional amines, acids, phenols, alcohols, and thiols. These coreactants are often referred to as hardeners or curatives. Reactions of polyepoxides with themselves or with polyfunctional hardeners form polymers that may have strong mechanical properties as well as high temperature and chemical resistance, and are thus widely used. The epoxy resin itself may be formed by a reaction between the substances bisphenol A diglycidyl and epichlorohydrin; these account for about 75 % of the epoxy resins used worldwide. Depending on the producer, the coreactants vary. Polyepoxides as highly reactive substances are known to be associated with work-related allergic contact dermatitis and occupational asthma . A possiA 58-year-old man presented with multiple lesions of the skin on his right forearm from the hand to the elbow. His medical history did not include any diseases associated with granulomatous inflammation or immunosuppression, and there was no family history of granulomatous disease. Prior to the patient’s current work, he had worked with epoxy resins for 4 years, repairing concrete constructions, in the period from 1988 to 1992. He had no exposure to epoxy in the period from 1992 to 1998 and had since then worked for 15 years as an industrial flooring specialist constructing epoxy floors. He had daily contact with polyepoxides, which he first would use as a coating on concrete floors. After hardening, he would use a primer over the first layer and add a final layer of epoxy compound. The patient blended the compounds together directly before he applied the mixture to the floor with a mason’s trowel. He reported sporadic use of gloves and a filter mask while working with epoxy compounds.In August 2013, he accidentally cut himself on the second digit of the right hand with the mason’s trowel contaminated with unhardened epoxy. During the following 10 days, he developed swelling with redness and pain of the digit, which spread to his palm. Because of the increasing symptoms, the patient contacted his general practitioner, 20 days after the accident. The level of C-reactive protein (CRP) was then < 5 mg/L. The wound was cleaned but the patient reported that some of the hardened compound remained in the depth of the wound. The patient was given dicloxacillin (500 mg × 4). The wound swabs did not show any growth of aerobic or anaerobic pathogens. In the following days, after another visit to the emergency ward because of an increase of the swelling, he was given erythromycin (250 mg × 2). At this point, the level of CRP was still < 5 mg/L. Thereafter, during the next weeks, the swelling and pain of his finger and palm disappeared. At the same time, multiple nodules (Fig. During the period of medical investigation, the patient did not work and had no further exposure. He reported that he manipulated the granulomas by squeezing, whereby they would disappear some days later. The granulomas did not spread proximal of his right elbow. All granulomas had disappeared six months after the day of intradermal deposition of epoxy resins Fig. . HoweverEpoxy resins can cause both immediate and delayed allergic reactions, but immediate reactions are rare –10. It iIn the present case, allergy to epoxy was suspected and standard patch testing performed. However, no allergy against epoxy was revealed and the patient returned to his workplace. His IgE level increased between two visits in our department. This may indicate an allergic response, but could not be directly attributed to the epoxy exposure because we had no evidence of a specific allergic reaction. Most commonly, epoxy exposures will result in irritant or allergic contact dermatitis that will not result in elevations of total IgE. Exposure to low molecule weight chemicals such as epoxy can, however, result in mixed immunological responses which may be IgE-mediated .It is known that testing for epoxy allergy can be challenging and that epicutan tests may give false-negative results, thus testing with workplace substances is recommended , 13. UnfSince specific IgE was not examined, and taking the limitations of the skin prick test into consideration, it is possible also that that the patient’s rhinitis was caused by another work related exposure. His work as an industrial flooring specialist may have involved mixed and multiple chemical exposures. For our patient, early recognition of an association between the dermal reactions and exposure probably could have secured the use of personal protective equipment or other means to stop or reduce exposure. Instead, the worker returned to exposure and developed severe work related upper airway symptoms. It is difficult to know if avoidance of exposure at an earlier stage could have prevented the development of respiratory symptoms for this worker. However, after the establishment of the diagnosis, the patient avoided further exposure to epoxy products and reported improvement of symptoms from the nose.We report an unusual case of multiple subcutaneous granulomas and severe work related rhinitis after a small injury with an epoxy-contaminated tool. It remains unclear whether the development of rhinitis in our patient may be a result of sensitization after intradermal deposition of epoxy or other irritants, or represents a delayed response to long-term airborne occupational exposure. However, the case highlights the challenges of establishing the association between exposure and dermal reactions. It may also be a reminder of the potential major role for the skin in respiratory allergy and that efforts should be made to prevent sensitization through skin exposure.CRP, C-reactive protein; IgE, immunoglobulin-E; SiC, specific inhalation challenge test


While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using  In multi-cellular organisms, cell-to-cell junctions are force-bearing and highly dynamic, both critical functional requirements for embryogenesis and tissue homeostasis. Proper cell-cell adhesion requires cells to respond to and withstand the mechanical forces that are exerted from neighboring cells. The actin-myosin contractile network exerts force on the sites of cell-cell adhesion, and is an integral component in strengthening adhesive structures. Therefore, how actin-myosin generated forces alter the protein organization at cell-cell contacts is an important detail in the regulation of cell-cell adhesion.The role of the actin cytoskeleton in cadherin-mediated cell-cell adhesion has been extensively studied. The cadherins, a family of calcium-dependent cell-cell adhesion proteins, play fundamental roles in cell organization during physiological and pathological processes in multi-cellular organisms. The canonical binding partners, α-catenin and β-catenin, are the key regulatory proteins in the cadherin complex. While β-catenin is a well-known component of Wnt pathway, α-catenin recently emerged as a critical player in regulating the actin network at the sites of cadherin mediated cell-cell adhesion.Recent studies uncovered a unique mechanism by which α-catenin regulates the actin cytoskeleton. The protein sequence of α-catenin contains an actin binding site at the C-terminus, and an overlapping sequence containing a β-catenin binding site and a homo-dimerization site at the N-terminus. Originally, α-catenin was described as a scaffolding protein that links the cadherin complex to the actin cytoskeleton . Interesin vitro. For example, in live cells, the application of mechanical stress via cadherins stiffens the cell junctions [in vitro reconstituted cadherin complex has been shown to interact with actin filament in a force-dependent manner [These biochemical analyses suggest that α-catenin is unlikely to stably link the cadherin complex and the actin cytoskeleton. While other actin-binding proteins that localize to the sites of cadherin mediated cell-cell adhesion have been proposed as potential linkers , detectiunctions  and recrunctions ,9 and viunctions , suggestunctions . Recentlt manner . TogetheOne mechanism of force-sensitivity at cell junctions is through mechanically-induced conformational changes that expose cryptic sites. For example, in integrin-mediated cell-extracellular matrix adhesion, a number of proteins possess allosteric properties that are regulated by the application of external force . In in vitro to isolate protein complexes, and thus, the inherent nature of the assay prevents detection of force-dependent protein interactions. An alternative approach to traditional immuno-precipitation is to detect proximal proteins by in situ biotin labeling [Identification and analysis of mechano-responsive proteins rely on methods that often focus on single proteins, such as the use of conformation-sensitive antibodies, FRET, or single molecule force spectroscopy. In contrast, traditional biochemical analysis relies on stable protein-protein interactions labeling . In thisin situ biotin labeling exceeds that of standard optical microscopy. Since the purification of proximal proteins does not rely on the stability of protein interactions between binding partners, it is possible to identify mechanically-induced protein interactions. Our analysis demonstrates in situ biotinylation as a potential method to screen force-sensitive protein interactions.Using a mutant BirA tagged epithelia specific isoform of α-catenin and a cell stretch device, we sought to identify proteins that may associate with α-catenin in a force-dependent manner. Interestingly, the proteins that co-localize at E-cadherin positive cell-cell contacts with immuno-fluorescence are not always biotinylated, suggesting that the spatial resolution of To identify force-sensitive protein-protein interactions, we designed a fusion protein with a mutant biotin ligase (denoted as BirA*) and epithelia-specific α-catenin. The BirA* contains an arginine to glycine point mutation at residue 118, and has promiscuous biotinylation activity due to the reduced affinity for an intermediate biotin donor, bioAMP . The bioThe BirA*-α-catenin proteins localized to cell-cell contacts in MDCK cells , and aloTo analyze the proximal biotinylation by BirA*-α-catenin, we first isolated biotinylated proteins from cell lysates using streptavidin conjugated beads. Using cells plated on a standard P150 dish, the cells were scraped and lysed in 0.2% SDS RIPA buffer, then sonicated and centrifuged to separate soluble and insoluble pools. Note that the majority of biotinylated proteins remained in the soluble pool . Based o2 vs ~150 cm2 of P150 dish). Under the standard purification protocol, BirA*-α-catenin was isolated with the streptavidin-conjugated beads, but endogenous α-catenin was not, consistent with the isolation from a P150 dish , αE-catenin , α-tubulin , E-cadherin , β-catenin , non-muscle myosin IIA , vinculin  and β-actin . Filamentous actin was labeled with AlexaFluor-568-conjugated phalloidin (Invitrogen), and biotinylated proteins were labeled with AlexaFluor-568-conjugated streptavidin. For western blotting, the signals on the nitrocellulose membrane were detected using a Western Quantum Bright chemiluminescence kit . Following pharmacological reagents were used in this study: biotin (Sigma), (-)-blebbistatin , and cComplete protease inhibitor cocktail .5’-CGAGCTCAAGCTTCGAAGGACAACACCGTGCCC (Forward) and 5’-GCAGTCATGGTGGCGGCCTTCTCTGCGCTTCTCAGG (Reverse), then, using SalI and EcoRI, the PCR product was inserted between GFP and epithelial-α-catenin in the pEGFP-C1-α-catenin vector. The GFP-BirA*-α-catenin sequence was then inserted into the PiggyBac vector using NheI and SacII sites . The Piggybac GFP-BirA*-α-catenin plasmid and Piggybac Transposase expression plasmid (System Biosciences) were transfected into MDCK cells using Lipofectamine2000 (Invitrogen) and selected with 500 μg/ml G418 (Invitrogen). G418-resistant cells were sub-cloned and selected by confocal microscopy and western blotting to obtain homogeneous cell populations.MDCK cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum , penicillin and streptomycin (Invitrogen). The mutant BirA R118G  was PCR amplified using the following primers: MDCK cells were fixed using either 1.5% (for myosin IIA staining) or 3%  para-formaldehyde and 0.3% Triton X-100 in PBS for 10 minutes and blocked with 1% BSA and 0.3% Triton X-100 in PBS. Primary antibodies were added and incubated for 1 hour at room temperature, and detected using AlexaFluor-568-conjugated secondary antibodies or AlexaFluor-568-conjugated streptavidin (Invitrogen). Samples were imaged with a Zeiss Axio Observer equiped with Yokogawa spinning confocal system (CSU-10), 40x C-Apochromat water immersion objective, 488/561 nm solid state laser system, and a CoolSNAP HQ camera. The microscope system was controlled by Slidebook software . The alignment of actin bundles was manually analyzed using ImageJ. The overlap coefficients for co-localization analysis of α-catenin and myosin IIA were calculated based on Manders et al. . If the 2.Mechanical stretch device consists of a linear stage for uniaxial stretch driven by a servo motor  and a servo controller  with a customized stage for stretch chambers . BirA*-α5 cells on p150 dishes or 2×105 cells on PDMS membrane chamber and cultured for 20–24 hours in complete media supplemented with 50 μM biotin, and if indicated, 50 μM blebbistatin. After PBS washes, cells were lysed in RIPA buffer  and sonicated  on ice. After centrifugation at 16.1x103g for 20 min, cell supernatants were collected as a lysate sample. Protein concentrations were measured by Bradford assay. The lysates were diluted with TN buffer (150 mM NaCl and 25 mM Tris–HCl pH 7.5) to decrease the SDS concentration to 0.1%. The diluted lysates were incubated with magnetic streptavidin-beads  for 1.5 hours. Using magnetic collection stands, streptavidin-beads were washed with RIPA buffer with either 2% or 0.1% SDS. In some experiments, the samples were further washed with wash buffer 2 , wash buffer 3  and wash buffer 4  for one time each, but similar results were obtained regardless. Purified proteins were dissolved in SDS sample buffer and separated by SDS-PAGE in a 4–15% gradient precast gel . The identical protocol was used for immuno-precipitation using E-cadherin, GFP, and myosin IIA antibodies except that antibodies were cross-linked to Protein A magnetic beads (Invitrogen).Cells were seeded 2×10S1 Fig5 cells/ml medium. 25 µl of cell suspension was seeded onto glass-bottom dishes, inverted upside-down, and incubated at 37°C. Cell suspensions were then triturated through a pipette tip 30 times and the cluster sizes were quantified using ImageJ. The data are represented as mean cluster size ± standard error of the mean.(A) Subcellular localization of BirA*-α-catenin and E-cadherin in sub-confluent and confluent cell monolayer. The overlay images of BirA*-α-catenin and E-cadherin staining are shown in the last panel (Merge). For the confluent cell monolayer, 3D stack images were reconstructed to visualize the lateral membrane localization of BirA*-α-catenin and E-cadherin. Scale bar 10 μm. (B) Hanging drop assay to test cell aggregation potential of wildtype and BirA*-α-catenin expressing cells. Cells were suspended at a density of 2.5 x 10(TIF)Click here for additional data file.S2 Fig(A) The SDS concentration in wash buffer. Streptavidin-conjugated beads were washed with solutions containing different SDS concentration (%), then the wash solutions and the bead fractions for each SDS concentration were collected and analyzed using Western blot with streptavidin–HRP. While high SDS concentrations (0.5–2%) removed a significant amount of biotinylated proteins from the beads, the removal of biotinylated proteins were minimal for 0.1% SDS wash solution. (B) Relative detection sensitivity of α-catenin and vinculin antibodies. The lysates from MDCK cells expressing GFP-tagged α-catenin or vinculin were loaded onto a SDS-gel and analyzed with Western blot using GFP, α-catenin or vinculin antibodies. The blot analyzed with the GFP antibody shows relative loading of GFP-tagged proteins (left). The same sample volumes were loaded onto the adjacent lanes and analyzed with α-catenin or vinculin antibodies under the identical exposure of the blot (right). The identical antibody dilution as main figures (1:1000 for both antibodies) was used in this experiment. The α-catenin and vinculin antibodies detected the exogenous GFP-tagged α-catenin and vinculin, respectively, as well as the endogenous proteins. The relative intensities of GFP-tagged proteins in the GFP blot (left) and α-catenin or vinculin blot (right) are similar, suggesting that the detection sensitivity of vinculin antibody is similar to that of α-catenin antibody. Therefore, the lack of vinculin bands in streptavidin bead purified samples see Fig  and 4B i(TIF)Click here for additional data file.S1 MovieThe images were taken at 0.5 micron spacing denoted by the values in the upper left corner.(MOV)Click here for additional data file.


Little is known regarding the expression or clinical significance of δ-catenin, a member of the catenin family, in colorectal cancer (CRC). The present study examined the expression of δ-catenin using immunohistochemistry in 110 cases of CRC, including 70 cases with complete follow-up records and 40 cases with paired lymph node metastases. In addition, δ-catenin mRNA and protein expression were compared in 30 pairs of matched CRC and normal colorectal tissues by reverse transcription quantitative polymerase chain reaction and western blot analysis. δ-Catenin was weakly expressed or absent in the cytoplasm of normal intestinal epithelial cells, whereas positive δ-catenin expression local-ized to the cytoplasm was observed in CRC cells. The rate of positive δ-catenin expression in CRC  was significantly higher than that in normal colorectal tissues . In addition, δ-catenin mRNA and protein expression were significantly increased in CRC tissues compared to those in their matched normal tissues . The expression of δ-catenin in stage III–IV CRC was higher than that in stage I–II CRC, and the expression of δ-catenin in the tumors of patients with lymph node metastases was higher than that in patients without lymph node metastases. Kaplan-Meier survival curves demonstrated that the survival time of patients with positive δ-catenin expression was shorter than that of patients with negative δ-catenin expression (P=0.005). Furthermore, Cox multivariate analysis indicated that the tumor, nodes and metastasis stage (P=0.02) and positive δ-catenin expression (P=0.033) were independent prognostic factors in CRC. The present study therefore indicated that δ-catenin may be a suitable independent prognostic factor for CRC. Colorectal cancer (CRC) is one of the most common malignant tumor types, and the incidence of CRC is increasing by 2% per year worldwide. Although the prognosis for CRC has greatly improved with the continuous development of diagnostic techniques, the mortality rate remains high . The majet al  repeats. Initially, δ-catenin was considered to only be expressed in cerebral neurons, where it has been shown to bind with presenilin to exert a function in the development of Alzheimer's disease –6. Howevet al  verifiedl  blocks from 110 cases of CRC and 30 normal colorectal tissue specimens were obtained from the Department of Pathology, Shengjing Hospital of China Medical University . None of the patients had received radiotherapy, chemotherapy or immunotherapy prior to tumor excision. In total, 67 of the patients were male and 43 were female . The patients' age at the time of surgery ranged from 31 to 87, with an average age of 61 years.Complete follow-up data were available for 70 of the cases of primary CRC, which were surgically excised between May 2004 and July 2005. In addition, lymph node metastases were present in 40 of the 110 cases. To evaluate the tumor, nodes and metastasis (TNM) stage, at least 12 lymph nodes were obtained during surgical resection. All tumors were classified according to the TNM staging system, as revised by the International Union Against Cancer (UICC) in 2002  were obtained from 30 cases of CRC, immediately frozen in liquid nitrogen and stored at −70°C for RNA and protein analysis. The present study was conducted in accordance with the regulations of and was approved by the Institutional Review Board of China Medical University. Informed consent was obtained prior to surgery from all enrolled patients.µm sections and mounted on poly-L-lysine-coated slides . The sections were de-paraffinized in xylene, re-hydrated using a graded ethanol series, incubated with 3% hydrogen peroxide in methanol for 20 min, and then washed in phosphate buffered saline (PBS) for 5 min (All from MaiXin). Antigen retrieval was conducted using enzymatic antigen retrieval solution for 15 min at room temperature, and then the sections were washed in PBS for 5 min and blocked using normal blocking serum from the Ultrasensitive™ S-P kit  for 30 min. The sections were incubated with anti-δ-catenin primary antibody  at 4°C overnight. Following washing with PBS, the sections were stained using diaminobenzidine tetrahydrochloride (MaiXin) as a chromogen, lightly counter-stained with hematoxylin (MaiXin), dehydrated and mounted. For the negative controls, the primary antibody was replaced with PBS.FFPE tissue blocks were cut into 4-et al  following the manufacturer's instructions.RT-qPCR was performed following the MIQE guidelines  published by Bustin et al . Total Rµl using the primer sequences from Takara listed in −ΔCq method with the SDS 2.4 software package (Applied Biosystems) following the method of Schmittgen and Livak  with SYBR Premix Ex Taq II . Amplifications were performed in a total volume of 10 nd Livak .Total protein was extracted from the colorectal tissues using lysis buffer  for 30 min at 4°C, the lysates were centrifuged at 14,000 ×g for 30 min at 4°C and the supernatants were collected. Aliquots containing 50 mg protein were separated by 8% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred to PVDF membranes  at 100 V for 2.5 h at 4°C. The protein concentration was determined using the bicinchoninic acid assay (Beyotime Institute of Biotechnology). The membranes were blocked in 5% skimmed milk for 2 h, and the proteins were detected by incubation with mouse monoclonal antibodies against δ-catenin  or GAPDH  overnight at 4°C, followed by anti-mouse immunoglobulin G conjugated to horse radish peroxidase at a dilution of 1:2,000 for 2 h at room temperature. The bands were visualized using the EC3 Imaging System  and the optical densities were normalized to GAPDH using ImageJ software, version 1.44 .in vitro experiments were performed at least three times and values are expressed as the mean ± standard deviation. Pearson's χ2 test was used to analyze the correlation between δ-catenin expression and clinicopatho-logical features of CRC. The overall survival probabilities were calculated using the Kaplan-Meier method and compared using the log-rank test. To determine the significant factors associated with overall survival, a multivariate Cox proportional hazard model was created. All statistical analyses were performed using SPSS 13.0 for Windows . P<0.05 was considered to indicate a statistically significant difference between values.All Negative or weak δ-catenin immunoreactivity was observed in the cytoplasm of normal colorectal mucosal epithelial cells . Based oThere was no significant association between positive δ-catenin expression and the age, gender, lesion location or tumor size in CRC patients (P>0.05); however, positive δ-catenin expression was closely associated with the degree of differentiation, TNM stage and lymph node metastasis in CRC . The ratFor the 70 CRC patients with complete follow-up data, the five-year survival rate was 64.29% (45/70). Kaplan-Meier survival analysis demonstrated that the mean survival time of patients with positive δ-catenin expression was significantly shorter than that of patients with negative δ-catenin expression  than those in the matched normal colorectal tissues (3.25±0.88; t=7.137, P<0.0001; The correlation between δ-catenin mRNA expression and the clinicopathological features of CRC was examined in 30 patients . A signiFinally, the δ-catenin protein expression levels were determined in 30 cases of CRC and the paired adjacent normal colorectal tissues using western blot analysis . The aveet al (Initially, δ-catenin mRNA was considered to be mainly expressed in the brain. δ-Catenin can bind presenilin ,6 and iset al  providedet al . δ-catenet al . In addiet al , and δ-cet al .Analysis of human expressed sequence tags demonstrated that δ-catenin mRNA may be expressed in multiple tumor types, including esophageal, ovarian and breast cancer ; howeveret al (et al (The immunohistochemical analysis in the present study demonstrated that δ-catenin was mainly expressed in the cytoplasm of CRC cells, with only small amounts of δ-catenin observed at the cell junctions, in agreement with the localization of δ-catenin in prostate cancer and lung cancer as reported by Lu et al  and Zhanl (et al . It is kl (et al ; howeveret al (et al (Zhang et al  and Dai l (et al  demonstrIt has been reported that increased expression of δ-catenin in tumor tissues may be linked to overexpression of the transcription factors E2F1 and Pax6 . HoweverIn conclusion, the presents study showed that δ-catenin protein is overexpressed and mainly localizes to the cytoplasm in CRC. Positive expression of δ-catenin was associated with poor differentiation, higher TNM stage, lymph node metastasis and poor prognosis in CRC. Expression of δ-catenin mRNA was upregulated in CRC compared to the corresponding adjacent normal tissues, and the δ-catenin mRNA expression levels were positively correlated with the tumor stage and lymph node metastasis in CRC. Hence, δ-catenin may represent a potentially clinically useful independent prognostic factor in CRC.


Background: Dissociation of the Wnt/β-catenin pathway effector Dishevelled from its complex with nucleoredoxin is a redox-sensitive process, yet the ROS sources remain elusive.Results: Mitochondrial Ca2+ influx stimulates endogenous ROS production and mediates Wnt/β-catenin pathway activity.Conclusion: Ca2+-mediated ROS production modulates the signaling efficiency of the Wnt/β-catenin pathway.Significance: Metabolic states influence fundamental and developmental signaling to drive cell differentiation. 2+ from the endoplasmic reticulum stores. Ca2+ subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca2+ uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/β-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca2+-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/β-catenin signaling output.Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/β-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/β-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca Excessive production of reactive oxygen species (ROS)3–et al.  (dishevelled segment polarity protein 2), and β-catenin revealed their activation already within the first 4 h of differentiation , immortalized human neural progenitor ReNcell VM197 cells (hereafter hNPCs) differentiate within 3 days into neurons and glial cells A 12, 13, 13A (12A) , 15. A d2+ from endoplasmic reticulum stores through the inositol 1,4,5-triphosphate receptor, type 1 (ITPR1). Subsequently, a fraction of Ca2+ flows into the mitochondria via the mitochondrial calcium uniporter (MCU). This increase in mitochondrial Ca2+ is required for elevated ROS production. The inhibition of Ca2+ efflux via ITPR1 or Ca2+ influx via MCU attenuates the ROS metabolism and prevents the dissociation of DVL2 from its inactive pool sequestered by NRX in the cytoplasm. Moreover, the robust activation of DVL2 is blocked as we observe a significant decrease in the β-catenin nuclear accumulation, attenuated expression of Wnt/β-catenin signaling target genes, and impeded neuronal differentiation. Our data reveal that Ca2+-mediated mitochondrial ROS metabolism is directly involved in the regulation of early events of Wnt/β-catenin transduction and imply that the cellular metabolic state has an integral role in the Wnt/β-catenin pathway.Here, we provide evidence that in hNPCs, endogenous mitochondrial ROS production is markedly increased as a result of GF depletion at the onset of neural differentiation and that ROS production precedes the activation of the Wnt/β-catenin pathway. We find that GF depletion stimulates the release of Cal-glutamine, heparin, and gentamycin)  as described previously (i.e. medium without growth factors). Treatment of cells with 0.5 or 10 μm ruthenium red (RuR) (Sigma) was performed for 3 h as follows: 1 h of pretreatment with the reagent prior to the induction of differentiation, followed by a post-treatment up to the 2nd h of differentiation; to reverse the drug effect, the drug-containing differentiating medium was replaced by a drug-free medium after Hanks' balanced salt solution rinsing. Proliferating cells were also pretreated with lithium chloride  and N-acetyl-l-cysteine  (both Sigma), followed by post-treatment up to 24 and 72 h after the induction of differentiation, respectively. Cytotoxicity was assessed by trypan blue exclusion assay (Invitrogen) or MTT test (Sigma). Cells seeded in Petri dishes were trypsinized and stained with trypan blue; the cell counting was performed in triplicate and each condition assessed in duplicate. For the MTT assay, cells were seeded in 96-well plates and incubated for 2 h with 50 μg/ml MTT prior to lysis; absorbance at 550 nm was measured with 8 wells per experimental conditions for each experiment. Data were obtained from three independent experiments.The immortalized human neural progenitor cell line ReNcell VM197 (ReNeuron) was derived from the ventral midbrain of 10-week-old human fetal neural tissues. Cells proliferate in laminin (R&D Systems) pre-coated flasks under human bFGF (Invitrogen) and human EGF (Sigma) stimulation in proliferating medium , SMARTpool Accell inositol 1,4,5-triphosphate receptor, type 1 (ITPR1), and Accell nontargeting pool (negative control). Briefly, 1 μm siRNA was mixed to Accell siRNA delivery medium  supplemented with EGF, bFGF, and B27 neural cell supplement and incubated with cells for 72 h prior to the induction of differentiation.Cells were transfected with following human siRNAs as per the manufacturer's instructions : SMARTpool Accell mitochondrial calcium uniporter  were kept constant for all comparative sets of experiments. With these settings, no photobleaching was detected after several repeated measurements on the same microscopic field. Brightness/contrast adjustments were applied equally to every pixel in the images (i.e. maximum projections) and for each comparative set (e.g. proliferation versus differentiation) using Fiji/ImageJ. Adjustments were performed on individual color channels before merging images. No change to γ settings was applied. Regions of interest were set individually, i.e. for each cell of the population in the images, based on cell boundaries to calculate the mean fluorescence intensities (ratio of the sum of fluorescence intensity emitted in the regions of interest to the amount of pixels in the regions of interest). For each image, the background fluorescence was subtracted, and values were normalized. All data were obtained from at least three independent experiments. For each time point or treatment, at least 10 images per experiment were recorded. Results are shown as means ± S.D. arbitrary units.Fluorescent images of fixed cells were acquired with LSM 710 NLO (Zeiss) and TCS SP5 and TCS SP8 (Leica) confocal microscopy systems. Live cell imaging was performed using TCS SP2 (Leica) and A1 (Nikon) confocal microscopes. Live cell samples were observed in a 37 °C humidified incubation chamber supplied with a CO2DCFDA    or tubulin TrackerTM Green   allowed discriminating the cell boundaries for quantification of the mean fluorescence intensities. Nuclei were stained 10 min with 2 μm Hoechst 32258 (Sigma). All stainings were done at 37 °C, 5% CO2, in the dark. Between each staining three washing steps were achieved with pre-warmed culture medium. For the short term kinetic experiments (i.e. 1 h time line), proliferating cells were pre-loaded with the appropriate dyes and then the differentiation was initiated by GF removal, and the mean intensities were measured every 10 min. For the long term measurements (i.e. up to 3 h), the dyes were loaded right after the differentiation was induced, and measurements were performed every 30 min.Live cell imaging of intracellular redox state and mitochondrial ROS metabolism were, respectively, performed with TCS SP2 and A1 confocal microscopes, using the redox indicator carboxy-Hμm, 1 h)  and Mito 45 min) , respect2DCFDA or dihydrorhodamine 123  (Invitrogen), a nonspecific indicator for intracellular ROS . Mean fluorescence intensities in a total of 104 events (i.e. cells) were determined in each sample using EPICS XL-MCL flow cytometer system (Beckman Coulter). An unstained cell sample was carried along as a control for autofluorescence. Data were analyzed for four independent exposure experiments measured in duplicate. 1–3 mm hydrogen peroxide (H2O2) (Invitrogen) and 5 μm phorbol 12-myristate 13-acetate (Sigma) were used as positive controls.Flow cytometry was performed to determine intracellular redox state and ROS levels. Cells were stained with carboxy-Hular ROS . Stainin2+ fluxes were monitored with A1 confocal microscope using mag-fluo-4 AM     was used to assess cytosolic Ca2+ levels (m caffeine (CAF)  was used as positive control. Fura red, although presented as a ratiometric Ca2+ indicator through a dual excitation wavelength mode , is rarely used this way because of the weak quantum yield of the Ca2+-bound form (i.e. the Ca2+-unbound form. An increase of cyto-Ca2+ levels leads to a decrease of the measured fura red intensity due to the binding of Ca2+ (i.e. reduction of the pool of Ca2+-unbound form of the dye). Results were presented as F0/F ratio .Endoplasmic reticulum (ER) and mitochondrial Ca, 0.5 h)  and x-rh, 0.5 h) , respect+ levels , 21 withProtein extraction from the cells was achieved using Triton lysis buffer supplemented with protease and phosphatase inhibitor mixtures (Sigma). Cell lysates were loaded with Laemmli buffer, centrifuged for 10 min, and boiled for 5 min. Proteins were separated by SDS-PAGE using 10% SDS-polyacrylamide gel and transferred in a wet condition onto a nitrocellulose membrane (Amersham Biosciences). After blocking with 5% nonfat dry milk, proteins were labeled overnight with primary antibodies as follows: goat anti-NRX ; rabbit anti-DVL2 ; and rabbit anti-GAPDH  used as stable housekeeping protein. Proteins were probed for 1 h with HRP-conjugated secondary antibodies as follows: anti-goat  and anti-rabbit . Signals were detected with ECL Western blot detection reagent  and quantified with Fiji/ImageJ. Relative signal intensities were normalized to GAPDH.d-lysine and then with laminin were fixed 20 min with 4% paraformaldehyde and 4% sucrose in PBS followed by 10 min of quenching with 50 mm NH4Cl and 5 min of permeabilization with 0.2% Triton X-100. Nonspecific binding sites were blocked for 1 h with 1% gelatin . Cells were loaded 1 h with primary antibodies as follows: rabbit anti-DVL2 ; goat anti-NRX ; mouse anti-α-tubulin ; mouse anti-β-catenin-Alexa488-conjugate ; mouse anti-glial fibrillary acidic protein-Cy3-conjugate ; mouse anti-βIII-tubulin-FITC-conjugate ; and mouse anti-Hu antigens C and D . After rinsing with 0.2% gelatin, secondary antibodies were loaded during 45 min as follows: anti-rabbit or anti-mouse Alexa488; anti-goat Alexa594; and anti-mouse Alexa647 . Nuclei were stained with Hoechst 32258 . A post-fixation step was performed with 2% paraformaldehyde for 10 min to prevent any dissociation of secondary antibodies, followed by a quenching step with 50 mm NH4Cl for 5 min. Coverslips were mounted using prolong gold antifade reagent (Invitrogen). Images of DVL2 and NRX were acquired using a TCS SP8 confocal microscope. Localization of β-catenin or neuronal markers was assessed using LSM 710 NLO confocal system.Cells grown on glass coverslips precoated with poly-i.e. direct acceptor excitation and donor emission bleed-through) from the FRET specimen (cell sample labeled with both donor and acceptor). Following this procedure, the fully corrected FRET image was obtained (according to the recommendations in Ref. i.e. FRET efficiency (FRETeff), was calculated using the equation of Van Rheenen et al.  for every condition. Measurements were performed for three independent experiments.To assess physical protein-protein association between DVL2 and NRX, we used FRET microscopy  through n et al. . FRETeff18 primers and generated from 1 μg of template RNA with Moloney murine leukemia virus reverse transcriptase using first strand cDNA synthesis kit (Thermo Scientific). RT-PCR was performed as follows: 5 min at 25 °C, 60 min at 37 °C, and 5 min at 70 °C. Real time PCR quantitation was performed by mixing 100 ng of template cDNA with TaqMan gene expression master mix and following TaqMan gene expression assays : MCU (Hs00293548_m1); ITPR1 (Hs00181881_m1); AXIN2 (Hs00610344_m1); MAP2 ; RPL13A . Contents were transferred into 96-well PCR plates (Thermo Scientific) with a final concentration of cDNA of 5 ng/μl in each well. Amplifications were performed using iQ5 real time PCR detection system (Bio-Rad) as follows: 2 min at 50 °C for activation of the uracil-N-glycosylase; 10 min at 95 °C for polymerase activation; 40 repeats of two-step cycling . Relative expression values were obtained by normalizing tC values of the tested genes in comparison with tC values of ribosomal protein L13a  using the ΔtC method  according to the manufacturer's instructions. Residual contaminating genomic DNA was digested by DNase I recombinant RNase-free (Roche Applied Science). cDNA synthesis was primed with oligo(dT)t test. *, p ≤ 0.05, significantly different from the control (untreated proliferating cells). Data are presented as means ± S.D. and averaged from at least three independent experiments.Statistical analyses were performed using two-tailed unpaired Student's 2DCFDA (B–D). Proliferating cells showed only a faint signal . Upon induction of differentiation, the signal increased within 30 min , reached its maximum after 1 h, and returned to baseline after 2.5 h . In parallel, we quantified the cellular redox state using flow cytometry and obtained similar results .The onset of neural differentiation in hNPCs is regulated in a narrow time range by the tight activation of Wnt/β-catenin pathway components. Whether endogenous ROS levels could increase after GF depletion and play any role in DVL2-NRX dissociation is unclear. To assess the changes in intracellular ROS levels, we first monitored the cellular redox balance state using the redox indicator carboxy-H2DCFDA  at 10-mi2DCFDA , B–D. PrF). The 0.5-h incubation of proliferating cells with pro-oxidant agent H2O2 (1 mm) resulted in the increased redox imbalance, as anticipated (D and E). Although H2O2 cytotoxicity depends on various factors, including the dose or the incubation time (2O2 (3 mm) was noticeable only 2 h after treatment (41%). Consistent with the reported data . Thus, our data attribute the increase in the intracellular redox imbalance in the first 3 h of hNPCs differentiation solely to GF depletion.Exchange of cell culture medium can impose oxidative stress ; howeverFig. 1ion time , the cytted data , no visiA, 0h). mito-ROS production led to an increased fluorescence within 0.5 h of differentiation  and dropped to baseline by 3 h . Using flow cytometry, we obtained similar results with cells stained with another ROS-sensitive dye, dihydrorhodamine 123  (2O2 (1 mm) or phorbol 12-myristate 13-acetate (5 μm), induced a significant increase of the fluorescent signal after 0.5 and 1 h, respectively (C and D).The changes in the redox state could reflect the rise in ROS production in the mitochondria. We examined the mitochondrial ROS (mito-ROS) metabolism using the MitoTracker red marker . The dyeFig. 2Fig. 2D) . TreatmeOur data show the dynamic changes in the redox state at the early phase of hNPC differentiation and reflect the alteration in the cellular physiology. They further reveal the existence of a switch in mitochondrial ROS production within 30 min after GF depletion.2+ is a ubiquitous messenger involved in various aspects of cell physiology ; after 0.5 h of differentiation, the signal significantly decreased indicating the Ca2+ release from the ER stores . Similarly, a 1-h treatment of proliferating cells with 1 mm CAF, which stimulates Ca2+ release from the ER stores via the ryanodine receptors . Of note, in agreement with reports showing CAF cytotoxicity only after longer exposures .The ER-Ca+ levels . The mag+ levels A, 0h; afeceptors , 36, strxposures , a 2-h C2+ (cyto-Ca2+) levels after GF depletion using ratiometric fura red. The dye fluoresces in its Ca2+-unbound form, so an increase in cyto-Ca2+ concentration diminishes the signal  and returned to baseline at 3 h of differentiation . These data corroborate the inverse relationship between ER and cytosolic Ca2+ levels. Although intracellular Ca2+ levels remained unchanged in proliferating cells after the sequential exchanges of proliferative medium (F), 1 h of CAF treatment led to an increase in cyto-Ca2+ levels, comparable with the increase at 0.5 h in differentiating cells (E and G).Next, we assessed the changes in cytosolic Cae signal , 21. Cone medium F, 1 h ofng cells , E and G2+ levels, we used x-rhod-1, a dye that preferentially accumulates in mitochondria and fluoresces proportionally to the Ca2+ levels in these organelles , and decreased by 3 h of differentiation . CAF treatment of proliferating cells also increased mito-Ca2+ levels (C) corroborating the data observed in differentiating cells. Altogether, these data indicate that GF depletion leads to an increased Ca2+ efflux from the ER to the cytosol and a subsequent Ca2+ influx into the mitochondria within the first 30 min of differentiation.To assess the mito-Caganelles . The mit+ levels C corrobo2+ dynamics occur concomitantly with the changes in mito-ROS metabolism and cellular redox state . RuR reportedly blocks the mito-Ca2+ uptake by inhibiting the MCU, the main conduit for mitochondrial Ca2+ influx , and we monitored Ca2+ levels within mitochondria for 3 h. The effect of RuR was dose-dependent; at 0 h, the high dose (10 μm) strongly reduced the signal indicating the decrease of baseline mito-Ca2+ levels, whereas the low dose (0.5 μm) did not cause any significant change in comparison with untreated cells . At 1 h, Ca2+ accumulated within the mitochondria in untreated cells, whereas 0.5 μm RuR prevented mito-Ca2+ uptake . At 3 h, the mito-Ca2+ levels dropped to baseline in the untreated cells. Conversely, after 0.5 μm RuR removal, mito-Ca2+ levels significantly increased indicating that the drug effect is reversible . The effect of the high dose was long lasting, and the mito-Ca2+ levels were decreased for the entire examined period .The observed intracellular Ca+ influx , 39. We 2+ and ROS metabolism at the onset of hNPC differentiation, we examined the mito-ROS production after inhibiting Ca2+ influx into the mitochondria. At 0 h, mito-ROS levels in 0.5 μm RuR-treated cells were comparable with the untreated cells . After 1 h of differentiation, the low dose prevented the rise in ROS levels . However, 1 h after the drug removal, its effect was reversed, and the mito-ROS production was elevated . The high dose drastically reduced the signal and completely abolished mitochondrial metabolism, presumably due to its cytotoxicity (G and H). The low dose did not cause any significant cell death (H).To assess the functional link between mito-Catoxicity , G and Hll death H.m RuR is nonlethal and reversible, and inhibits both mito-Ca2+ uptake and ROS increase. Moreover, our findings suggest that mito-ROS metabolism is triggered by a rise in mito-Ca2+ levels at the onset of differentiation.These data demonstrate that the effect of short term treatment with 0.5 μ2+ fluxes can tune the activation of the Wnt/β-catenin cascade in hNPCs. First, we verified the reported data showing the early kinetics of DVL2 activity in these cells (A and B). Markedly, we found that NRX protein levels followed the same pattern; the NRX accumulated within 1 h, and its levels decreased by 3 h (A and B). If the DVL2 and NRX accumulation were mediated through the increase in intracellular ROS, the treatment of proliferating cells with pro-oxidant agent would result in higher DVL2 and NRX protein levels. Conversely, inhibiting the ROS production would prevent the DVL2 and NRX increase. Indeed, although DVL2 and NRX proteins were significantly elevated after 1 mm H2O2 treatment of proliferating cells (A), the incubation of differentiating cells with 0.5 μm RuR delayed the accumulation of both DVL2 and NRX proteins . These data suggest that preventing mitochondrial ROS formation affects cytoplasmic DVL2 as well as NRX protein levels.The GF depletion results in the activation of Wnt/β-catenin pathway that is required for induction of hNPC differentiation , 15, 40.se cells , A and Bd by 3 h , A and Bng cells A, the ineff) significantly decreased . The H2O2 treatment of proliferating cells also resulted in decreased FRETeff . Markedly, 0.5 μm RuR treatment inhibited the dissociation of DVL2-NRX complexes. FRETeff between DVL2 and NRX was high in RuR-treated cells 1 h after differentiation . Drug removal promoted dissociation of DVL2 from NRX as the FRETeff decreased . These data show that the mito-Ca2+-mediated physiologic increase in ROS levels may facilitate fast dissociation rate of DVL2 from the initial pool of DVL2-NRX complexes. DVL2 and NRX proteins accumulate in the cytoplasm as a result of their reduced physical interactions. In the absence of elevated ROS production, DVL2 is kept inactive in the cytoplasm by binding to NRX.To address whether the altered redox and metabolic cell states could mediate the dissociation of DVL2 from NRX, we examined the physical association between the two proteins by fluorescence resonance energy transfer (FRET) microscopy for a period of 3 h after GF removal , 24. At 2+-mediated ROS metabolism would decrease the pool of unbound DVL2 and inactivate the transduction cascade. We therefore examined the ability of β-catenin to localize into the nucleus in RuR-treated cells. At 0 h, the nuclear β-catenin signal was identical between untreated and RuR-treated cells . After 3 h of differentiation, β-catenin accumulated in the nuclei of untreated cells  confirming previous reports . We monitored the kinetics of β-catenin nuclear localization by quantifying its mean fluorescence intensities along the first 4 h of differentiation . The nuclear β-catenin signal was significantly enhanced in untreated cells from 2 h of differentiation onward . Frequency distribution analysis revealed that at 3 h of differentiation, two cell populations emerge as follows: the first one, in which β-catenin levels are comparable with proliferating cells, and the second one with twice as high mean intensities representing the cells with the accumulated nuclear β-catenin . RuR treatment prevented the two cell populations to emerge , but a moderate increase in the nuclear mean intensities of β-catenin occurred after drug removal . These data indicate that Ca2+-mediated ROS metabolism enhances the nuclear β-catenin accumulation and may affect the rate of its nucleo-cytoplasmic shuttling.As a result of Wnt/β-catenin pathway activation, β-catenin accumulates in the cytoplasm and shuttles to the nucleus . In the 41– reports . In cont2+-mediated ROS metabolism modulates the Wnt/β-catenin signaling, we genetically perturbed the ER and mitochondrial Ca2+ fluxes and examined their effects on Wnt target gene expression during hNPC differentiation. We reduced ITPR1  (A). Next, we examined the functional effects of ITPR1 and MCU silencing. At 0 h, the ER-Ca2+ levels were comparable between control cells and cells with the reduced ITPR1 (B). At 1 h of differentiation, ER is depleted of Ca2+ by 50% in control cells , in contrast loss of ITPR1 completely blocks ER-Ca2+ release after GF depletion (B). Similarly, Ca2+ influx into mitochondria is increased after 1 h of differentiation in control cells (by ∼80%)  but is significantly suppressed in the absence of MCU. Of note, the basal levels of mito-Ca2+ are mildly affected in proliferating cells transfected with MCU siRNA. These changes in Ca2+ fluxes have a profound effect on ROS metabolism. Although in control cells ROS levels significantly increase 1 h after differentiation , loss of both ITPR1 and MCU affect the ROS metabolism already in proliferating cells and diminish the mito-ROS production after induction of differentiation , indicating that Ca2+ flux through MCU is critical for cellular bioenergetics as suggested previously  to inhil influx . We firsl cells) A. Next, ed ITPR1 B. At 1 hepletion B. Similaeviously . The dat2+-mediated ROS metabolism is perturbed. We tested the expression levels of two Wnt/β-catenin pathway target genes: AXIN2 and MAP2, a neuron-specific microtubule-associated protein 2 (AXIN2 and 4-fold increase in MAP2 expression 2 days after differentiation in untreated and control cells (A); a similar increase in Wnt target gene expression was reported previously . In contrast, RuR treatment as well as loss of ITPR1 and MCU significantly reduced the AXIN2 and MAP2 mRNA levels at this time point (A). Taken together, our findings demonstrate that Ca2+ release from the ER at the onset of hNPC differentiation depends, at least in part, on ITPR1 activity. Subsequent Ca2+ influx into the mitochondria requires MCU and stimulates ROS production. Moreover, these data show that reduced Ca2+-mediated mitochondrial ROS metabolism has a negative impact on Wnt target gene expression.We then addressed whether Wnt/β-catenin signaling is affected when Carotein 2 , 47. We ol cells A; a simieviously . In cell agonist ), AXIN2  20-fold A. In conme point A. Taken 2+-mediated ROS metabolism enhances the Wnt/β-catenin signaling output infer that the neuronal yield of differentiating hNPCs could be modulated by the mitochondrial Ca2+ influx and ROS production. To test the effect of Ca2+-mediated ROS metabolism perturbations on hNPC neural fate commitment, we quantified the neuronal yield after 3 days of differentiation in cells with disrupted ROS metabolism. We treated the cells either with 0.5 μm RuR or 10 mm NAC, a potent ROS scavenger that protects against oxidative stress (B and C). Inhibiting mito-Ca2+ uptake with RuR had a profound effect on the neuronal yield. Only 6% of the cells differentiated into neurons (B and C). Similarly, NAC also reduced the neuronal yield to 6% (B and C). NAC treatment efficiently blocked the ROS increase at the onset of hNPC differentiation, indicating its high antioxidant capacity (D). Conversely, stimulating Wnt/β-catenin signaling with LiCl significantly enhanced the neuronal yield up to 17%, confirming that the induction of the Wnt/β-catenin pathway promotes the neuronal differentiation in our cell model. Of note, the proportion of differentiated neurons of the RuR-treated cells at 3 days was comparable with that observed with untreated cells at 2 days after GF depletion indicating delayed and ineffective differentiation . Altogether, we demonstrate that a change in Ca2+-mediated mitochondrial ROS metabolism at the onset of hNPC differentiation is required for fine-tuning the Wnt/β-catenin signaling efficiency, which in turn controls the decision of the cells to commit to their neuronal fate.Our findings that Cae stress . Upon GFe stress , B and C neurons , B and Cld to 6% , B and Ccapacity D. Conver2+ release through ITPR1. Upon GF removal, the inhibition is alleviated and Ca2+ floods the cytoplasm. The increased intracellular Ca2+ concentration drives a number of responses, one of which involves the opening of MCU at the outer mitochondrial membrane. The increased mitochondrial Ca2+ influx changes the oxidative state of the mitochondria and triggers the endogenous ROS production. Importantly, loss of ITPR1 or MCU abolished Ca2+ influx into the mitochondria and subsequent increase in ROS metabolism.In our study, we provide direct evidence that an endogenous increase in mitochondrial ROS metabolism accelerates the rate of DVL2 dissociation from its complex with NRX and augments Wnt/β-catenin signaling efficiency. We further unravel the mechanisms that regulate ROS production in the initial phase of neural differentiation in hNPCs. At steady state, GF suppresses ER-Ca2O2, by exogenous insult such as surgical amputation, or by induction of expression of NADPH oxidases , their withdrawal leads to ITPR1-dependent Ca2+ release from the ER to the cytosol and initiates neural differentiation (right panel). Ca2+ then accumulates in mitochondria through MCU resulting in the change in the electron transport chain activity, which significantly enhances ROS metabolism. ROS interacts with redox-sensitive targets such as NRX. In the proliferating cells, NRX binds DVL2 and keeps it inactive in the cytoplasm. Upon ROS elevation, NRX releases DVL2, which becomes available to further activate the downstream signaling cascade. As a consequence, cytosolic β-catenin accumulates and shuttles to the nucleus to drive specific expression of target genes involved in neuronal differentiation. At the steady state, mitochondrial Ca2+ returns to baseline level and the stimulation of mitochondrial ROS production is alleviated.The following model summarizes the spatio-temporal dynamics of the successive events : while t2+-mediated ROS metabolism, we observed not only a delayed dissociation of DVL2 from NRX but also a slower rate in β-catenin nuclear accumulation and lowered Wnt/β-catenin target gene expression. Consequently, the neural differentiation is attenuated as quantified by the reduced neuronal yield. Releasing the block on ROS production led to partial recovery in the protein levels of Wnt/β-catenin pathway effectors. Already 1 h after the exchange with drug-free medium, we showed a significant increase in both mito-Ca2+ and ROS levels with simultaneous rise in DVL2 and NRX cytoplasmic protein levels. In addition, we also detected elevated nuclear β-catenin 2 h after the drug depletion suggesting a partial rescue. These data indicate that the response amplitudes of Wnt/β-catenin pathway effectors could be modulated by physiologic changes in ROS metabolism.The metabolic switch induced by GF removal provides the cells with a cue to change from their proliferative to the differentiated state. Although Wnt/β-catenin signaling induces differentiation through regulation of target gene expression, we show that the increased ROS metabolism is required for modulating the extent of the initial signaling response. The advantage of our model system is that we can directly determine the Wnt/β-catenin signaling efficiency at the different steps of the transduction cascade. When we inhibited the CaOur data imply ROS in modifying the cellular decisions, in our case the neural commitment of hNPCs and their differentiation into neurons. Furthermore, the findings also demonstrate that the timing of the elevated ROS production is crucial. ROS are able to tune the intensity and the magnitude of the Wnt/β-catenin pathway responses right at the onset of neural differentiation. In conclusion, this study shows for the first time that endogenously induced changes in mitochondrial ROS metabolism positively regulate Wnt/β-catenin signal transduction and demonstrates that endogenous ROS act as permissive signals in modulating the extent of the Wnt/β-catenin signaling output. Wnt/β-catenin signaling is not the only developmental pathway regulating the neural commitment of hNPCs . Given tin vitro studies of molecular and cellular mechanisms regulating neurogenesis but also encourages the exploitation of these cells in studies directed toward stem cell therapies. Our findings imply that modulating intracellular Ca2+ fluxes and/or ROS metabolism in hNPCs prior to their neuronal fate commitment phase facilitates their neurogenesis. This could be particularly beneficial for enhancing in vitro neuronal yields and for using hNPCs as a potential neuronal source for cell replacement and regenerative therapies in the treatment of neurodegenerative diseases. Conversely, our finding that a potent ROS scavenger NAC negatively regulates neuronal differentiation prompts the re-evaluation of the therapeutic administration of antioxidants, at least when applied in the context of neuronal development as suggested previously (The ability of hNPCs to promptly differentiate into neurons within 3 days upon GF removal significantly reduces not only the time range for 2+ has been shown to interact with the Wnt signaling pathway at many different levels; its role as a second messenger downstream of DVL-mediated Wnt signaling is well established (2+ fluxes through regulation of L-type Ca2+ channel conductance (2+-dependent events are essential for robust activation of DVL and positively regulate Wnt/β-catenin signal transduction ensuring more efficient and sustained signaling. The data infer that ionic and metabolic cues are an integral part of the Wnt/β-catenin signaling pathway and may fine-tune the temporal element of signal transduction as well as magnitudes of responses of the pathway components.Caablished . Recentlductance . In this


Background. The increasing demand for endoscopic procedures coincides with the paradigm shift in health care delivery that emphasizes efficient use of existing resources. However, there is limited literature on the range of endoscopy unit efficiencies. Methods. A time and motion analysis of patient flow through the Hotel-Dieu Hospital  endoscopy unit was followed by qualitative interviews. Procedures were directly observed in three segments: individual endoscopy room use, preprocedure/recovery room, and overall endoscopy unit utilization. Results. Data were collected for 137 procedures in the endoscopy room, 139 procedures in the preprocedure room, and 143 procedures for overall room utilization. The mean duration spent in the endoscopy room was 31.47 min for an esophagogastroduodenoscopy, 52.93 min for a colonoscopy, 30.47 min for a flexible sigmoidoscopy, and 66.88 min for a double procedure. The procedure itself accounted for 8.11 min, 34.24 min, 9.02 min, and 39.13 min for the above procedures, respectively. The focused interviews identified the scheduling template as a major area of operational inefficiency. Conclusions. Despite reasonable procedure times for all except colonoscopies, the endoscopy room durations exceed the allocated times, reflecting the impact of non-procedure-related factors and the need for a revised scheduling template. Endoscopy units have unique operational characteristics and identification of process inefficiencies can lead to targeted quality improvement initiatives. The need for screening and surveillance procedures parallels the increase in an aging population and drives the demand for endoscopic procedures in a resource-constrained setting. In 2008/2009, 492,888 gastroscopies, 132,701 sigmoidoscopies, and 969,307 colonoscopies were performed across Canada [Audits of endoscopy units have revealed significant underutilized resources . HoweverEndoscopy efficiency has been challenging due to lack of reliable methodology for efficiency metrics, which is the cornerstone for improving endoscopy value and will facilitate benchmarking best practices , 13. TheTo develop a process to quantitate and analyze potential inefficiencies within the endoscopy unit, it was first necessary to characterize the different components of the workflow process. As depicted in The present study was undertaken into two distinct stages. During stage 1, a research assistant collected data from December to March 2014 in three distinct settings: the preprocedure/recovery room, endoscopy room, and the overall endoscopy unit room utilization. Procedures were directly observed and the time elapsed for different components from patient registration to exit from recovery room was recorded. The quantitative data were then analyzed for mean times and the 95% CI was calculated.The second stage included semistructured interviews with the endoscopy unit staff based on standardized interview questions that were drafted for each group . Participants were recruited via email; their interviews were recorded and the transcripts were analyzed for thematic content using NVivo , a qualitative software program.The Queen's University Health Sciences Research Ethics Board  approved the present study and the need for individual patient consent was waived.For the purpose of the present study, time intervals representing the endoscopy workflow processes were analyzed; their definitions are outlined in The HDH endoscopy unit is an outpatient facility that operates from 08:00 to 16:00 on weekdays and consists of administrative offices, a waiting room, a 10-bed preprocedure/recovery room, and three endoscopy rooms. It has a closed access scheduling system with block scheduling of procedures and operated as group lists. The preparation/recovery room beds are shared once per week for nonendoscopic procedures such as paracentesis and intravenous infliximab infusions. The daily endoscopy unit staff consists of two to three endoscopists, a variable number of gastroenterology fellows, six nurses, and two technicians. For three days of the week, it is a “two physicians in three rooms” operational model and the other days are staffed with “three physicians in three rooms.” The annual procedure volume of the unit is approximately 5500 cases.From December to March 2014, 137 procedures in the endoscopy room, 139 procedures in the preprocedure room, and 143 procedures for overall endoscopy unit utilization were collected for 28 nonconsecutive days. All data were analyzed for mean times and 95% CIs (Tables The mean duration of time spent by the patient in the endoscopy room before the start of the endoscopic procedure was 14.22 min (95% CI 12.17 min to 16.28 min). The average procedure duration for an esophagogastroduodenoscopy (EGD) was 8.11 min (95% CI 6.64 min to 9.57 min); 34.24 min (95% CI 29.51 min to 38.98 min) for a colonoscopy; 9.02 min (95% CI 7.00 min to 11.05 min) for a flexible sigmoidoscopy; and 39.13 min for a double procedure (95% CI 30.59 min to 47.66 min). The mean overall time spent by the patient in the endoscopy room was 31.47 min (95% CI 26.82 min to 36.12 min) for an EGD; 52.93 min (95% CI 48.11 min to 57.75 min) for a colonoscopy; 30.47 min (95% CI 25.51 min to 35.43 min) for a flexible sigmoidoscopy; and 66.88 min (95% CI 58.64 min to 75.12 min) for a double procedure. The mean room turnover time was 7.9 min (95% CI 7.66 min to 8.14 min). The patient spent an average of 71.21 min (95% CI 63.95 min to 78.48 min) in the preprocedure room and the average recovery room time was 56.27 min (95% CI 53.90 min to 58.64 min). The mean time spent in the endoscopy unit was 2.73 h (95% CI 2.55 h to 2.90 h).P ≤ 0.00).With respect to start time delays, the mean time for patient registration was 39.22 min ahead (95% CI −44.76 to −33.68) of their scheduled start time. The mean scheduled start delay was 19.07 min (95% CI 13.98 min to 24.15 min). The mean procedure start delay was 38.37 min (95% CI 31.34 min to 45.40 min), while the mean endoscopist start delay was found to be 7.51 min (95% CI 5.15 min to 9.86 min). Both the scheduled start delay and procedure start delay were significantly shorter with the two rooms per endoscopist when compared with the one room per endoscopist model  behind scheduled time. Furthermore, the mean start time of the first case of the day was approximately 15 min behind the time of entry of the first patient into the endoscopy room .Focused interviews were conducted with four endoscopists, one nurse sigmoidoscopist, nine nurses, five technicians, and one scheduling assistant. The endoscopy staff work experience varied with the endoscopists ranging from four months to 28 years, four months to 24 years in gastrointestinal endoscopy for the nurses, and five months to 20 years for the technicians. The present time-motion study observing patient flow through an academic endoscopy unit followed by qualitative interviews yielded baseline endoscopy workflow metrics and process inefficiencies. These data are essential for future resource optimization. We found the cumulative time spent in the endoscopy room was within the allocated time frame for an EGD but far exceeded the allocated times for colonoscopy, flexible sigmoidoscopy, and double procedure. This was despite the procedure times for all except colonoscopies being within reasonable expectations, indicating that non-procedure-related factors are strong determinants of time consumption. This concurs with findings from previous studies that procedure time is rarely rate-limiting and that nonprocedural operational flow processes are instead crucial targets for improvement . EndoscoRecent studies have reinforced that the preprocedure and recovery room are key areas in the endoscopy centre , 11. GivPatient entry into the endoscopy room was approximately 38 min behind scheduled time. This was independent of further delay contributed by endoscopist arrival approximately 8 min later than scheduled start. The start time delays begin with the first case of the day and these incremental delays likely perpetuate a downstream effect on the subsequent scheduled patients. From these data, it was notable that factors such as endoscopist arrival and on-time starts for the first case of the day are modifiable and relatively simple to reinforce to optimize patient flow. The interviews with the endoscopy staff reinforced the observational phase finding that a major shortcoming was related to scheduling and procedure durations exceeding allocated times contributing to downstream patient waiting times. Furthermore, there was objective concordance with staff preference for two rooms per endoscopist given that it was associated with significantly shorter delays.Operational errors are the product of poorly designed systems as elucidated by the Institute of Medicine's report entitled “To Err is Human: Building a Safer Health System” . Our datTo address the complexity of variables involved in the endoscopic workflow process, the methodology was specifically designed with a time-motion study to provide a comprehensive assessment of patient flow through the unit. This was supplemented with qualitative information from the entire endoscopy staff to bridge information gaps. This enabled provision of baseline performance metrics and identification of process inefficiencies from multiple perspectives. Some of the limitations of the present study are that it was based at a single ambulatory endoscopy centre and did not directly address some other factors recognized to impact efficiency such as inpatient endoscopic procedures, impact of trainee involvement, or the range of endoscopist proficiencies on the efficiency of the endoscopy unit , 18. LasFuture directions of our research will entail targeted interventions based on the inefficiencies identified and the follow-up effects these have. Correlation with the patient perspective on endoscopy process inefficiencies will also be assessed.The present study has identified individual process inefficiencies and endoscopic workflow metrics to facilitate institutional quality control. These baseline data will be essential to move forth from productivity to efficiency and have facilitated the identification of unique opportunities for process improvement at the HDH endoscopy centre.


Nuclear E-cadherin was a negative regulator of Wnt/β-Catenin-elicited promotion of the CSC phenotype. Using immunohistochemistry on lung cancer tissue microarrays, we found that changes in subcellular location of E-cadherin may be described by tumor grade and stage, suggesting cellular redistribution during lung tumorigenesis. Furthermore, nuclear E-cadherin expression was more significantly inversely correlated with CD133 (a lung CSC marker) expression (P<0.005) than total E-cadherin expression (P<0.05), suggesting that lung cancer as defined by nuclear E-cadherinLow/nuclear β-cateninHigh/CD133High biomarkers has superior prognostic value over total E-cadherinLow/nuclear β-cateninHigh/CD133High.Several studies suggest that Wnt signaling contributes to reprogramming and maintenance of cancer stem cell (CSC) states activated by loss of membranous E-cadherin expression. However, E-cadherin's exact role in Wnt/β-catenin-mediated promotion of the CSC phenotype remains unclear. Recently, a significant positive correlation has been observed between the expression of nuclear  E-cadherin and β-catenin in gastric and colorectal carcinomas. Here we conducted a series of  CSCs are defined by their ability to self-renew, form tumor spheres and seed new tumors in a xenotransplant system, and they are intrinsically resistant to therapy.12 and other tissues.13 Incidentally, important evidence of Wnt signaling has been emphasized in cancer cells14 and the Wnt/β-catenin signaling cascade is a critical regulator not only of normal stem cells but also of CSCs.15 In non-small cell lung cancer, β-catenin and adenomatous polyposis coli (APC) mutations are uncommon, but Wnt signaling is important in non-small cell lung cancer cell lines, and Wnt inhibition reduces tumorigenic potential.16 Roles for Wnt signaling have been described in a variety of CSC settings, including colon, breast and cutaneous CSCs, as well as in hematopoietic stem cells.19 For example, stem-like colon cells with a high level of β-catenin signaling have a much greater tumorigenic potential than counterpart cells with low β-catenin signaling.17 More specifically, a recent study provides evidence that prostate cancer cells may be regulated by Wnt activity to enhance self-renewal capacity with stem cell characteristics.20The Wnt/β-catenin signaling pathway is crucial for the maintenance and activation of normal stem cells, as clearly illustrated with the intestinal mucosa epithelia, epidermis, mammary gland15 thus, it may be correlated with tumor grade and stage.22 Some evidence shows that the nuclear accumulation of β-catenin is correlated with the expression of CD44 the CSCs markers.23 This indicates that activation of β-catenin is required for the sustention of CSC-like traits generated by loss of E-cadherin expression first. E-cadherin can be downregulated through a variety of mechanisms including mutation, epigenetic silencing and transcriptional repression. E-cadherin-inactivating mutations were first described in diffuse gastric cancer24 and loss of the E-cadherin locus on the long arm of chromosome 16 (16q22) occurs frequently in hepatocellular carcinomas, breast and esophageal cancer.25 However, in colorectal cancer E-cadherin does not always show absent or reduced expression, but often there is redistribution from cell membrane to the cytoplasm.26 Failure to localize to the membrane in spite of normal expression may be due to posttranscriptional modification of the complex.28 Mutations or loss of heterozygosity within the E-cadherin gene are rare in sporadic colorectal cancer (CRC).29 A panel of 49 human colorectal carcinoma cell lines has been screened and it was found that only 7% exhibited inactivating E-cadherin mutation.30Loss of E-cadherin expression often results in the disruption of cell–cell adhesion and an increase in nuclear accumulation of β-catenin. The latter is kept at a low level through a destruction complex;in-vitro and in-vivo tumor model systems. Although this is the first report of nuclear E-cadherin in human lung cancer, aberrant staining of E-cadherin in the nucleus has been reported in other types of cancer including Merkel cell carcinoma, signet ring cell carcinoma and solid pseudopapillary tumors of the pancreas.33 Furthermore, those cancers with nuclear E-cadherin also show increased β-catenin levels in the nucleus.33 It is unclear whether an aberrant nuclear localization of E-cadherin has an anti- or pro-oncogenic role, for example, via modulating β-catenin-mediated signaling. In this study, we attempted to dissect the molecular mechanisms through which nuclear E-cadherin may have an important role in generating cells with CSC properties. We found that the β-catenin/TCF4 interaction was abolished by E-cadherin and correlated with its nuclear localization, and consequently decreased β-catenin/TCF4 transcriptional activity. Nuclear E-cadherin was a negative regulator of Wnt/β-catenin-elicited promotion of the CSC phenotype. In clinical use, nuclear E-cadherinLow/nuclear β-cateninHigh/CD133High biomarkers have superior prognostic value over total E-cadherinLow/nuclear β-cateninHigh/CD133High.Together, this evidence suggests a possible link between cancer stem-like properties and loss of E-cadherin expression. The graded loss expression of E-cadherin correlates with the aggressiveness of numerous carcinomas and the worsening of prognosis, whereas ectopic expression of E-cadherin suppresses tumor initiation and invasion in various 35 consecutive slides were examined from 39 primary  and 10 metastatic human lung cancer specimens. Low-grade (stage IA and IB) primary lung cancers showed low levels of nuclear E-cadherin, nuclear β-catenin and CD133, and high levels of membranous E-cadherin and membranous β-catenin. Alternately, moderate-grade (stage IIA and IIB) primary lung cancers showed low levels of CD133 and low-to-moderate levels of nuclear β-catenin, and high-to-moderate levels of nuclear E-cadherin  expression in lung cancers correlated inversely with nuclear β-catenin expression  staining and scoring;cadherin . In cont P<0.01; , whereas P<0.01; . InteresP=0.004; . As expeP<0.001; , suggestHigh fraction, the invasion abilities  or mutant (FOP) TCF-binding sites were used to drive the expression of enhanced green fluorescent protein. Next, we isolated the highest and lowest 10% of TOP-GFP-expressing cell fractions by flow cytometry of TOP-GFP-transduced cell lines with . LM cells were functionally fractionated by invasive assays, 5 times (HM5 cells), 10 times (HM10 cells) and 20 times (HM20 cells). in-vitro invasive, in-vivo metastatic abilities, in-vitro proliferative potential, in-vivo tumor growth, apoptotic abilities and SP cells were determined. After serial selection, cells revealed increased invasive abilities  and HM20 (E-cadherin deficient) cells, the expression of nuclear β-catenin was determined after stimulation with LiCl, Wnt3a, or Wnt5a for 4 h. Western blotting demonstrated that nuclear β-catenin was consistently and strongly overexpressed in HM20 cells after stimulation with Wnt3a and LiCl as compared with cells treated with control medium or Wnt5a . Further36 We therefore tested whether Wnt3a treatment enhanced the dephosphorylation of β-catenin. The amount of active β-catenin (dephosphorylated at Ser33 and Ser37) was increased by Wnt3a treatment in HM20 but not in LM cells (37 We checked in our cell systems whether β-catenin associated with TCF4 or other proteins. Co-immunoprecipitation (IP) experiments carried out in LM and HM20 cells showed that β-catenin did indeed associate with TCF4 and with E-cadherin as well (Degradation of β-catenin is dependent on its phosphorylation by GSK3β and CK1α while bound to axin.LM cells . It has  as well . In addi as well .LRP6 transcripts using short hairpin RNA (shRNA) or E-cadherin overexpression significantly abolished Wnt/β-catenin-mediated transcriptional activation in HM20 cells. However, knockdown of CDH1 transcripts using shRNA significantly increased transcriptional activation in LM cells  cells significantly increased TOPflash reporter activity, consistent with our data showing that nuclear β-catenin expression was responsive to Wnt3a stimulation . PretreaLM cells . To charLM cells  and signLM cells  after stSP cells . EquallySP cells .As shown in PROM1) that encode CD133 in LM  and HM20  cells after stimulation with Wnt3a. We found that PROM1 was prominently bound by β-catenin in HM20 cells whose CSC reprogramming was strongly β-catenin dependent , mitogen-activated protein kinase (PD98059) and protein kinase C (GF109203X) had little effect on the apparent endocytosis and subsequent nuclear translocation of E-cadherin. The blockage of those functions was accompanied by treatment with PP2, a Src kinase inhibitor (753YYY755→753AAA755) remained at cell–cell contacts and endocytosis was not significantly enhanced by Wnt3a , no tumor growth was evident at 4 weeks in the 102 cell mice; 3 of 6 mice from the 103 cell group had tumors and all 6 of the 104 cell mice developed tumors (2 cells (2 cells) significantly inhibited Wnt3a-elicited tumor initiation, whereas ectopic expression of endocytosis-deficient E-cadherin diminished effects against Wnt3a-elicited subcutaneous tumor growth , which interacts with the central ARM repeat of β-catenin, thereby blocking access to them.39 identified two classes of β-catenin-binding sites. The first class represents the majority of the DNA-bound β-catenin and co-localizes with TCF4, the prominent TCF/LEF family member in murine intestinal epithelium, Wnt-responsive colorectal cancer cells and HEK293 embryonic kidney cells. The second class consists of β-catenin binding sites that co-localize with a minimal amount of TCF4. The latter consists of lower affinity β-catenin-binding events, does not drive transcription and often does not contain a consensus TCF-binding motif. It might suggest that membrane/E-cadherin-bound and nuclear b-catenin represent a different pool of B-catenin and is not involved in Wnt signaling.Clevers and colleagues40 which has been linked to the activation of Wnt signaling.41 However, other studies demonstrate that tyrosine phosphorylation does not affect β-catenin abundance and/or its transcription activity.42 It has also been reported that Src becomes activated by Dvl2 in a Wnt-dependent manner.43 Recently, Davidson et al.44 proposed that LRP6 tyrosine phosphorylation by Src serves a negative regulatory function to prevent overactivation of Wnt signaling at the level of the Wnt receptor, LRP6. Src knockout cells show that a loss of LRP6 tyrosine phosphorylation is associated with a markedly increased responsiveness to Wnt3a stimulation, which suggests that Src is both necessary and sufficient for repressing Wnt/β-catenin signaling.44 In this study, we also found that Src kinase induced E-cadherin tyrosine phosphorylation, endocytosis and nuclear translocation, which functioned as a negative regulator of Wnt/β-catenin signaling. Taken together, Src, such as GSK3,45 is a multifunctional kinase in Wnt signaling and likely can function either in a positive or negative regulatory manner depending on cellular context.There are several reports of cross-talk between Src and Wnt signaling. β-Catenin can be phosphorylated by Src kinase and promote its release from adhesion complexes,46 β-catenin47 and CD1337 are used as markers of malignant prognosis in different cancers, whereas their relationship has not been clearly understood. Some studies suggest that loss of E-cadherin expression does not induce Wnt signaling, whereas in other studies a role of E-cadherin in modulation of Wnt-dependent gene expression has been suggested.49 In view of the close association between E-cadherin and β-catenin, we speculate that the nuclear localization of E-cadherin is related to β-catenin abnormalities in human lung cancer. In this study, we found that low-grade (stage IA and IB) primary lung cancers showed high levels of membranous E-cadherin and membranous β-catenin, whereas moderate-grade (stage IIA and IIB) cancers showed low-to-moderate levels of nuclear β-catenin and high-to-moderate levels of nuclear E-cadherin. In contrast, high-grade (stage III and IV) primary and metastatic lung cancers showed high levels of nuclear β-catenin and low levels of total (nuclear/membranous) E-cadherin. Loss of E-cadherin cell membrane expression may be accompanied by its detection in the nucleus, suggesting cellular redistribution during neoplasia. Furthermore, nuclear E-cadherin expression was more significantly inversely correlated with CD133 expression (P<0.005) than E-cadherin  expression (P<0.05), suggesting that lung cancer defined by nuclear E-cadherinLow/nuclear β-cateninHigh/CD133High biomarkers has superior prognostic value over total E-cadherinLow/nuclear β-cateninHigh/CD133High.Accumulating clinical evidence has demonstrated that E-cadherin,50 . Wild-type E-cadherin was generated by PCR amplification of the corresponding cDNA fragments using E-cadherin–GFP as a template. The E-cadherin tyrosine-to-alanine (753YYY755→753AAA755) mutant was generated by site-directed mutagenesis using wild-type E-cadherin as a template. The correct clone sequence was verified by sequencing. Lentiviral-TOP-dGFP-reporter plasmids were obtained from Addgene . A pLKO.1-shRNA encoding an shRNA with a scrambled sequence or sequences targeting human Sox15 and E-cadherin, purchased from the National RNAi Core Facility, Taiwan, was introduced into HEK293T cells using the lentiviral packaging vectors pMD.G and pCMV_8.91. Antibodies against the following proteins were used: E-cadherin ; histone H3 (Ab1791) and ABCG2 (Ab3380) ; active β-catenin ; total β-catenin (C2206), β-actin (A5441) and TCF4 (T5817) ; CD133 (3663) and phosphorylated β-catenin (phosphorylated at Ser33/37/Thr41) (9561) .The E-cadherin–GFP expression construct was a gift from Alpha YapM citrate, 2 mM EDTA, 0.05% Tween 20). Slides were blocked with 2% hydrogen peroxide for 10 min and primary antibody was used at 1:500 overnight at 4 °C. BioGenex 3,3'-diaminobenzidine and hematoxylin were used for chromogenic detection and counterstaining, respectively. A semi-quantitative method for calculating positive signals was used. Signals were counted in six fields per sample under a light microscope at × 400 magnification. The results were manually evaluated by two independent observers, to determine both the percentage of positive cells and the staining intensity as described.35 The observers were blind as to the stage of each sample. The IHC score was obtained by multiplying the staining intensity  by the percentage of positive cells  in the field. The maximum possible IHC score was 4 × 4=16.Sectioned human lung cancer specimens were obtained from SuperBioChips Laboratories . All staining procedures were performed using the Super Sensitive IHC Detection Systems kit . Slides were dewaxed by xylene for 3 min and rehydrated in graded ethanols from 100% to 75%. Antigen retrieval was performed by boiling slides for 20 min using citrate-EDTA retrieval buffer  using ultra-low attachment plates  and serum-free RPMI  supplemented with B27 , 20 ng/ml epidermal growth factor and 10 ng/ml basic fibroblast growth factor (BD Biosciences). Spheres with a diameter of >30 μm were then counted. For serial passages , spheres were collected and dissociated to single cells with trypsin and dissociated cells were re-plated in a 96-well plate (diluted to 1 cell/well in an ultra-low attachment plate) and cultured for 12 days. The spheres were then counted again. The individual spheres were found to be derived from single cells.53Spheres were generated as previously described.52SP cells were identified as previously described.54 LM cells were treated with Wnt3a-containing medium for 1 h, 4 h, and 15 days as indicated. Total cell lysates, endosomes (purified by sucrose gradient centrifugation) and nuclear (Nuc) fractions were subjected to western blotting. For internalization measured by flow cytometry, cells were incubated with sulfo-NHS-SS-biotin-conjugated anti-E-cadherin mAb at 4 °C for 1 h followed by further incubation with Wnt3a at 37 °C for 2 h. Cells were incubated with 0.1 M glycine in phosphate-buffered saline for 30 min at 4 °C, to quench the unreacted biotin . Surface-retained biotin was removed using reduced glutathione  of 10 N NaOH, for internalized E-cadherin detection). After cytoplasm membrane lysis, nuclei were isolated (for nuclear E-cadherin detection). Cells and nuclei were fixed and stained by avidin-Alexa 488 for flow cytometry analysis. The expression level of E-cadherin was calculated by setting the fluorescence intensity of cells after biotin labeling but without glutathione incubation as ‘total'. The expression level of internalized E-cadherin was calculated by setting the fluorescence intensity of cells after biotin labeling followed by glutathione incubation as ‘internalized'. The expression level of nuclear E-cadherin was calculated by setting the fluorescence intensity of cells after biotin labeling, glutathione incubation and cytoplasm membrane lysis and nuclei isolation as ‘nuclear'.The internalization and nuclear translocation assays were performed as described.54ChIP assays were performed as previously described.in-vivo tumorigenicity assay, mice were injected subcutaneously with 102–104 cells in 100 μl of a 1:1 mixture of Dulbecco's modified Eagle's medium (with or without Wnt3a)/Matrigel. Tumorigenicity was evaluated at 4 weeks after transplantation.The murine studies were conducted in accordance with the Institutional Animal Care and Use Committee guidelines and were approved by the Animal Care and Use Committee of National Tsing Hua University. Severe combined immunodeficient CB17 female mice (6 weeks old) were used. For the t-test using Microsoft Excel . Spearman's rank correlation coefficient was used to assess the association between two continuous variables. Differences were considered to be statistically significant at P<0.05.Statistical analysis of data was performed by Student's