Drosophila). Whereas β-catenin is crucial for cadherin function, the role of p120-catenin is less clear and appears to vary between organisms. We show here that p120-catenin has a conserved role in regulating the endocytosis of cadherins, but that its ancestral role might have been to promote endocytosis, followed by the acquisition of a new inhibitory role in vertebrates. In Drosophila, p120-catenin facilitates endocytosis of the dynamic E-cadherin–Bazooka subcomplex, which is followed by its recycling. The absence of p120-catenin stabilises this subcomplex at the membrane, reducing the ability of cells to exchange neighbours in embryos and expanding cell–cell contacts in imaginal discs.The intracellular functions of classical cadherins are mediated through the direct binding of two catenins: β-catenin and p120-catenin (also known as CTNND1 in vertebrates, and p120ctn in  Summary: In Drosophila, p120-catenin facilitates endocytosis of the dynamic E-cadherin, which is followed by its recycling. Consequently, p120-catenin regulates cell shape and promotes the ability of cells to exchange neighbours. Cell–cell adhesion physically links cells within stable cell sheets, while also contributing to dynamic morphogenetic events such as directional proliferation, cell sorting and cell shape regulation . TransmeDrosophila; hereafter E-cad) at cell–cell junctions in the embryonic epidermis of Drosophila  regulates cadherin endocytosis . These cells are elongated along the dorso-ventral axis of the embryo and have an asymmetric distribution of E-cad, with more E-cad at cell–cell borders perpendicular to the dorso-ventral axis than on those parallel to it  (Table S1). Previously, we have demonstrated that the fast process is diffusion, as its half-time depends on the size of bleached spot, and the slower process occurs through endocytosis, as it is abolished by expression of dominant-negative dynamin and does not depend on the size of bleached spot (Table S1). The recovery of ubi::E-cad–GFP in animals homozygous for 308p120-catenin (−p120ctn−/) or with this allele in trans to a deficiency (−p120ctn/Δp120ctn) showed identical phenotypes, best fitted by single exponential models containing only the rapid diffusive component (Table S1), indicating that the slow endocytosis-dependent recovery of E-cad is either absent or reduced to a level below what is detectable in these FRAP experiments. Thus, we conclude p120-catenin is required for E-cad recovery through endocytosis in both epithelia.We measured the FRAP of ubi::E-cad–GFP in the epithelia of wild-type embryos and embryos homozygous for the romoter) . Owing thed spot . In wingp120-catenin mutants Baz–GFP was present at adherens junctions and only the fast diffusion half recovered (Table S1).To discover the fate of the mobile E-cad that no longer exchanged in the absence of p120-catenin, we examined Baz, as previous work has shown that at stage 15 it is only associated with mobile E-cad and not with immobile E-cad . This might be explained by p120-catenin regulation of E-cad transcription or mRNA levels (Ubiquitin-63E (ubi) promoter and found that the amount of E-cad–GFP in p120-catenin mutants was indistinguishable from wild type , the number of intracellular vesicles containing anti-E-cad antibody reached 0.026±0.001 vesicles/µm3  by 30 min and was modestly increased at 60 min (Table S2); this data was best fitted by an exponential model with an 18-min half time, consistent with the FRAP slow-process half time . In p120-catenin mutants, the number of vesicles was significantly reduced (P<0.0001): very few were detected at 30 min , and although the number doubled by 60 min, it remained substantially lower than that for the control (Table S2), and therefore it was not possible to calculate the half time of the endocytosis rate. However, comparing vesicle number at 30 min and the rate of increase between 0 and 30 min suggests that the endocytic rate is reduced at least fourfold in p120-catenin mutants (Table S2). The effect of removing p120-catenin was specific for E-cad, as Notch was internalised normally . A similar increase in vesicle number at 60 min was observed in p120-catenin mutants . Given that similar amounts of E-cadherin are slowly targeted for degradation in both wild type and p120-catenin mutants, this confirms that our predicted p120-catenin-independent pathway internalises E-cad for degradation. It is not possible to tell whether the few vesicles detected without chloroquine in p120-catenin mutants are recycling endosomes or vesicles on the degradation route. If they are the former, then this would suggest some recycling does occur in the absence of p120-catenin. A fourfold reduction of the endocytic rate will appear as no recovery in the FRAP timeframe (Table S1). Therefore, we conclude that the endocytosis of E-cad–Baz for recycling is strongly reduced, perhaps eliminated, in the absence of p120-catenin.Next, we tested whether some E-cadherin was targeted for degradation in the absence of p120-catenin, as suggested by the normal amount of ubi::E-cad–GFP in g the pH . In wildThus, our experiments reveal two independent pathways for removal of E-cad from the cell surface. The first pathway endocytoses E-cad–Baz for recycling and involves p120-catenin. The second is p120-catenin-independent and targets E-cad for lysosomal degradation G. The laDrosophila E-cad was not increased in the absence of p120-catenin. The simplest way to explain this difference is that Drosophila E-cad lacks the endocytic motif. Two sequence motifs, LL and DEE, are crucial for internalisation of vertebrate cadherins . The DEE motif is replaced in Drosophila E-cad by a similarly acidic EDE . Fusion of the core p120-catenin-binding region of Drosophila E-cad to IL-2R promotes its internalisation in human endothelial cells . Taken together, we favour the idea that p120-catenin has an ancestral role of linking cadherins to the endocytic machinery, and additional endocytic motifs arose within the p120-catenin-binding site on cadherins during vertebrate evolution.Although we lack evidence for p120-catenin repressing endocytosis in -adaptin . This mep120-catenin mutants  and more cells aberrantly cross segment boundaries . We examXenopus embryos , suggesting that overall tissue packing was unaffected. Similar changes did not occur in the embryonic epidermis . The increased apical area and rounding up following stabilisation of surface E-cad–Baz could arise from increasing the fraction of E-cad engaged in adhesion or increasing its association with the cortical actin cytoskeleton.Next, we tested whether the absence of p120-catenin caused defects in wing discs, measuring apical cell surface area, elongation (the ratio of the lengths of the long and short axes of the best-fit ellipse) and the neighbouring cell number. Apical cell surface area and elongation were best fitted by lognormal distributions. Loss of p120-catenin increased apical cell surface area, changing the centre of the distribution from 3.13±0.04 µmDrosophila – that p120-catenin promotes endocytosis of E-cad. Furthermore, we have found that E-cad endocytosis induced by p120-catenin is followed by E-cad recycling, and is required for cell shape regulation and cell rearrangements during morphogenesis.To summarise, we have demonstrated that at least one function of p120-catenin is similar between vertebrates and 308p120ctn, Df(2R)M41A8/ SM1 (Δp120/ SM1), 1numb/CyO, Df(2L)γ7/ CyO (Δnumb/ CyO), en::Gal4, UAS::myr-GFP and C8LR1CycA/TM3 , ubi::E-cad-GFP  and UAS::Baz-GFP  at late stage 15, following dorsal closure completion, and in the posterior-dorsal quadrant of wing pouches from third-instar larval imaginal discs. Live imaging was performed in embryos as described in http://uk.mathworks.com/), which are available upon request. The values for each type of border in a single animal were averaged and used as individual data points to compare datasets with the non-parametric Mann–Whitney test. The binned distributions of apical cell areas and elongation were fitted with lognormal distributions, and cell neighbour numbers with normal distributions using GraphPad Prism software (http://www.graphpad.com/). An F-test was used to compare datasets.Rat anti-E-cad  and Cy3-conjugated anti-rat-IgG (Invitrogen) were used. All imaging was performed with an Olympus FV1000 upright confocal microscope using 100×/1.40 Oil UPlanSApo objective. A total of 21 embryos and 19 wing discs of each genotype were used. Quantifications were performed using Packing Analyzer v2.0  and custDiscs were dissected in M3 medium (Sigma), incubated with anti-E-cad  or anti-Notch  antibodies in M3 medium at 4°C for 1 h. Then, the antibody was washed out with cold M3 medium, and discs were incubated in fresh M3 medium with or without 200 µM chloroquine . Five discs per time point (four discs in control without chloroquine) were fixed in 4% formaldehyde in PBS after labelling and stained with Alexa-Fluor-488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Quantification of vesicle numbers was performed with the 3D ObjectCounter Plugin in Fiji.The number of cells outside of the segment boundaries and types of cell–cell contacts formed by the cells at segment boundaries was analysed as described in


We analyzed the pathological consequences of abnormal Wnt/β-catenin signaling in endothelial cells of brain vessels using a murine model of Cerebral Cavernous Malformation (CCM) disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report increased transcription activity of β-catenin in CCM3-knockout endothelial cells in in-vitro and in-vivo models. Such activation is cell-autonomous, independent of Wnt-receptor stimulation, does not induce canonical Wnt/β-catenin signaling and represents an early response to CCM3 ablation that initiates the expression of EndMT makers before the onset of Tgf-β/BMP signaling which is required for the progression of the pathology, as we have previously described. We also show that the NSAIDs sulindac sulfide and sulindac sulfone, which attenuate β-catenin transcription activity, significantly reduce the number and dimension of vascular lesions in the central nervous system of mice with endothelial cell CCM3 knockout. These NSAIDs thus represent pharmacological tools for inhibition of the formation of vascular lesions, particularly with a view to patients affected by the genetic variant of CCM, who continue to develop new malformations over time.


Nuclear accumulation of β-catenin is important for cancer development and it is found to overlap with p68 (DDX5) immunoreactivity in most breast cancers, as indicated by both clinical investigations and studies in cell lines. In this study, we aim to investigate the regulation of p68 gene expression through β-catenin/transcription factor 4 (TCF4) signaling in breast cancer.Formalin-fixed paraffin-embedded sections derived from normal human breast and breast cancer samples were used for immunohistochemical analysis. Protein and mRNA expressions were determined by immunoblotting and quantitative RT-PCR respectively. Promoter activity of p68 was checked using luciferase assay. Occupancy of several factors on the p68 promoter was evaluated using chromatin immunoprecipitation. Finally, a syngeneic mouse model of breast cancer was used to assess physiological significance.in vitro in breast cancer cell lines and in vivo in an animal breast tumour model.We demonstrated that β-catenin can directly induce transcription of p68 promoter or indirectly through regulation of c-Myc in both human and mouse breast cancer cells. Moreover, by chromatin immunoprecipitation assay, we have found that both β-catenin and TCF4 occupy the endogenous p68 promoter, which is further enhanced by Wnt signaling. Furthermore, we have also established a positive feedback regulation for the expression of TCF4 by p68. To the best of our knowledge, this is the first report on β-catenin/TCF4-mediated p68 gene regulation, which plays an important role in epithelial to mesenchymal transition, as shown Our findings indicate that Wnt/β-catenin signaling plays an important role in breast cancer progression through p68 upregulation.The online version of this article (doi:10.1186/s13058-014-0496-5) contains supplementary material, which is available to authorized users. Compelling evidences indicate that the Wnt/β-catenin signaling is implicated in different stages of mammary gland development and is also important for mammary oncogenesis when aberrantly activated -5]. Gen. Gen5]. 593 by platelet-derived growth factor (PDGF) was shown to be associated with cellular transformation and epithelial to mesenchymal transition (EMT) in colon cancer by promoting nuclear translocation of β-catenin, and upregulation of its target genes like cyclin D1 and c-myc . In. In593 bnd c-myc . It has nd c-myc , breast nd c-myc , prostatnd c-myc , head annd c-myc . Serum-ind c-myc .p68 regulates the expression of several oncogenes through co-activation of β-catenin-mediated transcription, and controls tumour growth and metastasis. Although, there is an expansive evidence of literature deciphering the central role of p68 with respect to β-catenin in the architecture of intracellular signaling networks, little is known about its transcriptional regulation that may contribute to cancer development. Moreover, cellular consequences due to the modulation of its expression are not yet completely understood. Such knowledge might provide invaluable insights into the molecular mechanisms with respect to p68 in the context of oncogenesis.HEK293T (human embryonic kidney); MCF7, MDA-MB 231 (human breast cancer); 4T1 (mouse breast cancer); H1299 (lung adenocarcinoma) and HCT116 (colon cancer) cell lines used in this study were obtained from the American Type Culture Collection (ATCC). Cell culture and transfections were performed using standard procedure as described previously . Small iWnt3a-L cells were split at the ratio of 1:10 in 10 ml medium using standard procedure. Medium was collected from two consecutive batches of cells cultured for four days. Wnt-3a conditioned medium (Wnt3a-CM) was either freshly used with serum-free media (1:1) or stored at 4°C until further use.The human wnt3a gene was cloned in pcDNA4/TO and further sub-cloned into pcDNA3.1-myc-his. Cloning of c-myc in pcDNA3.1-myc-his was done previously . pEGFP-cFor ICC, cells were processed as described earlier . BrieflyPreparation of whole cell lysates (WCL), cytoplasmic and nuclear extracts and IB were performed as described previously . Either Total RNA was extracted and converted to cDNA, which was subsequently used for qRT-PCR analysis  using PoCells were transiently transfected with pGL3-p68 promoter luciferase reporter construct(s) along with Renilla luciferase plasmid (pRL-TK) and subjected to various treatments as indicated in the relevant figure(s). Luciferase activity was determined by luminometry using the GLOMAX 20/20 luminometer  by the dual-luciferase assay system (Promega), as specified by the manufacturer. Quantification was based on three technical replicates over at least two independent biological repeats.Sonicated cross-linked chromatin fragments were prepared from cells treated with formaldehyde as described earlier ,42]. Br. Br42]. t test using GraphPad software with level of significance P <0.001 .Colony formation assays in soft agar were performed in triplicate as described earlier . Images Formalin-fixed paraffin-embedded (FFPE) tissue sections derived from normal human breast (n = 10) and breast cancer samples (n = 20) were obtained from Indian patients after formal approval from ethical committee of both Council of Scientific and Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB) and Park Clinic (registered under WB Societies Act. 1961). All patients involved in the study agreed to participate and publish the research outcome. IHC was performed and H-scores were calculated as described previously . Primary5) in the mammary fat pad region. Growth of the tumour was observed for a period of 21 days after which the mice were sacrificed and the tumours were excised, fixed in 10% buffered formalin and embedded in paraffin for further IHC analysis.All animal care and experimentation conformed to the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Govt. of India) following internationally recognized guidelines. The animal ethics approval was granted by ‘IICB-Animal Ethics Committee (IICB-AEC), CSIR, Govt. of India’. To generate the tumour model, three- to four-week-old BALB/c mice were injected once with either EV or p68 knockdown 4T1 stable cells (1 × 10U test or Student’s t test (unpaired) was used. In all the experiments a value of P <0.05 was considered as statistically significant. Image quantification and densitometric scanning of immunoblots were done using Image J software .All statistical calculations were done using GraphPad QuickCalcs calculator, . For theCanonical Wnt signaling in human colon and breast cancer cells plays a key role for the aberrant activation of the β-catenin/TCF4 in tumour progression. Like β-catenin, p68 is an important transcriptional regulator, crucial for early growth and development and is also associated with cell proliferation ,47]. Ov. Ov47]. To address this issue, we first examined the expression of β-catenin, TCF4 and p68 in various established cancer cell lines and HEK293T cells Figure a. We havIt was interesting for us to discern the role of β-catenin in the regulation of the p68 gene expression. For this, we transiently overexpressed β-catenin in MCF7 cells and examined its effect on p68 expression, which increased with enhanced expression of β-catenin in a dose-dependent manner Figure a. Again,To further support these results we have investigated this β-catenin/TCF4-mediated p68 gene regulation in both mouse 4T1 and human HEK293T cells. Here, we observed that overexpression of either β-catenin or TCF4 leads to increased expression of p68 along with Wnt targets c-Myc and Cyclin D1 Figures .Hence, it can be stated that the transcriptional upregulation of p68 by ectopic expression of β-catenin or TCF4 indicates a direct involvement of the β-catenin/TCF4 transcription complex on p68 gene expression.To decipher the significance of c-Myc in the Wnt signaling-mediated tumourigenesis and its involvement in p68 gene expression, if any, we have knocked down either β-catenin or c-Myc in HEK293T cells followed by transiently overexpressing Wnt3a. We have observed that the resultant knockdown of either β-catenin or c-Myc reduces the Wnt3a-dependent increase in the expression of p68 as well as Wnt/β-catenin target c-Myc  cells . Since pWe have searched the interconnecting effect of these three crucial proteins in terms of their expression levels in serum-starved Wnt3a-MCF7 stable cells. Our results suggest that Wnt3a promotes expression of both p68 and TCF4 proteins and mRNAs  kept as a positive control. We have found occupancy of β-catenin on the p68 promoter as well as Cyclin D1 promoter (positive control) by ChIP assay in four different cancer cell lines and relatively more in the case of HCT116 and 4T1 Figure f. SimilaEMT is a process by which tumour-associated epithelial cells obtain mesenchymal features resulting in reduced cell-cell contact and increased motility; hence playing a critical role in metastasis. Wnt signaling plays a vital role in EMT ,53]. Al. Al53]. To assess the anchorage-independent growth and tumourigenic potential of Wnt3a-MCF7 cells, we conducted colony-formation assays. The results indicate that these cells form significantly more number of colonies in comparison to EV-MCF7 cells. Also, individual knockdown of β-catenin, TCF4, or p68 in Wnt3a-MCF7 cells using respective siRNAs showed that knockdown of any of these molecules resulted in reduced number of colonies, indicating that the tumour-promoting ability of β-catenin not only depends on TCF4 but also on p68 (a) Immunohistochemistry was performed in normal human breast samples (n = 10) to assess the status of E-cadherin, β-catenin, TCF4 and p68. (b) The mean H scores of the desired proteins for each normal breast tissue samples were represented by scatter plot. The images were captured at 100X magnification using BX61 microscope (Olympus). (JPEG 1 MB)Additional file 2: Figure S1.: Immunohistochemical analysis of p68, β-catenin and TCF4 in normal human breast tissues samples. Additional file 3: Figure S2.: Wnt signaling promotes p68 expression. HEK293T, HCT116 and H1299 cells were serum starved for 24 h before treatment with either control condition medium (EV-CM), Wnt3a conditioned medium (Wnt-CM) for another 24 h. Whole cell lysates (WCL) were prepared and analysed by IB to examine the levels of β-catenin and p68. (JPEG 112 KB)Additional file 4: Figure S3.: GSK3β inactivation regulates p68 expression due to β-catenin stabilization. MCF7 cells were serum starved for 24 h and treated with 20 mM Licl for the indicated periods. Whole cell lysates (WCL) were prepared and analysed by IB to examine the levels of β-catenin, p68 and Cyclin D1. (JPEG 87 KB)Additional file 5: Figure S4.: TC4 regulates p68 transcript level. HEK293T cells were transfected with either WT-TCF4 or control vector (ctrl). RNAs were isolated from 36 h post-transfected cells and subsequently analysed by qRT-PCR. (JPEG 80 KB)Additional file 6: Figure S5.: β-Catenin along with c-Myc regulates p68. HEK293T cells were transfected with β-catenin and c-Myc either alone or in combination. WCLs were prepared after 36 h of transfection and analysed by IB to examine the levels of β-catenin, p68 and c-Myc. (JPEG 77 KB)(a) Cross-linked chromatins of MCF-7, MDA-MB 231, 4T1, HCT116 cells were immunoprecipitated with anti-TCF4 antibody. (b) Cross-linked chromatins of 4T1 and HCT116 cells were transfected with either scrambled siRNA or β-catenin siRNA, and immunoprecipitated with anti-β-catenin antibody. The relative values in both (a) and (b) were normalised to negative control IgG. SEMs were calculated from two independent experiments. (JPEG 2 MB)Additional file 7: Figure S6.: β-catenin/TCF4 complex occupies the p68 promoter. Additional file 8: Figure S7.: c-Myc occupies the p68 promoter. Cross-linked chromatin of HCT116 cells transfected with either scrambled or c-Myc siRNA were immunoprecipitated with anti-c-Myc antibody as indicated and subsequently qRT-PCR was performed. The relative values were normalised to IgG (negative control). SEM was calculated from two independent experiments. (JPEG 3 MB)Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Authors’ original file for figure 7Authors’ original file for figure 8Authors’ original file for figure 9Below are the links to the authors’ original submitted files for images.


Furthermore, the fabricated biosensor had a good selectivity, reproducibility, and long-term stability, indicating that the novel CTS+PTFE/GOx/MWCNTs/PTH composite is a promising material for immobilization of biomolecules and fabrication of third generation biosensors.A novel glucose biosensor was fabricated. The first layer of the biosensor was polythionine, which was formed by the electrochemical polymerisation of the thionine monomer on a glassy carbon electrode. The remaining layers were coated with chitosan-MWCNTs, GOx, and the chitosan-PTFE film in sequence. The MWCNTs embedded in FAD were like “conductive wires” connecting FAD with electrode, reduced the distance between them and were propitious to fast direct electron transfer. Combining with good electrical conductivity of PTH and MWCNTs, the current response was enlarged. The sensor was a parallel multi-component reaction system (PMRS) and excellent electrocatalytic performance for glucose could be obtained without a mediator. The glucose sensor had a working voltage of −0.42 V, an optimum working temperature of 25°C, an optimum working pH of 7.0, and the best percentage of polytetrafluoroethylene emulsion (PTFE) in the outer composite film was 2%. Under the optimised conditions, the biosensor displayed a high sensitivity of 2.80 µA mM Glucose has always been an analyte that has received much attention because of its vital effect in clinical and environmental analysis. Since the first concept of an enzyme-based biosensor was proposed by Clark and Lyons However, for the flavin adenine dinucleotide (FAD) that is the active centre of glucose oxidase (GOx) and is deeply embedded in the molecular cavity Multiwalled carbon nanotubes (MWCNTs) have unique electronic, mechanical, and thermal properties, which leads to their potential application in the fabrication of biosensors. Many recent reports have demonstrated that the integration of MWCNTs and conducting polymers 3COOC12H10N3S) with two amino groups symmetrically distributed on each side of its aromatic rings Recently, conducting polymers have been extensively employed for the construction of electrochemical biosensors due to their conductive nature and superior stability In this study, we investigated the step-wise immobilizations of PTH, MWCNTs, GOx, and chitosan-PTFE composite films onto a glassy carbon electrode to build a biosensor for measuring glucose. A mediator-free GOx-based glucose biosensor was constructed though a layer-by-layer self-assembly approach. Electrochemical impedance spectroscopy was used to monitor the biosensor modification process. DET between the active site of the GOx and the electrode was achieved, and its behaviour as a biosensor and its enzyme stability were investigated by electrochemical techniques.Glucose oxidase  and bovine serum albumin (BSA) were purchased from MP Biomedicals Co., Ltd. (Shanghai). The multiwalled carbon nanotubes  were obtained from the Chengdu Organic Chemicals Co., Ltd. of the Chinese Academy of Science . Uric acid (UA) was purchased from Sigma. D-glucose, chitosan, glacial acetic acid, glutaraldehyde, vitamin C, L-cysteine (L-cys), and the polytetrafluoroethylene emulsion (60% PTFE) were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd .Amperometric experiments and cyclic voltammetric experiments were performed on an Autolab AUT72230 electrochemical work station (Metrohm China Ltd.). A conventional three-electrode system was applied: the modified or unmodified GCE electrode (3 mm diameter) acted as the working electrode, and a saturated calomel electrode (SCE) and a platinum wire served as the reference and counter electrodes, respectively. All of the electrochemical experiments were carried out at room temperature. Scanning electron microscopy (SEM) images were taken using a Tescan 5236 SEM (Tescan China Ltd.).2O3 slurries successively and was then rinsed with water, acetone, absolute ethanol, and doubly-distilled water. According to the method described by Wang −1).The glassy carbon electrode  was polished with 1.0 and 0.05 µm α-Al−1 GOx aqueous solution.Two mg MWCNTs was dispersed by ultrasonication in 1 mL 0.5% chitosan (CTS) solution. One mg GOx and 7.5 mg bovine serum albumin (BSA) were added to 100 µL 0.1 M PBS to prepare a 10 mg mLPreparation of the outer chitosan-PTFE composite film: the 60% (w/w) PTFE solution was dispersed by ultrasonication in a 0.5% chitosan solution, which was finally diluted to different ratios of 1%, 2%, 5%, and 10% (w/w) chitosan/PTFE suspension (CTS+PTFE).−1 MWCNTs, 6 µL 10 mg mL−1 GOx aqueous solution , and 5 µL 2% (w/w) chitosan/PTFE suspension onto the pre-treated PTH/GCE. Each casting was performed after the previous cast had been air-dried. Finally, the modified GCE was immersed in PBS with a pH = 7.0 to remove the loosely absorbed GOx and was stored at 4°C under dry conditions. For comparison, MWCNTs/GCE,PTH/GCE,MWCNTs/PTH/GCE, and GOx/MWCNTs/GCE coatings were also prepared according to the same procedure. The assembly process of CTS+PTFE/GOx/MWCNTs/PTH is showed in A CTS+PTFE/GOx/MWCNTs/PTH/GCE coating was prepared facilely by successively casting 6 µL 2 mg mLDuring the process of thionine electropolymerisation , the curEIS can give more detailed information on the impedance changes during the modification process. When presented in a Nyquist plot, the impedance spectrum includes a semi-circular portion corresponding to the electron-transfer-limited process and a linear part resulting from the diffusion process Compared with bare GCE , the dia2 GOx redox cofactor pa1/ipc1≈1) and ΔEp1≈30 mV; however, that ratio for PTH is slightly greater than 1 (ipa2/ipc2>1) and ΔEp2≈20 mV. These results further demonstrated the good reversibility of PMRS.From −1  catalysed the reduction of oxygen dissolved in the solution. The relative consumption of GOx (FADH2) and generation of GOx (FAD) directly led to the changes in the peak currents of GOx. Because GOx and PTH were both in the PMRS of the electrode, the peak currents of PTH had the same tendency to change, which can be used as an indicator of the change in GOx in turn.When in an air-saturated solution, the electrochemically formed GOx (FADHIn 2), leading to a continuous decrease in the cathodic and corresponding anodic peak currents of both GOx and PTH.The equation indicates that GOx (FAD) is directly involved in the glucose oxidation reaction. As the glucose concentration increases, GOx (FAD) is competitively employed by glucose and increasingly converted into GOx  were obtained from the amperometric response (−1) C (mM)–0.01 (µA) (R = 0.997) was derived from the calibration curve and revealed that the glucose sensor has a detection limit as low as 5.0 µM (S/N = 3).The calibration curve (current vs. glucose concentration) and the Lineweaver Burk plot . The biosensor has an optimum working voltage of −0.42 V, an optimum working temperature of 25°C, and an optimum working pH of 7.0. In addition, the best percentage of PTFE in the outer composite film was 2%.To evaluate the selectivity of the biosensor, many interfering biomolecules, including uric acid (UA), ascorbic acid (AA), and L-cysteine (L-cys), that normally co-exist with glucose in real samples (human blood) were investigated . The curWe put three sensors in PBS solution, which had a glucose concentration of 1.0 mM, to perform current tests. Then, the sensors were saved in the refrigerator at 4°C to perform stability tests. As In The current response of CTS+PTFE/GOx/MWCNTs/PTH is from the competitive reaction of glucose. Glucose competitively spreads to DET-GOx and reacts with it, thereby the DET-GOx is reduced, and thus the response current is decreased.DET-GOx influences detection performance. The MWCNTs embedded in FAD are like “conductive wires” connecting FAD with electrode, reduce the distance between them and are propitious to DET. Combining with good electrical conductivity of PTH and MWCNTs, the current response is enlarged. According to response current, glucose concentration can be detected.2) and PTH. The parallel multi-component reaction system (PMRS) of the electrode played an important role in facilitating the electron transfer between GOx and the electrode surface. To the best of our knowledge, this is the first report about the direct electrochemistry of GOx based on PMRS. Furthermore, the potential use of the resulting electrode as a third generation glucose biosensor was confirmed by the amperometric response for successive additions of glucose. The biosensor showed a high sensitivity, fast amperometric response and low detection limit. Possible interfering species in blood, such as uric acid, ascorbic acid, and L-cysteine, did not influence the glucose determination. In addition, the CTS+PTFE film had good permeability and could greatly improve glucose detection range and stability. The MWCNTs embedded in FAD were like “conductive wires” connecting FAD with electrode, reduced the distance between them and were propitious to DET. Combining with good electrical conductivity of PTH and MWCNTs, the current response was enlarged. These results demonstrate that the CTS+PTFE/GOx/MWCNTs/PTH is an attractive material for the fabrication of efficient amperometric biosensors. The method presented can be used for the immobilization and evaluation of DET of other enzymes or proteins.In this work, we successfully introduced a promising glucose biosensor based on the immobilization of GOx onto a MWCNTs/PTH nanocomposite film. The evaluation of the CTS+PTFE/GOx/MWCNTs/PTH/GCE demonstrated that DET of GOx was achieved on this nanocomposite film. Cyclic voltammetry showed three pairs of well-defined redox peaks corresponded to DET of GOx (FAD/FADH


Aberrant regulation of Wnt signaling is a common theme seen across many tumor types. Decades of research have unraveled the epigenetic and genetic alterations that result in elevated Wnt pathway activity. More recently, it has become apparent that Wnt signaling levels identify stem-like tumor cells that are responsible for fueling tumor growth. As therapeutic targeting of these tumor stem cells is an intense area of investigation, a concise understanding on how Wnt activity relates to cancer stem cell traits is needed. This review attempts at summarizing the intricacies between Wnt signaling and cancer stem cell biology with a special emphasis on colorectal cancer. Dedicated populations of stem cells ensure self-renewal of most adult epithelial tissues . These cRNF43 that encodes an E3 ubiquitin ligase that negatively regulates Wnt signaling [Over the past decade, the mutational landscape of CRC has unfolded and it became apparent that most, if not all, CRCs display genetic alterations in the Wnt pathway. Although the nature of these mutations may be distinct, they ultimately result in the stabilization of β-catenin, the key transducer of canonical Wnt signals. The most prevalent mutations are truncating events that inactivate the tumor suppressor Adenomatousis Polyposis Coli (APC) , a proteignaling ,8.KRAS, BRAF, TP53, and SMAD4 [As mentioned above, Wnt activating mutations occur early during colon tumorigenesis whereas the progression of the disease is often accompanied by other genetic alterations, most commonly seen in nd SMAD4 ,10. AlthAPC. Administration of doxycycline resulted in activation of Wnt signaling, intestinal hyperproliferation, and a block in differentiation. Importantly, this phenotype was completely reversible when endogenous levels of APC were restored after doxycycline removal. Of note, this was also true even in the presence of other CRC canonical oncogenic mutations, suggesting that APC loss and hyperactivation of Wnt signaling is crucial for tumor maintenance [In a recent elegant mouse study, Dow et al.  addressentenance .KRAS, BRAF, and/or SMAD4 [APC mutations are very early events whilst in serrated neoplasia RNF43 mutations are selected for at a much later stage [In a separate study, a functional blockade of R-spondins in RSPO fusion tumors caused tumor regression even in the presence of mutations in or SMAD4 . Altogeter stage . Thus, dAXIN1 and/or AXIN2 [The role of Wnt signaling in carcinogenesis extends beyond CRC. Mutations of pathway components are also frequently detected in a subset of hepatocellular carcinoma. There, hyperactivation of Wnt signaling is mediated by loss of function and inactivation of the Wnt negative regulators or AXIN2 . Early sor AXIN2 . Soon afor AXIN2 . Importaor AXIN2 . Anotheror AXIN2 . Patientor AXIN2 . Since MAPC truncation causes an inherited autosomal condition called Familial Adenomatousis Polyposis (FAP), which almost exclusively results in an increased risk in developing colon cancer but with only a limited increase in the incidence of other malignancies [It is intriguing to note that both the type of Wnt activating mutations and the level of activation of the pathway seem to be selected in a tissue specific manner. For example, germline gnancies . Conversgnancies . As we wgnancies .Apc is induced in the stem cell compartment [Apc mutation, for example, to become fixed—that is, to enable that mutated stem cell to populate the entire crypt—is ~60%, which still means that in a large fraction of cases the mutated cell will be replaced by one of its wild-type stem cell neighbors and will be lost [Apc deletion is not sufficient to induce tumorigenesis [Apc-deficient tuft cells could form colonic tumors [Bcl-2, which is most pronounced in the intestinal stem cell compartment, greatly facilitates oncogenic transformation following Apc-inactivation by alleviating Apc-loss induced pro-apoptotic signals [Bcl-2 in more differentiated cells also rendered these cells susceptible to generating oncogenic outgrowth following Wnt pathway activation. Of further interest is the high level of telomerase activity detected upon β-catenin activation, potentially explaining why Wnt active cells are more permissive to tumorigenesis when mutations occur [Cancer develops from a single founding cell, the identity of which is still highly disputed. Self-renewal capability of most adult tissues is restricted to stem cells and therefore these cells are likely to be a prime target for cellular transformation. In addition, adult stem cells are usually long-lived, which is a prerequisite to accumulate sufficient genetic events, especially in the case of epithelial tissues undergoing high turnover. Support for the stem origin of intestinal cancer comes from mouse models of intestinal tumorigenesis that have confirmed that adenomas develop preferentially when loss of partment . More repartment ,22. If o be lost . For the be lost ,24. Unfo be lost . More re be lost . Anotherigenesis . Only inc tumors . Altogetc tumors . Express signals . Of notens occur . Besidesns occur .There are several lines of evidence to support the notion that high Wnt activity identifies cancer cells endowed with stem cell capabilities. In CRC, despite the presence of mutations that constitutively turn on Wnt signaling, remnant regulation of the transduction cascade occurs which results in various degrees of Wnt signaling output within a clonal population of cancer cells. Using a Wnt reporter construct, we have reported that human colorectal CSCs can be defined on the basis of high Wnt signaling activity, and these cells preferentially localize to a myofibroblast niche . AlreadyAPC gene rarely result in a complete null genotype. Instead, there seems to be a particular selection for alleles that retain some level of β-catenin regulation. This has formed the basis for the “just-right” hypothesis, which postulates that a finely tuned β-catenin/Wnt activity level is required to optimally endow incipient tumor cells with tumorigenic potential. As we will elaborate below, there are various levels at which regulation of Wnt activity operate. Both intrinsic as well as microenvironmental regulation have been reported but altogether they seem to converge towards a similar phenotypic quality: tumor cells displaying high Wnt activity are functionally related to cancer stem cells (CSCs).As we have summarized, Wnt activating mutations have been shown to be essential for the initiation of several tumor types and tumor maintenance is also dependent on Wnt activity irrespective of the presence of additional driver mutations . InteresAPC mutations that inhibit β-catenin degradation and thereby enhance Wnt pathway activity. Yet, despite this global increased level of activity in all cancer cells, additional regulation occurs, and cells with distinct activity levels can be identified and associated with clear biological significance. This regulation takes place at several levels and displays clear resemblance to regulation of the Wnt pathway in normal stem cell compartments  mediated fashion. Following Wnt ligand binding, the β-catenin degradation complex, comprising AXIN, APC, and GSK3β, translocates to the plasma membrane where its activity is inhibited by several mechanisms: the stability of components of the complex is diminished and GSK3β kinase activity is reduced resulting in relative stabilization of β-catenin. β-catenin accumulates, translocates to the nucleus, and enhances Wnt target gene expression in a TCF/Lymphoid Enhancer-Binding Factor (LEF) dependent manner.Sources of Wnt ligands in normal tissues are the stem cell-supporting cells comprising the cellular niche. For example, Paneth cells in the intestine produce Wnts in support of the crypt base columnar stem cell population. Additionally, in the hematopoietic stem cell niche Wnt ligands are pivotal in the regulation of stem cell function and hematopoiesis . The impIn estrogen receptor negative (ER negative) breast cancers Wnt3a has been demonstrated to increase the sphere forming ability . SimilarLgr5 is both a Wnt signaling component as well as a prominent Wnt target gene. Lgr5 is a cell surface G-protein coupled receptor (GCPR) that binds R-spondins, which activate the Frizzled-LRP5/6 complex together with Wnt ligands. As a result, both Lgr5 and R-spondins augment Wnt ligand mediated Wnt signaling. Further evidence has indicated that R-spondin proteins inhibit the E3 ubiquitin ligases Rnf43 and Znrf3 that cause stabilization of Frizzled receptors, thereby enhancing Wnt signaling B.These insights suggest that the R-spondin-Lgr5-Rnf43 axis enhance Wnt activity and thereby likely promote stem cell properties. Indeed, Lgr5 has been reported to be a CSC marker in many malignancies which might both reflect the high Wnt activity levels of these cells and also the functionality of Lgr5 in promoting stem cell functions. In agreement, in breast cancer the overexpression of Lgr5 or its inhibition results in increased and decreased stem cell functionality, respectively . Also, iAPC truncating mutations or β-catenin/CTNNB1 stabilizing mutations, heterogeneous Wnt activity levels can be detected as evidenced by various degrees of nuclear localized β-catenin in individual cancers. This suggests that Wnt signaling can be modulated by extrinsic features in otherwise homogenous populations of cancer cells all harboring Wnt activating mutations. This notion became known as the “β-catenin paradox” and in recent years many factors have been identified that are accountable for this observation [The tumor microenvironment is characterized by an influx of activated cell types, including cancer-associated fibroblasts (CAFs) and various types of immune cells. These cells, together with the cancer cells, produce a plethora of cytokines and signaling molecules of which many have a direct impact on Wnt signaling activity. As we described above, in CRC it was long known that despite activating mutations in the Wnt pathway that occur early in tumor development, such as ervation . In the ervation . Indeed,ervation ,52. Thiservation . IntriguBesides regulation of the Wnt pathway that is exerted by extracellular factors additional cancer cell intrinsic features can also contribute to Wnt modulation and thereby CSC biology. These factors are genes or microRNAs (miRs) that are up- or down-regulated following genetic aberrations that occur during tumor development and might be related to long-lasting input from the microenvironment, or potentially represent epigenetic features associated with the cell of origin of the malignancy. In many cases the exact mechanism by which these internal regulators of Wnt activity are established remains unknown, but their downstream effect of Wnt signaling is evident. Knowledge of these factors is critical as they are both informative in elucidating the intricacies of the Wnt cascade in cancer but also might constitute important therapeutic targets. In contrast to the extracellular Wnt regulators, the means by which these types of regulators dictate the CSC vs non-CSC distinction is less clear-cut. However, it can be envisioned that these features increase or decrease global Wnt activity levels thereby impacting on the proportion of CSCs in that cancer . At the AXIN2, APCDD1, and DKK1 [With respect to intrinsic regulators of Wnt activity levels, it was recently established that proliferative cell nuclear antigen (PCNA) associated factor (PAF) is upregulated in breast cancer as compared to healthy mammary tissue . PAF is and DKK1 ,75,76. Iand DKK1 . Similarand DKK1 ,78,79,80Similarly, a large number of miRs have been implicated in CSC maintenance via modulation of the Wnt signaling axis. To name a few: miR-21 has been established to be upregulated in CRC and promotes stem cell properties. Mechanistically, miR-21 suppresses transforming growth factor β receptor (TGBR2), which results in activation of the Wnt pathway, and increases c-Myc and cyclin D1 levels ,82. miR-Another example of non-coding RNAs in Wnt signaling regulation relevant for CSC biology comes from hepatocellular carcinoma (HCC). In HCC long noncoding RNA (lncRNA) lncTCF7 that is highly expressed in this malignancy is required for CSC maintenance . lncTCF7To conclude this section, we wish to highlight that direct genetic events also impact on the structure of the CSC hierarchy by means of Wnt pathway modulation. Obviously, it is expected that specific Wnt activating mutation results in distinct degrees of Wnt pathway activation, and that these different basal activity levels impact on stem cell properties in a similar fashion, as has been detected along the intestinal tract . UnfortuAs we have discussed in this review, there is an extensive body of evidence supporting the concept that Wnt signaling defines CSCs and is key to their biology. Hence, targeting this pathway is an attractive therapeutic target for cancer. However, the characteristics of the signal transduction cascade, and the molecular mechanisms by which its activity is regulated, make it a very difficult task. The field has proven to be very good in generating inhibitors of kinase activity and antibodies that bind either growth factors or their receptors. Drugs that fall in these categories have been proven to be clinically most successful. Unfortunately, the regulation of the Wnt pathway relies heavily on protein-protein interactions, and blocking these has proven to be much more challenging. Furthermore, kinases involved in the pathway predominantly exert an inhibitory effect , therefore devising direct kinase inhibitors of pathway components is problematic.APC mutations. Indeed, tankyrase inhibition has been shown to reduce the growth of APC mutant CRC tissue in xenograft models [Apc-loss driven mouse model [APC wild type only under serum deprived conditions [Yet, accumulating knowledge in the regulation of the pathway has led to novel putative targets that can, in principle, be effectively modulated by drugs. For example, tankyrase inhibitors prevent poly(ADP-ribosyl)ation-dependent AXIN degradation, thereby promoting β-catenin destabilization. Critically, these agents also exert their Wnt inhibitory properties in the presence of t models , as wellse model . This rese model . Furtherse model ,93. Tanknditions , induce nditions , and inhnditions . APC or CTNNB1. Instead, cancers that rely on upstream Wnt activating events, including the recently reported fusion events involving RSPO2 and RSPO3 in a small percentage of APC wild type CRCs [ASCL2 and LGR5. Similarly, OncoMed Pharmaceuticals reported anti-RSPO treatment of RSPO producing ovarian, colon, lung, and pancreatic cancers attenuated cancer growth and was again associated with reduced expression of CSC marker genes [Another Wnt component in the spotlight for Wnt inhibitor development is Dvl. The PDZ domain of Dvl acts as a critical mediator of the Dvl-Frizzled interaction and downstream transduction of the Wnt signal. PDZ domain inhibitors have been discovered that inhibit the Wnt pathway ,98. Alteype CRCs ,101 or Rype CRCs . Indeed,ype CRCs . In addiype CRCs . Alternaype CRCs . Intriguer genes . This saer genes . Secondler genes . This ager genes . Phase 1As indicated, upstream targeting of the Wnt pathway is unlikely to show efficacy in cancers with genetic activation of the pathway by mutations in more downstream components. However, a multitude of strategies have been employed to impede β-catenin activity. PRI-724 disrupts the complex β-catenin forms with CBP (cyclic AMP response element binding protein), which reduces the expression of a subset of Wnt target genes that are particularly important for (cancer) stem cell proliferation ,107. SevAdditional opportunities to reduce Wnt pathway activity in cancer focus on targeting the growth factors and cytokines that modulate the pathway. Examples of these include HGF inhibition, which reportedly reduces colon cancer tumor growth , potentiLGR5, CD44, and PROM1 (CD133) [As our review of the literature advocates, there is an overwhelming amount of evidence to support the critical role of the Wnt pathway in CSC biology, thereby mirroring the even larger body of studies advocating the critical function of this signaling cascade in normal stem cells. Wnt pathway activity is associated with the stem cell population in many malignancies and the most prominent CSC markers used to identify this population are, in fact, direct Wnt target genes including  (CD133) ,115,116. (CD133) ,118. It


Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%. Using large arrays of small molecules immobilized on a solid support , the reaction of a protein probe with thousands of distinct molecules can be characterized simultaneously in a single experiment. As a result, SMMs have emerged as one of the high-throughput screening platforms for probing of protein-ligand interactions  in appliThe success of microarray-based screening platforms hinges on efficient immobilization of a wide variety of small molecules on solid supports. A number of immobilization schemes exist for SMM fabrication, based on selective or non-selective attachment methods. Examples of selective attachment schemes include immobilization of biotin groups on strepavidin functionalized surface ,11,12,13One issue concerning the isocyanate capture method is the relatively low immobilization efficiency for some molecules due to their low reactivity with isocyanate. Among nucleophilic residues, primary amine, aryl amine, and thiol have high reactivity to isocyanate; primary alcohol, phenol, and secondary amine have medium reactivity to isocyanate; carboxylic acid, secondary alcohol, and tertiary alcohol have low reactivity to isocyanate. The immobilization efficiency of molecules with carboxylic acid and secondary alcohol is less than 10% that of molecules with primary amine . In screIn this paper we report experimental studies on immobilization efficiency and morphological quality of printed small molecules on isocyanate functionalized surface as functions of (1) nucleophilic residues on small molecules for surface attachment; (2) penultimate chemical groups after terminal isocyanate residues on the isocyanate functionalized surface; (3) the length of polyethylene glycol (PEG) as the spacer between the penultimate chemical group and the glass surface; and (4) post-printing treatment. We identified a set of conditions that enable immobilization of small molecules with a wide range of nucleophilic residues, with an overall immobilization percentage of at least 73%. 1 was from PolyPeptide Group  and (PEG)6 was from Shanghai Plus Bio-Science and Technology Company . (PEG)12, (PEG)24, and (PEG)36 were from Biomatrik Incorporated . (PEG)2, N,N-Diisopropylethylamine (DIPEA), piperidine, pyridine, baicalin, hexamethylene diisocyanate (HDI), 1,4-Phenylene diisocyanate (PPDI), (benzotriazol-l-yloxy) tripyrrolidinophosphonium hexafluoro phosphate  biotin-(PEG)2-NH2, biotin-(PEG)6-OH and D-biotin were from Aladdin Industrial Corporation . Biotin-NH-NH2 and biotin4-nitrophenyl ester were from Shanghai Yuanye Bio-Technology Company . N,N-Dimethylformamide (DMF), tetrahydrofuran (THF) were from Sinopharm Chemical Regent Company . Dimethyl sulfoxide (DMSO) was from Shanghai Lingfeng Chemical Reagent Company . Bovine serum albumin (BSA) was from Sigma-Aldrich  and mouse anti-biotin antibody was from Jackson ImmunoResearch Laboratories . Streptavidin (SAVD) was from Life Technologies . Biotin conjugated BSA was from Beijing Bo Sheng Bio-Technology Company Limited .Amine functionalized glass slides were purchased from CapitalBio Corporation . Polyethylene glycol (n = 1) or (PEG)n solution for 10 h with stirring. The (PEG)n solution contained 1 mM Fmoc-NH-(PEG)n-(CH2)2-COOH, 2 mM PyBOP and 20 mM DIPEA dissolved in DMF. (PEG)n of varying lengths  were used on respective slides. Second, the protecting Fmoc group was removed by incubating the PEG-treated glass slides in a solution of piperidine (v/v 1%) in DMF for 12 h with gentle stirring. Third, the terminal isocyanate group was added through incubation of de-protected glass slides in a solution of isocyanate for 1 hwith stirring. Two isocyanate solutions at the same concentration of 60 mM were used to add isocyanate residues with different penultimate chemical groups to the (PEG)n spacer: the HDI solution in DMF was used to add hexyl-isocyanate residues and the PPDI solution in THF was used to add phenyl-isocyanate residues 6-OH, and D-biotin (with intrinsic COOH group), were printed on as-prepared isocyanate functionalized glass slides with a contact microarray printer . Each compound was diluted in DMSO and separately in a mixture of DMSO with ddH2O (v/v 50%) to 4 concentrations of 1 mM, 4 mM, 8 mM, and 10 mM. Three control compounds  were diluted in DMSO at a concentration of 5 mM. The biotinylated compounds were each printed in triplicate. There were a total of 135 printed spots over an area of 3.75 × 2.5 mm2 that formed one microarray. We printed 6 identical microarrays on one slide. The averaged diameter of printed spots was 100~150 μm and the center-to-center spot separation was 250 μm. After printing, 4 distinct post-printing treatments were used on respective glass slides to facilitate covalent attachment of nucleophilic residues to isocyanate functionalized slides: (1) drying at room temperature only; (2) drying at 45 °C only; (3) catalyzation in pyridine vapor at room temperature; and (4) catalyzation in pyridine vapor at 45 °C. After post-printing treatment, the microarrays were stored in a −20 °C freezer until use.Biotinylated compound microarrays were fabricated as follows. Five biotinylated compounds, biotin-(PEG)2-NH2 at a concentration of 5 mM in DMSO were also printed as the inner and the outer borders of SMMs to serve as position markers and positive controls. There were 148 columns and 64 rows in each SMM over an area of 37 × 16 mm2. The SMM was dried at 45 °C for 24 h and were then stored in a −20 °C freezer until binding reactions with proteins.A small molecule microarray of 3375 bioactive compounds (FMSCL: Fudan MolMed-Selleck Compound Library), including 1053 natural compounds from Traditional Chinese Medicine (most of them from herb), 1527 drugs approved by Food and Drug Administration (FDA) and 795 known inhibitors, was prepared as follows. Each compound was dissolved in DMSO to a concentration of 10 mM and was printed in duplicate on both phenyl-isocyanate functionalized slides and hexyl-isocyanate functionalized glass slides. Biotin-BSA at a concentration of 7600 nM in 1× phosphate-buffered saline (PBS) and biotin-(PEG)in situ using an optical biosensor, scanning oblique-incidence reflectivity difference (OI-RD) microscope, whose working principle has been reported previously  is the concentration of protein. Since KD between unlabeled mouse anti-biotin antibody and biotin is ~1 nM [D) is close to unity as the concentration of anti-biotin antibody [c] is 86 nM. As a result, the coverage of anti-biotin antibody can be used as the measure of the immobilization efficiency of biotinylated compounds on hexyl-isocyanate functionalized surface. To quantify immobilization efficiencies of compounds with different nucleophilic residues, we calculated the coverage of antibodies as the ratio of the surface mass density of antibodies captured by the compounds to that of one monolayer of antibodies in upright or “end-on” configuration. The latter is roughly 1.3 × 10 23.5 nm ,39. The is ~1 nM , [c]/ inimized ,42. We s 24, 36) . ClearlyUsing a contact-angle measurement system , we measured the contact angles of DMSO on hexyl-isocyanate functionalized surfaces as a function of the PEG spacer length . The coni.e., post-printing thermal annealing treatment at 45 °C alone yields the highest immobilization efficiency among all four treatment conditions.We further examined the effects of post-printing treatments of compound microarrays on immobilization efficiency. In addition to catalyzation in pyridine vapor at room temperature, we explored three other treatment conditions: (1) drying printed microarrays at room temperature without catalyzation in pyridine vapor; (2) annealing printed microarrays to 45 °C without catalyzation in pyridine vapor; (3) catalyzation of printed microarrays in pyridine vapor at 45 °C . The immCompared with immobilization on a hexyl-isocyanate functionalized surface followed by catalyzation in pyridine vapor at room temperature, immobilization efficiencies of compounds are improved significantly on a phenyl-isocyanate functionalized surface followed by thermal annealing at 45 °C alone, as shown in The stability of a functionalized glass slide is important for producing reproducible binding assays as one may not use the slide immediately after fabrication. To explore this issue experimentally, we measured immobilization efficiencies of compound microarrays fabricated on phenyl-isocyanate functionalized slides that have been stored for various lengths of time before printing the compounds on the slide surface. On Day 0, we fabricated a number of phenyl-isocyanate functionalized slides. These slides were stored in a −20 °C freezer. On the day as indicated on the x axis, biotinylated compounds were printed on a slide (taken out from the freezer and thawed to room temperature) and annealed at 45 °C for 24 h. Immobilization efficiencies of the printed compounds as functions of slide storage time  shows thi.e., amine, phenol, carboxylic acid) are highlighted in color rectangles in SMMs of 3375 bioactive compounds are prepared on hexyl-isocyanate functionalized glass slides and on phenyl-isocyanate functionalized slides followed by thermal annealing at 45 °C for 24 h. From OI-RD images of printed microarrays before washing , 2982 coi.e., ligands of streptavidin) are listed in To examine whether the improvement in immobilization efficiency of a compound collection indeed leads to a corresponding increase in the number of protein ligand discovered on the SMM platform, we use streptavidin as a protein probe and incubate microarrays of 3375 compounds printed on hexyl- and phenyl-isocyanate functionalized slides in the solution of streptavidin at 154 nM for 1 h. Both microarrays are treated with thermal annealing at 45 °C for 24 h before the reaction. From changes in OI-RD image , 15 compWe investigated several surface chemistry parameters in fabrication of SMMs on isocyanate functionalized glass slides for optimal immobilization efficiency, spot morphology and reproducibility. We find that both the penultimate group next to the isocyanate reside and post-printing treatment have significant impacts on immobilization efficiencies of small molecules, particularly those having residues with inherently low reactivity with isocyanate. Both presumably facilitate covalent attachment of nucleophilic groups to the isocyanate residues on the functionalized surface. In particular, we find that a phenyl-isocyanate functionalized surface is more efficient for small molecule immobilization than a hexyl-isocyanate functionalized surface by almost a factor of two. We further find that thermal annealing of printed small molecule microarrays at 45 °C yields higher immobilization efficiency by almost another factor of two than the method of catalyzation in pyridine vapor at room temperature ,27. As an spacer with n = 6 is optimal in yielding high-quality SMM spot morphology while minimizing non-specific protein binding to the surface. Furthermore, we find that isocyanate functionalized surfaces degrade after storage in −20 °C freezer for a period of time. Ambient moisture reacts with surface isocyanate residue slowly and causes the surface density of the isocyanate residues to diminish in time. It is therefore important to prepare isocyanate functionalized glass slides and use them immediately. On the other hand, water present in the printing solution has little effect on the immobilization efficiency of small molecules, presumably due to rapid evaporation of deposited solution droplets. Insensitivity of immobilization efficiency to water in a printing solution is a pre-requisite for fabrication of SMMs, as ambient moisture is easily absorbed into DMSO during long printing runs.In addition we find that a (PEG)6 as the spacer between the phenyl-isocyanate residues and the solid surface is optimal for fabrication of SMMs. The high immobilization percentage (over 73%) and spot morphology of SMMs on such surfaces should improve the quality and the range of SMM applications in high-throughput drug lead discovery.In conclusion, we find that in combination with post-printing thermal annealing treatment at 45 °C, a phenyl-isocyanate functionalized glass surface with (PEG)


Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery. Adaptor proteins act as molecular scaffolds in various intracellular processes . UsuallyEndocytic adaptor proteins participate in all stages of endocytosis, including internalization of cargo and its subsequent intracellular sorting between endosomal and lysosomal compartments . Some adTollip (Toll-interacting protein) is an example of an endocytic adaptor protein. This ubiquitously expressed protein of 274 amino acids localizes to endosomes via interactions with Tom1, ubiquitin and clathrin . It contIn contrast, the proposed role of Tollip in control of sumoylation and nucleocytoplasmic shuttling of proteins is likely unrelated to its endocytic function. Tollip was shown to bind the components of the sumoylation machinery and to colocalize with SUMO-1 in the nuclear PML bodies . Such a Adaptor proteins perform multiple roles in intracellular signal transduction. Among such processes, Wnt signaling involves a complex network of pathways governing cell proliferation, differentiation and migration, as well as stem cell maintenance , 22. In Here we report the results of a small-scale unbiased RNAi screen of endocytic adaptors which identified Tollip as a potential regulator of Wnt signaling. We further demonstrate that Tollip can function as an inhibitor of canonical Wnt signaling in cultured mammalian cells, both normal and cancerous, acting independently of dynamin-mediated endocytosis. We also reveal a role of Tollip in Wnt signaling in embryonic development of zebrafish: interfering with Tollip function in fish results in phenotypes reminiscent to those of canonical Wnt signaling mutants. Cumulatively, our results point to a previously unknown function of Tollip in intra- and intercellular signaling.No specific ethics approval under Polish, Swiss and EU guidelines was required for this project, as all zebrafish used in this study were between 0 and 5 days old. Fish embryos were obtained in-house, as both the International Institute of Molecular and Cell Biology and the University of Geneva have institutional licenses to house and breed fish.ntl plasmid from Dr. Carl-Philipp Heisenberg . CCND1-Luc and AXIN2-Luc luciferase reporters were provided by Dr. Frank McCormick (Addgene plasmid 32727)  or Renilla Buffer . The amount of light emitted in the reaction was determined in a microplate luminometer Centro XS3 LB960 (Berthold Technologies). The firefly luciferase activity derived from Super8xTOPFlash or Super8xFOPFlash reporter was normalized to its respective Renilla luciferase activity as a control for the transfection efficiency. Results are presented as the fold of untreated control in cells without stimulation and transfected with an empty vector or non-targeting RNAi, along with the luciferase reporter plasmids. If esiRNA was used, the pathway activity was normalized to the average of the four non-targeting controls . All values are mean ± SEM from at least three independent experiments, each with three or four independent transfections performed in parallel.In overexpression experiments HEK293 cells were transfected with 200 ng of firefly luciferase reporter , 50 ng of pRL-SV40 reporter vector and maximum 1 μg of the tested plasmid in a 24-well format . The amount of transfected DNA in all tested samples was kept constant by addition of an empty vector. In silencing experiments, RNAi at a final concentration of 33 nM were co-transfected with 50 ng of Super8xTOPFlash and 10 ng of pRL-SV40 reporter vectors in a 96-well format. In case of Wnt3a stimulation, cells were treated for the last 18 h before lysis, unless otherwise indicated. 24–72 hours post transfection cells were lysed in Passive Lysis Buffer (Promega) and either luciferase assay or Western blot analysis was performed. Luciferase reporter activity was measured in Firefly Buffer [50 mM Tris pH 7.8, 12 mM MgClesiRNAs targeting 80 selected human genes encoding endocytic proteins, esiRNAs against β-catenin, EGFP and 3 different regions of β-galactosidase were prepared as described above. HEK293 cells were transfected in a 96-well format with 33 nM esiRNA and reporter plasmids for 72 h, stimulated with Wnt3a-conditioned medium for the last 18 h prior to cell lysis and the luciferase assays were performed and analyzed as described above.HEK293 cells were treated with Wnt3a-conditioned medium or control-conditioned medium for 1.5–18 h (depending on the assay) prior to cell lysis. LiCl was used at the final concentration of 50 mM for 5 h prior to cell lysis. Stimulation with Wnt1, Dvl2 and β-catenin S33Y was obtained by 24 h co-transfection of 25 ng of the respective plasmids in a 96-well format. To inhibit dynamin-dependent endocytosis cells were either transfected for 48 h with plasmid encoding the dominant-negative dynamin2-K44A mutant or treated with 30 μM dynasore (Merck) for 30 min and then for 5 h with Wnt3a-conditioned medium and dynasore.FGF9 5’-ACCCGCAGTCACGGACTTGGA-3’ (forward), 5’-TGCTGACCAGGCCCACTGCTA-3’ (reverse), NRP1 5’-CCTGATGTTGGCCCTCACAT-3’ (forward), 5’-TGTCGGTGTAAAAAACCATGGA-3’ (reverse), NKD1 5’-AGAGCCCGGAAGGTGACA-3’ (forward), 5’- AGCGTGTCTCTCAACACGTC-3’ (reverse), DKK1 5’-CTCGGTTCTCAATTCCAACG-3’ (forward), 5’-GCACTCCTCGTCCTCTG-3’ (reverse) [ACTB 5’-CAGGTCATCACCATTGGCAAT-3’ (forward), 5’-TCTTTGCGGATGTCCACGT-3’ (reverse), B2M 5’- GGAGGCTATCCAGCGTACTC -3’ (forward), 5’- GAAACCCAGACACATAGCAATTC -3’ (reverse). The PCR reaction was performed with KAPA SYBR FAST qPCR kit (Kapa Biosystems) and 7900HT Fast Real-Time PCR thermocycler (Applied Biosystems) according to manufacturer’s recommendations. qPCR data were analyzed by the ΔΔCt method implemented in the Data Assist software (Applied Biosystems). The data were normalized according to the level of ACTB and B2M. All values are relative expression levels (fold change) compared to controls from cells incubated with control-conditioned medium and transfected with an empty plasmid . Results are presented as the mean fold change ± SEM from 3 independent experiments.Total RNA was isolated from HEK293 cells at 48 h of Tollip overexpression with the last 18 h of treatment with Wnt3a-conditioned medium, using GenElute Mammalian Total RNA Miniprep (Sigma-Aldrich). For cDNA synthesis random nonamers, oligo(dT)23 and M-MLV reverse transcriptase (Sigma-Aldrich) were used according to manufacturer’s instructions. To measure expression of Wnt target genes, the following pairs of oligonucleotides were used: reverse) , ACTB 5’Samples of 10–50 μg total protein were subjected to SDS-PAGE. Resolved proteins were transferred to nitrocellulose membrane (Whatman), which was blocked in 5% skim milk, probed with specific antibodies and detected by enhanced chemiluminescence on ImageQuant LAS 4000  or Odyssey infrared imaging system (LI-COR Biosciences).The following primary antibodies were used: anti-Tollip , anti-ubiquitin , anti-ubiquitin Lys48-specific , anti-ubiquitin Lys63-specific , anti-myc , anti-HA , anti-β-catenin , anti-active β-catenin , anti-dynamin , anti-α-tubulin , anti-β-actin , anti-GAPDH  and anti-vinculin . All primary antibodies were used at dilution of 1:1000 for immunoblotting. Secondary peroxidase-conjugated antibodies were purchased from Jackson ImmunoResearch (#115-035-062 and 111-035-144) and fluorophore-conjugated from LI-COR Biosciences (#926-680-23 and #926–32212). All secondary antibodies for immunoblotting were used at dilution of 1:10000. For immunofluorescence, Alexa-conjugated secondary antibodies  were used at dilution of 1:500.Cells were seeded on 100 mm culture plates and the next day transfected with plasmids encoding Tollip and HA-tagged ubiquitin . For immunoprecipitation of β-catenin cells were grown for 24 h and treated for the last 4 h before lysis with 5 μM MG132 (Enzo Life Sciences). For immunoprecipitation of Tollip, transfected cells were grown for 48 h and incubated for the last 18 h prior to lysis with Wnt3a-conditioned medium in the presence or absence of 5 μM MG132. After 24 or 48 h cells were washed once with PBS and lysed using RIPA buffer  containing 5 mM N-ethylmaleimide (Sigma-Aldrich) at 4°C. Lysates were clarified from cell debris by centrifugation and DNA was sheared using Qiashredder columns (Qiagen). After preclearance for 3 h with nonspecific immunoglobulins, bound proteins were depleted using Protein A or G agarose (Roche) for 1 h. Precleared cell lysates were then incubated overnight at 4°C with the indicated antibodies  coupled to Protein A or G agarose. The agarose beads were washed four times using IP buffer  before elution with 0.1 M glycine of pH 2.5. Finally samples were analyzed by SDS-PAGE and immunoblotting.4 cells/well for overexpression experiments; 104 cells/well for silencing experiments). The coverslips were incubated in 0.2% gelatin  for 30 min at 4°C prior to plating. The next day cells were transfected with DNA using Lipofectamine2000 or with siRNA using HiPerFect (Qiagen) according to the manufacturer’s instructions. After 24 h (overexpression) or after 72 h (silencing) cells were stimulated with Wnt3a-conditioned medium or control-conditioned medium for the indicated time, rinsed with PBS containing 1 mM CaCl2 and 0.5 mM MgCl2 and fixed with 3.6% paraformaldehyde (PFA) in PBS for 15 min at room temperature. Next, cells were permeabilized with 0.1% Triton X-100 and blocked using 10% fetal bovine serum in PBS for 30 min. Cells were further incubated overnight with primary antibodies against: β-catenin , Tollip  or myc tag . Next day, cells were incubated with appropriate Alexa-conjugated secondary antibodies (30 min) and DAPI in 5% fetal bovine serum in PBS. For quantitative analysis at least ten randomly selected regions were imaged for each experimental condition. Single confocal plane images were acquired with Zeiss LSM710 microscope using 40x/1.30 oil immersion objective and 1024x1024 pixel resolution in ZEN 2009 software. Images were exported as TIFF and for visual presentation arranged in Adobe Photoshop CS4 Extended software with only linear adjustments. For quantification of β-catenin in the nucleus, 8-bit images were analyzed in ImageJ. Nuclei were identified as regions of interest based on DAPI staining. Fluorescence of β-catenin in the nuclei was measured and normalized to the nucleus area. Average β-catenin intensity in control cells plus standard deviation served as a cut-off for identification of β-catenin-positive nuclei. On average, over 200 nuclei per condition were analyzed in each experiment. The data are average percentage of β-catenin-positive nuclei or mean fluorescent intensity of nuclear β-catenin from 3 independent experiments ± SEM.HEK293 cells were seeded on 12-mm gelatin-coated coverslips in a 24-well plate  diluted 1:250 overnight. Primary antibody was detected using Peroxidase Vectastain ABC Kit (Vector), according to the manufacturer’s recommendations. Secondary antibodies were detected by incubation in DAB  solution for 15 min and next in DAB solution plus 0.01% H2O2. The embryos were washed to stop the reaction and mounted in glycerol.Zebrafish embryos collected at different stages post fertilization were manually dechorionated, fixed with 4% paraformaldehyde and permeabilized in a graded series of methanol dilutions: 30%, 60%, 100% for 5 min each at RT and in 100% methanol for at least 1 h at -20°C. One dpf embryos were additionally treated with proteinase K at the concentration of 10 μg/ml for 5 min. Activity of endogenous peroxidases was inhibited by treatment with 0.3% Hztollip mRNA were obtained from Gene Tools: ATG1 5’-GTGCTTATTGTTGTCGCCATTCTGC-3’, UTR1 5’-TTATGATATGACGGACAGCGCCTGT-3’ , ATG2 5’- CTGCTGCTGAGTCGGCATGATCCTC -3’ . Dose-response experiments were carried out for all MOs to determine the highest nontoxic dose for use in further experiments. Up to 4 ng MO was microinjected into the yolk of 1-cell stage embryos . In double knock-down ISH experiments half of the MO amount was used. To determine the effects of gene silencing, a combination of the ATG2 and the UTR1 MO was used. MO-injected embryos were then manually dechorionated and either their phenotype was analyzed or they were subjected to ISH. To knock down Rab5 function, 1-cell stage embryos were injected with 4 ng of morpholino oligonucleotide directed against the coding region of the zebrafish rab5a (5'-GACAGTTGTCAATCACCCCGTCTTC-3'). The β-catenin2 (ctnnb2) morpholino was previously described: 5’-CCTTTAGCCTGAGCGACTTCCAAAC-3’ [Translation-blocking morpholino (MO) oligonucleotides complementary to 5’UTR and ATG regions of CAAAC-3’ .In situ hybridization was carried out according to standard procedures [cmlc2, fgf8, goosecoid and ntl RNA synthesis the appropriate plasmids were linearized and in vitro transcribed. Zebrafish embryos were fixed with 4% paraformaldehyde. One dpf embryos were additionally treated with proteinase K at the concentration of 10 μg/ml for 20 min. Prehybridization was performed at 65°C for 1 h. Embryos were incubated with digoxigenin-labeled RNA probes at 65°C overnight and blocked in 2% Blocking Reagent (Roche) for 5 h. Incubation with anti-digoxigenin alkaline phosphatase-conjugated antibodies (Roche #11093274910) diluted 1:5000 was performed at 4°C overnight. Antibodies were detected by incubation in the alkaline phosphatase substrate BM Purple (Roche). Embryos were washed to stop the reaction and mounted in glycerol.ocedures . For antP≤0.05.Statistical analyses were carried out using the software STATISTICA 8.0 (StatSoft). Comparisons among groups were made by nonparametric Mann-Whitney U test or Wilcoxon signed-rank test. Data are expressed as mean ± SEM from 3–4 independent experiments. Differences were considered statistically significant for We set out to screen a subset of soluble endocytic proteins for a role in the regulation of canonical Wnt signaling, related to or independent of their function in endocytosis. The setup of a small-scale RNAi screen involved targeting 80 genes encoding adaptor and accessory proteins regulating endocytic internalization and cargo sorting. We employed a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) to downregulate expression of the selected genes in HEK293 cells stimulated with Wnt3a. As readout, we used the Super8xTOPFlash luciferase reporter measuring the activity of TCF/LEF transcription factors . The scrAmong the potential inhibitors of the pathway (i.e. increasing the reporter activity upon knockdown), Tollip knockdown consistently increased the reporter activity with three independent RNAi reagents  [FGF9 and NRP1 [NKD1 or DKK1 [Overexpression of Tollip decreased the activity of promoters of two Wnt target genes: AXIN2  and CCNDclin D1) , 42  might be important for Wnt signaling. Of the three domains of Tollip, the N-terminus is responsible for association with TOM1 protein, the middle C2 domain binds phosphoinositides, while the C-terminal CUE domain interacts with ubiquitin , 11.2) is produced at the plasma membrane upon Wnt3a stimulation to drive formation of LRP6 signalosomes [Deletion mutagenesis was performed and the resulting five mutants  were tesalosomes –54, whilalosomes .2 [We confirmed these results with a panel of previously characterized point mutations of Tollip . A doubl2 , 10. Whe2 , and/or 2 may be of some importance for the function of Tollip in Wnt signaling, consistent with the role of this lipid in Wnt signaling [FGF9 and NRP1 was tested, the ubiquitin-binding M240A/F241A mutant failed to inhibit expression of the two targets, in contrast to the wild-type and the K150E mutant  were unable to inhibit the reporter activity, although the levels of R78A overexpression were the lowest . The othignaling –54. Howeignaling , this woE mutant . FinallyE mutant . These dWe wished to identify components of the Wnt transduction pathway affected by Tollip. First, we assessed the impact of Tollip on the level of β-catenin, both total and its active (non-phosphorylated on Ser37/Thr41) form. In Wnt3a-treated cells, overexpression of Tollip reduced the pool of active β-catenin without altering its total levels   and treaLigand-independent stabilization of β-catenin leads to permanent activation of the Wnt pathway and is a common pathological mechanism of numerous diseases, including cancer , 24. To This conclusion was further supported by testing colorectal cancer cell lines in which canonical Wnt signaling is constitutively active due to mutations either in β-catenin (HCT116 line) or in APC (DLD1 line) , 61. IndOverall, our results suggest that an inhibitory action of Tollip in Wnt signaling can be exerted at two steps of the pathway. In ligand-stimulated cells, Tollip lowers the pool of active (non-phosphorylated) β-catenin without altering its total levels. Interestingly, in macrophages Tollip was reported to participate in the activation of GSK3β . GSK3β iIn general, our data support a widespread role of Tollip-mediated regulation of Wnt signaling, both ligand-induced and ligand-independent, in different cell types, including cancer cells. Consistently, decreased expression of Tollip was reported during oncogenic progression from normal colon mucosa to adenomas and carcinomas . ActivatIn the adult, Wnt signaling drives gene expression to maintain stem cell homeostasis and when aberrant leads to carcinogenesis. However, the Wnt pathway controls also body patterning during early vertebrate development . To addrDanio rerio harbors a Tollip ortholog (GenBank accession number NM_207061.1) which encodes a well-conserved protein of 276 amino acids sharing the same domain structure [The genome of tructure  and 82% ztollip mRNA in wild-type embryos. Upon injection of 200 pg of ztollip mRNA into 1-cell stage embryos , embryos displayed a reduction of the posterior body, a strong reduction (even lack) of somites, and partial fusion of the eyes  appeared slightly enhanced compared to control embryos (notail (ntl)  . Howevern absent . These pnjection . Indeed,pression  and in d embryos , indicatactivity , as obseactivity . These dztollip by using various non-overlapping, independent morpholinos (MOs). We capitalized on the existence of two zebrafish mRNA variants, which differ by their 5’ UTR and ATG regions . To block both mRNA variants, a combination of two morpholinos targeting each of them was used. Activation of Wnt signaling leads to pericardial oedema, smaller eyes, body curvature and failure of the heart to loop [ztollip resulted in twisted tail, pericardial oedema and smaller eyes  served as a positive control. esiRNAs targeting EGFP or 3 different regions of β-galactosidase served as negative controls. The pathway activity is presented relatively to the average of four non-targeting controls, which is normalized to one arbitrary unit. Average of non-targeting controls in cells without Wnt3a treatment (Ctrl NS) is shown to demonstrate the extent of stimulation. Data represent average of 3 independent experiments, error bars are SEM. Arrow indicates the results for Tollip knockdown.HEK293 cells were transfected for 72 h with 33 nM esiRNA targeting 80 indicated genes encoding soluble proteins regulating endocytosis. Cells were stimulated with Wnt3a-conditioned medium for 18 h before lysis and Super8xTOPFlash luciferase reporter assays were performed. Depletion of an established pathway activator β-catenin ((TIF)Click here for additional data file.S2 FigLysates from HEK293 cells transfected either with an empty plasmid (pcDNA) or HA-tagged Dnm2 K44A-expressing construct were blotted against Tollip and HA tag, with actin as a loading control. (B) Immunoprecipitation of overexpressed Tollip from lysates of HEK293 cells upon stimulation with Wnt3a or with control medium (CM) and proteasome inhibition by MG132. Western blots were probed for total ubiquitin and K48 or K63 ubiquitin subpopulations. Left panel; immunoprecipitates were probed with antibodies against Tollip, total ubiquitin (Ub), its K48-linked (Ub-K48) or K63-linked (Ub-K63) chains. Right panel; 10% of starting lysates taken for immunoprecipitation (input) were blotted against total ubiquitin (Ub) and Tollip, with GAPDH as a loading control.(TIF)Click here for additional data file.S3 FigHEK293 cells transfected for 24 h with myc-tagged Tollip: wild-type (wt), del.1 deletion mutant or M240A/F241A point mutant were stimulated with control medium  or Wnt3a-conditioned medium (B) for 8 h. Cells were immunostained for β-catenin and myc tag (overexpressed Tollip), with DAPI indicating cell nuclei. Scale bar 20 μm.(TIF)Click here for additional data file.S4 Figβ-catenin2 morpholino (MO) at different concentrations . Phenotypes of injected embryos at 48 hpf are shown, compared to an uninjected wild-type embryo. Lateral views with head to the left.Zebrafish embryos were injected at the 1-cell stage with (TIF)Click here for additional data file.


Elevated intraocular pressure (IOP), as well as fluctuations in IOP, is a main risk factor for glaucoma, but its pathogenic effect has not yet been clarified. Beyond the multifactorial pathology of the disease, autoimmune mechanisms seem to be linked to retinal ganglion cell (RGC) death. This study aimed to identify if intermittent IOP elevations in vivo (i) elicit neurodegeneration, (ii) provokes an immune response and (iii) whether progression of RGC loss can be attenuated by the B lymphocyte inhibitor Belimumab.n = 21) underwent 27 unilateral simulations of a fluctuating pressure profile. Nine of these animals received Belimumab, and additional seven rats served as normotensive controls. Axonal density was analyzed in PPD-stained optic nerve cross-sections. Retinal cross-sections were immunostained against Brn3a, Iba1, and IgG autoantibody depositions. Serum IgG concentration and IgG reactivities were determined using ELISA and protein microarrays. Data was analyzed using ANOVA and Tukey HSD test  or student’s independent t test by groups.Using an intermittent ocular hypertension model (iOHT), Long Evans rats (p < 0.001) and RGC  in iOHT eyes compared with fellow eyes. Belimumab-treated animals only showed slightly higher axonal survival and reduced serum IgG concentration (−29 %) after iOHT. Neuroinflammatory events, indicated by significantly upregulated microglia activation and IgG autoantibody depositions, were shown in all injured retinas. Significantly elevated serum autoantibody immunoreactivities against glutathione-S-transferase, spectrin, and transferrin were observed after iOHT and were negatively correlated to the axon density.A wavelike IOP profile led to a significant neurodegeneration of optic nerve axons  contains supplementary material, which is available to authorized users. Glaucoma is one of the leading causes of blindness worldwide, and the elevation of the intraocular pressure (IOP) is considered a major risk factor –3. HowevA minimally invasive animal model was recently established to induce intermittent ocular hypertension (iOHT) , 20. ThiIf it emerges that changes of the humoral immune response and a loss of RGC can be initiated by a wavelike profile of intermittent ocular hypertensions in vivo, the question of whether or not the subsequent humoral immune response affects the progression of RGC loss can be addressed. Moreover, a pharmaceutical modulation of the B lymphocyte population could have an influence on RGC survival. To test this, Belimumab, an inhibitor of the B lymphocytes activating component of the tumor necrosis factor family (BAFF), was applied to one experimental group before and during IOP elevations to determine the role of the humoral immune system in glaucomatous neurodegeneration.n = 7), iOHT (n = 12), and iOHT + Belimumab (n = 9).Animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and all experiments have been approved by the national investigation office in Koblenz, Germany (23 177-07/G 11-1-029). Eight-week-old male Long Evans rats were obtained from Charles River  and housed in climate-controlled rooms under 12-h light-dark cycles and received food and water ad libitum. Twenty-eight animals were assigned to three groups: CTRL . Rats were kept in a horizontal orientation with the head in a relaxed position. No pressure was applied to the head during measurement. The animals became habituated to the IOP measurements quickly. All measurements were performed by the same examiner, and a mean value was calculated from ten consecutive measurements. IOP values that were identified as inaccurate by the TonoLab device were not included in the analysis. The IOP measurements in anesthetized rats were performed immediately before and after onset or adjustment of the loop as well as every five minutes throughout the manipulation. The ΔIOP was defined as the integrated IOP to which the eyes were exposed during the intermittent periods of iOHT. It was calculated from the ∑ of areas under the curve after subtraction of IOP values of the fellow eye.In accordance with the previously described technique for minimally invasive short-term IOP elevations , 20, iOH2 inhalation followed by cardiac blood withdrawal and cardiac perfusion with 4 % paraformaldehyde/heparin  solution . Eyes were marked in the nasotemporal orientation, dissected, immersion-fixed in 4 % PFA, and embedded in paraffin. Optic nerves with the optic chiasm attached were harvested, preserved in 3 % glutaraldehyde/0.1 mol/l sodium cacodylate buffer  and processed for epoxy resin embedding.After the final in vivo experiments, all animals were euthanized by CO2.0.7 μm semithin transverse optic nerve sections, taken 2 mm distal to the optic chiasm, were stained with p-phenylenediamine (PPD), and quantification of the axon density was performed as described previously , 23. Bri2O2 solution for 30 min. RGC labeling, microglia, and immunoglobulin G (IgG) autoantibody detection were conducted in accordance with routine procedures [For each staining procedure, five 10 μm sagittal sections per eye were dewaxed, rehydrated, and blocked for endogenous peroxidase in 0.5 % Hocedures , 25. Braocedures , 27. SliFor immunolabeling of ionized calcium binding adaptor molecule 1 (Iba1), slides were blocked with 0.25 % NGS/0.5 % BSA/0.1 % Triton X-100/PBS after TRS antigen retrieval for 30 min at 90 °C. Rabbit anti-Iba1  primary antibody was diluted in 0.1 % Triton X-100/PBS. After overnight incubation at 4 °C, sections were washed in PBS followed by goat anti-rabbit IgG (H + L) chain specific HRP-conjugated  secondary antibody incubation.For IgG autoantibody staining, slides were boiled in TRS solution for 45 min at 90 °C and blocked in 1 % BSA/0.1 % Triton X-100/PBS solution for 10 min. A goat anti-rat IgG HRP-conjugated antibody  was diluted 1:500 in blocking buffer, and sections were incubated overnight.After several wash steps in PBS, 3,3′-diamicobenzidin  incubation for 10 min followed for color development. All sections were counterstained with hematoxylin, dehydrated, and coverslipped. For image acquisition, an Eclipse TS 100 microscope  with a DS-Fi1-U2 digital microscope camera (Nikon) and an ELWD 20x/0.45 S Plan Flour Ph1 ADM objective (Nikon) was used and recorded by the imaging software NIS Elements . Images were transferred to CorelDRAW X4 Graphic Suites .Data for each parameter was obtained from four representative cross-sections for each eye, containing the optic nerve head to ensure corresponding retinal position. The number of cells is calculated per 1-mm retinal length.Blood samples from the cardiac blood withdrawal were allowed to clot at room temperature and were centrifuged at 10 °C for 10 min and 4000 rpm. Individual serum IgG concentrations of all animals were determined using a rat IgG ELISA kit  according to the manufacturer’s protocol. Remaining serum samples were stored at −20 °C for further analysis of the antibody pattern.Using a non-contact array spotter , ten antigens  were analyzed with Imagene 5.5 . Technically faulty spots were identified by visual inspection and removed from the data set. For each spot, the local background intensity was subtracted from the median spot intensity. Intensity of the three technical replicates for each antibody was averaged and used for statistical analysis.t test. Data mining of microarray experiments was performed using one-way ANOVA and post hoc testing [P values <0.05 were considered significant. Data are given as mean ± standard deviation (SD) or as group median. All statistical analyses were performed by Statistica Version 12 .For multiple test schemes, Tukey’s HSD post hoc  tests were used. Comparison between two groups was performed using student’s independent  testing . The corp = 0.26).The IOP values of awake animals stayed within the normal physiological range of 10 and 13.3 mmHg and displayed no significant changes throughout the study  in iOHT injured eyes and −1.5 % in iOHT fellow eyes compared with control eyes. Belimumab-treated animals showed axonal loss of −1.9 % in iOHT + Belimumab fellow eyes and −7.5 % in iOHT + Belimumab injured eyes compared with control eyes. Injured eyes of the iOHT + Belimumab group revealed a slightly higher axonal density (+4.9 %) compared with iOHT group, even though this was not statistically significant , or to RGC numbers of control animals .Examination of the RGC density recapitulated the results of the optic nerve axon analysis. Eyes of control animals Fig.  and thosp < 0.03) and virtually identical to numbers found in animals which received iOHT + Belimumab treatment . Conversely, a significant reduction of microglia in the inner nuclear layer to 20 and 22 % in Belimumab-treated eyes occurred compared with iOHT fellow eyes (p < 0.01) or to iOHT injured eyes (p < 0.05). Apparently, a redistribution of microglia from the inner nuclear layer to the ganglion cell layer occurred in the eyes of Belimumab-treated animals  as well as in the inner nuclear layer 35 %). Despite an increase of the total number of microglia in the iOHT group, their distribution was as observed in control eyes. In the iOHT + Belimumab group, a larger fraction of microglia was found in the ganglion cell layer of the injured eye (60 %), as well as the uninjured control eyes  as well as in fellow eyes . Animals that received Belimumab showed higher, though not statistically significant, levels of IgG autoantibody depositions in iOHT eyes  as well as in their fellow eyes . The administration of Belimumab significantly reduced the IgG titer to 1.80 ± 0.24 ng/ml in comparison to control animals (p = 0.02) and to iOHT-treated animals (p = 0.003).The serum IgG titer of each group was quantified by ELISA. Control rats at the age of 14 weeks had a mean IgG titer of 2.55 ± 0.66 ng/ml. iOHT animals showed a similar level of IgG at 2.74 ± 0.38 ng/ml . Treatment of animals with the B lymphocyte inhibitor Belimumab led to significantly decreased immunoreactivity compared with iOHT animals , spectrin , and transferrin . All of these changes were statistically significant when compared between iOHT and controls , spectrin , and transferrin iOHT elicits neurodegeneration in the optic nerve and the retina, although the fluctuating IOP pattern does not have more destructive potential than constantly elevated IOP.(ii)Retinas of contralateral, uninjured eyes display pathological changes in comparison to untreated control eyes.(iii)Retinal microglia in iOHT eyes are enriched in the retinal ganglion cell layer and activated.(iv)iOHT eyes accumulate IgG autoantibody depositions in the retina.(v)Serum autoantibody reactivities against glutathione-S-transferase, spectrin, and transferrin significantly increased and might serve as biomarkers.(vi)Repeated injections of the B lymphocyte inhibitor Belimumab have very little effect on neurodegeneration, deactivation of microglia, and reduction of IgG autoantibody depositions despite lowering the total IgG serum concentration.This study allows the following six conclusions regarding retinal injury and the involvement of immune responses in this rat model of intermittent ocular hypertension:+ cells by −19.5 %. Interestingly, these findings are comparable to those from a previous study with similar parameters , but a static IOP elevation [The main risk factor for glaucoma is elevated IOP –3. In thlevation . Even thlevation . In thei+/MHC-II+ cells and rare Iba1+/CD68+ cells in the contralateral eye, suggesting an activation of the “M1” microglial phenotype. However, a decline in RGC density in the contralateral eye could not be determined in this study [Activation of retinal microglia is a characteristic finding in IOP-associated animal models of glaucoma –34. Inteis study . The theis study . All theIgG autoantibodies occur naturally in the healthy retina, but an increased number of retinal IgG autoantibody depositions have been observed in human glaucomatous retina , animal The overall aim of this study was the question whether or not a systemic immune response is initiated by RGC loss due to increased IOP and, more precisely, if the humoral immune response is correlated with optic nerve axon loss. Manipulation of the IOP elicits a significant increase of immunoreactivities against glutathione S-transferase, spectrin , and transferrin compared with control animals. Likewise, an increased immunoreactivity of these autoantibodies, except transferrin, has been described for patients suffering from glaucoma , 49, indGlutathione S-transferase (GST) belongs to a family of multifunctional enzymes that act alone or along with enzymes within most mammalian organs and their cells to reduce superoxide radicals, hydroxyl radicals, and peroxynitrites . The occSpectrin is a member of the F-actin-crosslinking protein superfamily and is equivalent to alpha-fodrin . This la3+) molecules into the cell via receptor-mediated endocytosis in a pH-dependent manner [Transferrin is a serum iron-binding protein that is produced in the liver. It interacts with the transferrin receptor located on the cellular surface for delivery of redox active iron , and transferrin. Immunomodulation using the BAFF-blocker Belimumab to hinder B lymphocyte activation led to a significant decrease of serum IgG concentration, a lower glutathione S-transferase-, spectrin-, and transferrin immunoreactivities but shows only limited ability to ameliorate the progression of axonal and RGC loss. This result limits the detrimental role of autoantibodies in inducing glaucomatous RGC loss but underlines the importance of changes of autoantibody reactivities as a valuable biomarker. The present study gives insight into the close relationship between IOP, the immune response, and the pathology of RGC loss. Nevertheless, more research is needed in the future to understand the origin of an altered humoral immune response with focus on cellular interactions in glaucoma and in other neurodegenerative diseases.Animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and all experiments have been approved by the national investigation office in Koblenz, Germany (23 177-07/G 11-1-029).