EC = 34,000 mJ/cm2) and sufficient expansion ratio (~148%).Epoxy foam adhesives are widely used for weight reduction, watertight property, and mechanical reinforcement effects. However, epoxy foam adhesives have poor impact resistance at higher expansion ratios. Hence, we prepared an epoxy composite foam adhesive with core–shell rubber (CSR) particles to improve the impact resistance and applied it to automotive structural adhesives. The curing behavior and pore structure were characterized by differential scanning calorimetry (DSC) and X-ray computed tomography (CT), respectively, and impact wedge–peel tests were conducted to quantitatively evaluate the resistance to cleavage of the CSR/epoxy composite foam adhesives under impact. At 5 and 10 phr CSR contents, the pore size and expansion ratio increased sufficiently due to the decrease in curing rate. However, at 20 phr CSR content, the pore size decreased, which might be due to the steric hindrance effect of the CSR particles. Notably, at 0 and 0.1 phr foaming agent contents, the resistance to cleavage of the adhesives under the impact wedge–peel condition significantly improved with increasing CSR content. Thus, the CSR/epoxy composite foam adhesive containing 0.1 phr foaming agent and 20 phr CSR particles showed high impact resistance ( An epoxy foam adhesive is an epoxy containing a foaming agent that generates a gas inside the epoxy resin or expands upon heat treatment. After heat treatment, the epoxy foam adhesive is cured and foamed, simultaneously filling the gap between two substrates and binding them ,2. ThrouAs the mechanical strength of an epoxy foam adhesive weakens through an increase in the expansion ratio , it is nRubber particles have been widely used to enhance the impact resistance of epoxy composites ,6,7,8. HThe pore structure of epoxy composite foams is typically characterized by two-dimensional analysis, such as scanning electron microscopy (SEM) ,13,14,15Further, the impact resistance of an epoxy composite containing CSR particles has been conventionally evaluated by a ballistic impact test , izod imIn this study, epoxy composite foam adhesives containing epoxy resin, a foaming agent, and CSR particles were prepared, and their pore structure was characterized by X-ray CT. We used an impact wedge–peel tester and their resistance to cleavage of the epoxy composite foam adhesive under impact condition. We investigated the effect of CSR particles on the pore structure and impact resistance of the epoxy composite foam adhesive containing different amounts of foaming agent and suggested an optimal content of CSR particles to achieve a high expansion ratio and impact resistance.3, 10CN, OMYA, Oftringen, Switzerland) and expandable microcell  were used as filler and a foaming agent, respectively. Detailed information of the materials employed in this work is presented in Two epoxy resins were used to prepare epoxy composite foams: Bisphenol-A diglycidyl epoxy resin  and urethane-modified epoxy resin . A dicyandiamide curing agent  and a latent accelerator  were blended with the epoxy resins for curing. The CSR particles were composed of butadiene rubber cores and poly(methyl methacrylate) shells. We used CSR particles (35 wt %) predispersed in bisphenol-A diglycidyl epoxy resin . Calcium carbonate  was performed to compare the curing behaviors of the epoxy composite foams. The heat flow of exothermic curing reaction was measured during the DSC run in the temperature range of 60–240 °C at a constant heating rate of 5 °C/min.Vpore and Vtotal represent the total volume of pores and the measured region, respectively. The pore structure was characterized by X-ray CT . The X-ray head (50 kV) was rotated around the epoxy composite foam and tomographic images were captured every 0.6°. These tomographic images were collected and converted into 3D images. The average pore size, standard deviation of pore size, and porosity were evaluated by the software , and the expansion ratio was calculated by Equation (1).2, thickness: 0.2 mm), and the force was measured when the adhesive layer was cleaved by the wedge at a velocity (v) of 2.0 m/s.An impact wedge–peel test was performed according to ISO 11343 standard to compare the resistance to cleavage of the CSR/epoxy composite foam adhesives under impact using a drop weight tester . Specimens for the impact wedge–peel test were prepared as shown in EC), which quantitatively represents the impact resistance. Displacement for cleavage (DC) is determined by the displacement at the finish point of the cleavage.As shown in Tmax (heat flow)) was plotted as a function of CSR content. Notably, as the CSR content increased, Tmax (heat flow) got higher. This indicates that the addition of CSR particles retarded the curing reaction of epoxy due to the steric hindrance effect of the CSR particles in the CSR/epoxy composite.The curing behavior of the CSR/epoxy composite was studied by DSC . As the The pore structures of the CSR/epoxy composite foam adhesives were analyzed by X-ray CT. As shown in Under the impact wedge–peel condition, the impact force was measured for 20 ms . With anDC). It indicates that the impact resistance deteriorated with increasing foaming agent content. As the CSR content increased, the impact resistance of the CSR/epoxy composite foam adhesive improved dramatically, resulting in an increase in DC and changing the type of crack growth from unstable to stable crack growth.As shown in EC), we quantitatively evaluated the resistance to cleavage of the CSR/epoxy composite foam adhesives under the impact wedge–peel condition. As shown in EC was significantly enhanced by the addition of CSR particles, indicating an improvement in impact resistance. However, as the foaming agent content increased to 1 phr, the EC hardly increased, which suggests that the impact resistance effectively improved at low foaming agent contents (0 and 0.1 phr).Additionally, by comparing the energy for crack growth  was more than two times that of the adhesive containing no CSR particle . In addition, the type of crack growth changed from unstable to stable crack propagation by the addition of 20 phr CSR particles into the sample containing 0.1 phr foaming agent. Moreover, the expansion ratio of the adhesive containing 0.1 phr foaming agent and 20 phr CSR particles increased, compared to the adhesive containing no CSR particle; this indicated that a simultaneous increase in both the expansion ratio (~148%) and impact resistance was achieved.Notably, for the CSR/epoxy composite foam adhesive containing 0.1 phr foaming agent and 20 phr CSR particles, the EC. It was significantly enhanced by the addition of CSR particles at 0 and 0.1 phr foaming agent. However, at 1 phr foaming agent, the EC was hardly improved by the addition of CSR particle, indicating that the improvement in impact resistance is effective only at low foaming agent contents (0 and 0.1 phr). For the CSR/epoxy composite foam adhesives containing 0.1 phr foaming agent and 20 phr CSR particles, a simultaneous increase in both the expansion ratio (~148%) and impact resistance (EC = 34000 mJ/cm2) was achieved. A limitation of this study is that we only focused on the impact resistance of the CSR–epoxy composite foam adhesives at room temperature. Since CSR particles can improve impact resistance at low temperatures, it is necessary to investigate the impact resistance of CSR/epoxy composite foam adhesives at low temperatures in future research.CSR/epoxy composite foam adhesives were prepared with different amounts of foaming agent and CSR particles. With increasing CSR content, the curing reaction retarded, which affected the growth of the pores. The pore structure, pore size, porosity, and expansion ratio were determined by X-ray CT. The expansion ratio for 1 phr foaming agent was higher than that for 0.1 phr foaming agent. At 5 and 10 phr CSR content, the pore size and expansion ratio increased by decrease in curing rate, but, at 20 phr CSR content, the pore size and expansion ratio decreased due to the steric hindrance effect of the CSR particles. The impact resistance of the CSR/epoxy composite foam adhesive was compared in terms of


Direct adsorption of multi-wall carbon nanotubes (MWCNTs) and subsequent covering with Nafion® layer was used for immobilization of Gox. The analytical figures of merit of the developed glucose (Glc) biosensor are sufficient for determination of Glc in body fluids in clinical analysis. From all tested heavy metals, mercury(II) has the highest inhibition effect. However, it is necessary to remember that cadmium and silver ions also significantly inhibit the catalytic activity of Gox. Therefore, the development of Gox biosensors for selective indirect determination of each heavy metal still represents a challenge in the field of bioelectroanalysis. It can be concluded that amperometric biosensors, differing in the utilized enzyme, could find their application in the toxicity studies of various poisons.The inhibition effect of the selected heavy metals (Ag Enzymes are organic catalysts produced within the living organisms. They speed up the biological reactions by lowering the activation energy. They can speed up the conversion of the substrate to the products in cellular metabolism up to 10 million times or more . To init2 was used as the mediator. Furthermore, the enzyme was covered by Nafion® membrane in order to increase the stability of the sensor. The developed sensor was applied for the concentration determination of hydrogen peroxide and Glc. Moreover, the possibility of using the developed sensor as an electroanalytical approach to study the heavy metal toxicity (inhibition of enzymes activity) was investigated. The effect of the heavy metals cations  on the Gox enzyme activity was investigated. It must be mentioned that screening of enzyme activity of other enzymes is possible using the same principle as well.The reaction monitoring brings about the indispensable information about the molecular speciation  and prov2O2 amperometric detection was studied using amperometry in a batch configuration. Nearly three times higher sensitivity was obtained in comparison with bare GCE at +0.8 V. Nafion® is better than chitosan for the covering of the electrode as it did not cause a significant decrease in the current compared to the GCE/MWCNTs. Moreover, the obtained amperograms were well-shaped (less noisy) when the electrode was covered with Nafion®  occurred at high potential values, around +0.8 V  ≤ 7.1 mM) the proposed GOx biosensor could be utilized in the clinical analysis [2O2 and Glc are presented in At first sight, it is evident that a noticeable increase in the sensitivity has been achieved using carbon nanotubes. A linear dependencies of current response  were nearly identical, but Vmax  was decreased in presence of the Hg2+, the inhibition effect of heavy metals is supposed to be noncompetitive [The nature of the inhibition of the enzyme activity by the heavy metals was also investigated. Two calibration curves of the Glc at the developed sensor were plotted in the presence of a given concentration of Hgodel see . Since tpetitive .2/GOx/Nafion® for 550 μM Glc (the first addition) with subsequent addition of 5 µM mercury(II) cation. Corresponding calibration curve for concertation range from 5 to 80 μM of Hg2+ is also presented  were selected to study their inhibition effect on GOx enzyme obtained from Aspergillus niger (EC 1.1.3.4). In principle, it is possible to compare the effect of heavy metals on the basis of a decrease in current response for a certain Glc concentration. In general, it can be assumed that the inhibitory effect of heavy metals could be studied for other enzymes using similar protocol. It should be noted that the enzyme-based biosensor is used to study the inhibition of enzyme activity by heavy metals. Therefore, some studies have demonstrated that GOx biosensors can be completely regenerated by the addition of ethylenediaminetetraacetic acid (EDTA) [Several heavy metals cations (Agd (EDTA) ,46.Despite the insufficient reproducibility (7.4% RSD) of five freshly prepared GOx biosensors, the study of heavy metals effect can be realized because the ratios between the current response of substrate and the decrease in current caused by the presence of the heavy metal are compared. A comparison of the obtained results is presented in 2+, Cd2+, and Ag+. From all tested heavy metals, Cu2+ does not have any significant inhibitory effect on GOx catalytic activity. The inhibitory effect of Ag+ was two time lower than that of the Hg2+. In fact, Ag+ inhibits the activity of enzymes that are involved in bacterial cells division and thus significantly slow their proliferation [feration .2+, the shapes of the amperograms are the same for all the studied heavy metal ions. A slower inhibition of GOx catalytic activity can be attributed to Cd2+ compared to Hg2+ and Ag+ due to its longest response time. For the initial concentration of 200 µM of Glc, an evident decrease of current response was only obtained for the first addition of 50 µM Cd2+. The subsequent additions did not cause any reduction in current magnitude. Thus, if cadmium(II) is indirectly determined using GOx biosensors, a very short linear range is expected in comparison with other heavy metals. An amperometric GOx biosensor modified with cobalt or copper hexacyanoferrate was also developed for monitoring heavy metals and similar behavior of cadmium(II) was also observed. The linear range for cadmium(II) determination with both types of GOx biosensors was 1.5–6.0 µM in the presence of 300 µM Glc [Except of the Cd0 µM Glc .Due to the high GOx enzyme activity inhibition by heavy metals, the development of GOx biosensors for selective indirect determination of a given heavy metal remains still a challenge in the field of bioelectroanalysis, especially in environmental analysis. It seems that amperometric biosensors could be used in the toxicity study of other poisons.® 117 membrane have been already studied by Malaysia scientists [® (anions) and heavy metals (cations). An accumulation of heavy metals on the membrane layer could increase the exposition of GOx enzyme to a higher concentration of heavy metals than what is actually added to the solution. These authors also tested Cu2+ which showed the highest affinity in the comparison with Co2+, Ni2+, Pb2+, and Ag+ [2+ cations did not cause any significant decreases in current response.Herein, it is necessary to mention that an adsorption of heavy metals cations from aqueous solutions on Nafionientists . They fo and Ag+ . Moreove® on the function of the developed sensor. Two biosensors (CPE/RuO2/GOx and CPE/RuO2/GOx covared by thin layer of Nafion®) were compared in measurements of glucose calibration when Hg2+ was presented in both cases , β-d-glucose, ruthenium oxide used as mediator, hydrogen peroxide (H2O2), multi-walled carbon nanotubes (MWCNTs), Nafion®, acetic acid (AA), chitosan (~50 kDa) and N,N-dimethylformamide (DMF), ethanol (99.98%), nitric acid (70%), ammonia solution (25%), and paraffin oil were purchased from Sigma-Aldrich . Inorganic salts of p.a. grade 2·H2O, and Cd(NO3)2·4H2O) from Lach-Ner, s.r.o.  were dissolved in deionized water for preparation of 0.01 M heavy metals stock solutions. This water (18.2 MΩ·cm) had been first double distilled by an Aqua Osmotic 02 from Aqua Osmotic  and then deionized using a Millipore RG . PB was prepared by mixing 0.2 M NaH2PO4 and 0.2 M Na2HPO4  in 1:1 volume ratio. The required pH value then was achieved by the appropriate addition of 1 M NaOH. The expanded graphite was purchased from Graphite Týn Ltd. .Glucose oxidase (GOx) obtained from 2/GOx/Nafion® was carried out at dimension FastScan Bio from Bruker  operating with Gwydion 2.52 for data visualization [The imaging of the developed GCE/MWCNTs amperometric transducer surface was carried out by the scanning electron microscope, Vega3 SB from TESCAN Brno, s.r.o. . Furthermore, energy dispersive spectroscopy (EDS) was utilized for elemental mapping of the transducer surface. The imaging was carried out by applying a potential of 15 kV at 18.4 mm working distance. Atomic force microscopy (AFM) for the electrode surface characterization of the developed GCE/MWCNTs-RuOlization . An AutoThe GCE; No. 6.1204.110 with a diameter 3 mm was purchased from Metrohm . It was polished on a polishing pad using alumina powder (0.3 and 0.05 μm) for 1 min. followed by sonication in deionized water for 1 min and in ethanol for another 1 min. Then, it was subjected to the various modifications to prepare different electrodes, as described below.2 was dispersed in 1.000 mL of DMF and put in ultrasonic bath  for 1 h with applied frequency 37 kHz, 10 µL of the dispersion was then dropped onto GCE surface and it was left to be dried at room. The content of 5% RuO2 (w/w) was incorporated into MWCNT due to better amperometric detection of hydrogen peroxide produced by biocatalytic oxidation [To improve the sensitivity of GOx biosensor, it was necessary to increase the active surface area of amperometric transducer. Therefore, the GCE surface was covered by a thin layer of MWCNTs (GCE/MWCNTs). In this case, a mixture of 2.0 mg of MWCNT and 0.1 mg RuOxidation .®) were tested for GOx immobilization. First GOx enzyme had to be directly adsorbed onto GCE/MWCNTs. A desired volume of the GOx solution (2.0 mg·mL−1 water solution) was dropped onto the prepared GCE/MWCNTs. When dried the electrode was further covered by the desired polymer.Two different polymers  was injected onto electrode surface and left to be air-dried under laboratory conditions. If no inhibition of heavy metals was measured, GOx biosensor (GCE/MWCNTs-RuO2/GOx/Nafion®) was stored in a refrigerator at 4 °C.The chitosan was prepared as 1% chitosan solution dispersed in 1% acetic acid containing 1.0 mL of HNOtructure . After t2/GOx) was prepared [2, and 45 mg glucose oxidase (GOx) for 20 min. The prepared paste was then filled into the Teflon® piston-like electrode holder (diameter 3 mm) [Additionally, a simple GOx sensor based on bulk modified carbon paste electrode (CPE/RuOprepared . The moder 3 mm) . The ele2+) on the developed GOx biosensor was investigated by the addition of 50 µL of their standard solution (0.01 M) into 10 mL of the measuring solution containing the Glc. In the case of Hg2+ the 5 µL of the stock solution was added to the 10 mL of the measuring solution. The added volume of the heavy metals was taken into account in plotting the calibration curves.Amperometry in a batch configuration at interval time 1.0 s, constant working potential +0.4 V and stirring speed 400 rpm was used. Due to the optimum biocatalytic activity of GOx , all amp2O2 and Glc. The LOD was calculated for to be 17.4 µM for Glc. Furthermore, through this electroanalytical study, the toxicity effect of the selected heavy metals cations on the GOx enzyme from Aspergillus niger (EC 1.1.3.4) was compared using a new amperometric biosensor. The developed biosensor can be applied for rapid and accurate monitoring of enzyme activity in industry and medical laboratories for preliminary tests. Unlike the other heavy metals studied herein, it was found that Cu2+ has a negligible inhibition effect on GOx catalytic activity. The mercury(II) had the highest inhibition effect among all selected heavy metals. The inhibitory effect of Ag+, and Cd2+ were also shown by the developed biosensor. In future, it can be assumed that non-competitive inhibition of various poisons  on the activity of other enzymes could also be studied using the same protocol. The developed sensor can be utilized in ore mining or screening wastewater of factories.An enzymatic Glc biosensor was developed which was successfully applied for determination of H


Xenopus embryos at gastrula stages. We show that caveolins and caveolin dependent endocytosis are necessary for proper gastrulation, most likely by interfering with Wnt5a/Ror2 signaling. Wnt5a regulates the subcellular localization of receptor complexes, including Ror2 homodimers, Ror2/Fzd7 and Ror2/dsh heterodimers in an endocytosis dependent manner. Live-cell imaging revealed endocytosis of Ror2/caveolin1 complexes. In Xenopus explants, in the presence of Wnt5a, these receptor clusters remain stable exclusively at the basolateral side, suggesting that endocytosis of non-canonical Wnt/receptor complexes preferentially takes place at the apical membrane. In support of this blocking endocytosis with inhibitors prevents the effects of Wnt5a. Thus, target genes of Lef1 interfere with Wnt5a/Ror2 signaling to coordinate gastrulation movements.The activation of distinct branches of the Wnt signaling network is essential for regulating early vertebrate development. Activation of the canonical Wnt/β-catenin pathway stimulates expression of β-catenin-Lef/Tcf regulated Wnt target genes and a regulatory network giving rise to the formation of the Spemann organizer. Non-canonical pathways, by contrast, mainly regulate cell polarization and migration, in particular convergent extension movements of the trunk mesoderm during gastrulation. By transcriptome analyses, we found caveolin1, caveolin3 and cavin1 to be regulated by Lef1 in the involuting mesoderm of  Activation of the canonical Wnt/β-catenin signaling pathway results in stabilization of β-catenin in the cytoplasm, which subsequently translocates into the nucleus2. There, it binds to transcription factors of the Tcf/Lef family and regulates expression of numerous target genes, which are essential for the formation of the dorso-ventral axis3. Thus, the Wnt/β-catenin branch is required prior to gastrulation for induction and positioning of the Spemann organizer.Different branches of the Wnt signaling network, including the Wnt/β-catenin and the β-catenin independent branches are tightly regulated to ensure proper cell differentiation and dynamics during early embryogenesis5 and central nervous system7 of developing Xenopus embryos. Interestingly, knock-down of Lef1 additionally impairs convergent extension (CE) movements at gastrula stages8. Thus, in addition to the β-catenin independent Wnt/Ca++ 8, Wnt/PCP10 and Wnt/Ror211 branches, a transcription factor of the Wnt/β-catenin pathway is essential for the regulation of these collective tissue movements.Consistently, knock-down of Tcf1 and Tcf4 impairs Wnt/β-catenin signaling in the mesodermXnr-3 has been shown to be relevant for CE movements8. Additional target genes might also be important, however, to provide the involuting mesoderm with the competence to react to non-canonical Wnt signaling to execute gastrulation movements. These β-catenin independent Wnt signaling pathways execute their function mainly via regulation of cell shape and behavior. At early gastrulation stages, Wnt11 triggers multipolar dorsal mesodermal cells to become bipolar12. Wnt5a, however, is responsible for migration of the polarized cells towards the dorsal midline. Polarization and migration result in a medio-lateral intercalation of axial mesodermal cells, causing medio-lateral narrowing (convergence) and anterior-posterior elongation (extension) of the dorsal trunk mesoderm12. Therefore, Wnt5a and Wnt11 are the main regulators of CE movements.Lef1 regulates the expression of Wnt/β-catenin target genes. Among them, 13, we asked whether Lef1 regulates CE movements by influencing the expression of non-canonical Wnt network components. However, in a transcriptome analysis of dorsal marginal zone (DMZ) explants we did not find core components of non-canonical Wnt signaling among the numerous putative Lef1 target genes. Instead, we identified caveolin1, caveolin3 and cavin1  as Lef1 regulated genes. Caveolin and cavin proteins form the structural core of caveolae. These cholesterol rich membrane invaginations become internalized and traffic as caveosomes along recycling pathways or end up in multivesicular bodies or lysosomes15. These findings prompted us to investigate whether non-canonical branches of the Wnt network are regulated by caveolin dependent endocytosis.In light of the many levels of crosstalk between Wnt/β-catenin and non-canonical Wnt signaling17. On the other hand, Wnt signalosomes are internalized in a caveolin dependent manner, resulting in a depletion of the cytosolic GSK3β pool, accumulation of β-catenin and activation of Wnt/β-catenin signaling17. The regulation of Wnt/β-catenin signaling, and also the regulation of other signaling pathways including fgf19 and vegf21 signaling, together with many other functions ascribed to caveolae and caveosomes implicate an important role of these subcellular structures for embryogenesis. However, cav1/cav3 double knock-out mice lacking any detectable caveolae are viable22, which suggests that mechanisms are activated in early mouse embryogenesis that can compensate for the loss of caveolae.So far it was shown that the activity of the canonical Wnt/β-catenin branch is regulated by endocytosis in at least two ways. On the one hand, dkk1/lrp complexes are internalized in a clathrin dependent manner, resulting in a decrease of membrane bound lrp and thus down-regulation of Wnt/β-catenin signalingXenopus development. Epistasis experiments revealed that these caveolae core components cooperate with Wnt5a in regulating CE movements. Wnt5a induces clustering of Ror2 receptor complexes, consisting of Ror2, Fzd7 and Disheveled (dsh). These receptor complexes are internalized both in a caveolin and clathrin dependent manner. In polarized cells of the animal ectoderm, internalization occurs predominantly at the apical surface. We provide evidence that internalization is essential for full activation of the Wnt5a response.Here we show that a controlled expression of caveolar core components and caveolin dependent endocytosis are essential for proper gastrulation movements during early 23. Here we show that Wnt5a cluster formation depends on the presence of Lef1. The Wnt5aeGFP clusters at the Gap43-mCherry marked membrane were determined by colocalization analysis using Manders coefficient M2. Due to the correlation with the membrane we can distinguish between membrane and non-membrane Wnt5aeGFP clusters. Interestingly, the proportion of Wnt5a clusters located in the membrane decreases in Lef1 morphants and Lef∆HMG expressing DMZ explants  and control morpholino injected embryos  until late gastrula (stage 12) and early neurula stages (stage 14). Gastrulation movements were only efficiently blocked if the inhibitor was present throughout gastrulation, from stage 9 until stage 12. Adding the inhibitor after onset of gastrulation (stage 10) or washing it off before blastopore closure (stage 11) did not impair blastopore closure or pbk, a target gene negatively regulated by Wnt5a24 is upregulated in genistein-treated cells, even in the presence of co-transfected Ror2 and Wnt5a  assay (split-YFP assay). mRNA of the Ror2YN- and Ror2YC-constructs were co-injected into the animal half of Xenopus tissue culture cells, we observed that the membrane fraction of Ror2 homodimers decreased with respect to the cytosolic fraction upon co-transfection with Wnt5a  and clathrin dependent endocytosisced Fig. . In the ced Fig. . This chced Fig. . The BiFced Fig. .Our data thus clearly reveal that Wnt5a induced redistribution of Ror2 homodimers is due to clathrin and caveolin dependent endocytosis of dimers at the apical membrane.We used the Ror2 clusters to enquire whether the Lef1 target genes of the caveolin family are involved in cluster formation and/or cluster internalization. Therefore, we counted clusters in the cytoplasm and plasma membranes of animal cap explant cells of caveolin morphants and caveolin overexpressing embryos Fig. . More thThe interplay between Lef1, caveolin dependent endocytosis and Wnt5a signaling shown here, lets us propose the following model Fig. : At earlcaveolin1, caveolin3 and cavin1 among the TOP100 Lef1 regulated genes. All of them were downregulated in Lef1 morphants, indicating that the expression of caveolar core components depends on β-catenin/Lef1 regulated transcription. In general, the Wnt/β-catenin branch of the Wnt signaling network is important for the induction and positioning of the Spemann organizer28. The non-canonical Wnt branches activated by Wnt5a and Wnt11 regulate distinct aspects of CE movements in Xenopus in a non-redundant manner24. Wnt11 is necessary for polarization of mesodermal cells, Wnt5a for migration of polarized cells towards the midline12. This separation into different pathways is simplistic, however, because it ignores that the different Wnt signal transduction pathways including the Wnt/β-catenin, Wnt5a/Ror2 and Wnt11/PCP branches regulate each other at several levels of signal transduction1.In a microarray based transcriptome analysis we found caveolin1, caveolin3 and cavin1 in Lef1 morphants is due to a change in cell fate/competence,Herein, we have provided evidence that CE movements are regulated by clathrin and caveolin dependent endocytosis. We have further observed that Wnt5a induces clathrin and caveolin dependent endocytosis of Wnt5a signaling complexes and that endocytosis is necessary for Wnt5a/Ror2 signal transduction. Thus, in addition to inducing the Spemann organizer, early Wnt/β-catenin regulated gene expression is a prerequisite for trunk mesodermal cells to respond properly to non-canonical Wnt signaling during gastrulation. This does not necessarily mean that caveolins are direct Lef1 targets; instead, it might reflect that Lef1 regulates the cell fate/competence of early mesodermal tissue and that the downregulation of 29 and Xenopus30, the expression of caveolin genes starts with the onset of gastrulation. In Xenopus, caveolin expression remains at low levels until neurula stages, with caveolin1 and cavin1 enriched in the notochord and caveolin3 distributed in a salt and pepper pattern in multiciliated cells of the epidermis. Epistasis experiments revealed that caveolin1, caveolin3 and cavin1 cannot compensate the loss of Lef1. Thus, further Lef1 target genes are involved in regulating CE movements. One obvious candidate might be Xnr-38. Our data demonstrates that caveolins and caveolin mediated endocytosis are involved in CE regulation. Thus, the low expression levels at early stages of development are of functional relevance. Treatment with endocytosis inhibitors revealed that caveolin and clathrin dependent endocytosis act in a redundant manner during Xenopus gastrulation. If this redundancy holds true for early embryogenesis of other vertebrates, it might explain why caveolin1/caveolin3 double knock-out mice are viable even though they lack caveolae22.In mouse31, who showed that endocytosis is important for bottle cell formation at the dorsal blatoporus lip, we asked if endocytosis is also involved throughout gastulation. Indeed, blocking endocytosis with chlorpromazine and genistein indicates that endocytosis is necessary until the end of gastrula stages.Based on the studies of Lee and Harlandvia clathrin mediated endocytosis26. Recently, Shojima and coworkers32 demonstrated that, for cancer cell activation, Wnt5a signals via endocytosis dependent and independent mechanisms. Using endocytosis inhibitors, we have shown here that Wnt5a dependent signaling in early embryogenesis needs internalization. Immunohistochemical analyses revealed that Wnt5aeGFP co-localizes with caveolin1 and early endosomal antigene (EEA), but not with clathrin23. In a proteomic screen, Zheng et al.33 identified Wnt5a among 148 caveolar proteins. Furthermore, Ror2, Fzd2 and Fzd5 co-precipitated with caveolin134. Thus, evidence is accumulating that caveolin-dependent internalization plays a role for at least some branches of the non-canonical Wnt signaling network, in addition to clathrin-dependent internalization.Fzd2 internalization after Wnt5a stimulation was shown to occur 11, Wnt5a/Ror2 signaling is not required for polarization of mesodermal cells early in gastrulation12 and Wnt5a/Ror2 signaling can be easily detected by fluorophore labelled Wnt5a/Ror2 clusters23. Most importantly, these clusters depend on Lef1 expression and thus are most likely regulated by Lef1 target genes. Furthermore, Ror2 clusters are specific towards Wnt5a because Wnt11, although inducing Fzd7 clusters in a similar manner as Wnt5a, hardly induces any Ror2 clusters (data not shown).We focused our study on the Wnt5a/Ror2 pathway, because Wnt5a/Ror2 signaling is essential for CE movements23 are Wnt5a induced multimeric protein complexes consisting of Wnt5a, Ror2, Fzd7 and dsh. These clusters are formed within a few minutes after adding Wnt5a protein, indicating that receptor clustering is an immediate response to Wnt5a. Interestingly, our BiFC experiments further revealed that Ror2 homodimers as well as Ror2/Fzd7 and Ror2/dsh heterodimers are formed also in the absence of Wnt5a, but cluster formation requires Wnt5a. Thus, in conflict with the model recently proposed by Curto et al.35, we provide evidence that Wnt5a is not necessary to form Ror2/dsh heterodimers, but instead induces or stabilizes multimeric ligand/receptor/adapter molecule complexes  were synthesized with DIG RNA labeling kit (Roche). Probes for ISH were chordin38, PAPC11, and the ORF of caveolin1, caveolin3 and cavin1 inserted into pGEMT. Templates for mRNA synthesis were: Lef∆HMG39, xLef140, Wnt5aeGFP23, Ror2mCherry41, Fzd7YN42, Wnt5a24. The ORFs of caveolin1, caveolin3 and cavin1 were inserted in pCS2. Ror2YN, Ror2YC, Wnt11eGFP, Ror2∆CRDYC, Ror2∆CRDYN, Fzd7mCherry, caveolin1mGarnet and caveolin1eGFP were generated by fusion PCR and inserted into pCS2 (primer upon request). DshYC was kindly provided by Susanne Kühl (Ulm). Ror2∆CRD was a gift from Alexandra Schambony (Erlangen).Capped mRNAs were transcribed from linearized DNA templates using mMESSAGE mMACHINE (Ambion). Digoxigenin labeled antisense probes for 11, Lef143, caveolin1, caveolin3, cavin1  were used: Wnt5a24. The microarray analysis was performed on the Xenopus 4 × 44 K gene expression Chip  by Atlas Biolabs . Data sets are deposited on http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84488. The TOP 100 spots of differentially expressed genes displayed more than 3.8-fold difference between morphants and control siblings at a significance level <0.05  according to the instructions of the manufacturer. For live imaging cells were seeded 24 h after transfection on fibronectin coated chamber slides and cultivated for additional 24 h. Cells were analysed with spinning disc microscopy  and Axiovision 4.8.2 software (Zeiss). Grey-value measurements of the subcellular distribution of fluorophore tagged proteins were carried out using ImageJ software (open source: http://rsbweb.nih.gov/ij/). Statistics of the grey-value distribution were done with Past3 software (open source: https://folk.uio.no/ohammer/past/).Xenopus embryos were obtained by in vitro fertilization and staged according to Nieuwkoop and Faber48. To target the axial mesoderm, both dorsal blastomeres of four-cell stage embryos were injected in the equatorial region. To target the animal cap, all four blastomeres of four-cell stage embryos were injected in the animal pole. To target one hemisphere of the developing embryo, one blastomere of two-cell stage embryos was injected in the animal hemisphere.in situ hybridization (ISH) embryos were fixed for one hour in 4% formaldehyde at the indicated stages. ISH was performed as described49. Statistical analyses of the phenotypes were done with Χ2 test.For RNA ® Plus RNA Purification Kit . Prior to the isolation the AC explants were collected and stored in RNAlater . After reverse transcription  cDNA was amplified according to standard protocols. Real time PCR was performed using iQ SYBR Green Supermix on an iCycler . Expression levels were calculated relative to ornithine decarboxylase (ODC) and normalized to wild-type explants. Results of four independent experiments were averaged, and statistical significance was calculated using Student’s t-test. Primer sequences and annealing temperatures are available upon request.RNA was isolated from 5 AC explants by use of the TRIzolDMZ explants were dissected at stage 10.25 and cultivated in petri dishes coated with 1% BSA in 1x MBSH until their siblings reached stage 12.Animal caps of stage 9 embryos were cut manually, placed on BSA coated glass cover slides and fixed with cover glass. To generate a cultivation chamber, the cover glass was separated from the cover slides by a thin layer of silicone. Recombinant Wnt5a protein (R&D Systems) was added by carefully changing MBSH in the cultivation chamber with MBSH containing 0,1 µg/µl Wnt5a and 0,1% BSA.Time-lapse movies and z-stacks were captured using the Z1 Cell Observer Spinning Disc inverse microscope . For the z-distribution of fluorophore-tagged proteins in animal cap explants ten optical sections with 1 µm distance were captured, starting from the basal side at position 0 µm, where cell protrutions were visible.Images were acquired at room temperature on an inverted microscope  equipped with an oil immersion objective . mCherry was excited by a solid state laser with wavelength 561 nm  by an illumination power of 2 W/cm². The laser beam was guided via dichroic mirrors  to an AOTF  for control of the laser intensities at the sample. After passing through the excitation dichroic , the emission light was split into two color channels by a dichroic beam splitter , filtered by 525/50 nm (center wavelength/width) and 607/70 nm filters for the eGFP-mCherry pair, and 525/45 nm and 641/75 nm filters for the YFP-mCherry pair . The two color-filtered images were focused side-by-side on an electron-multiplying CCD camera  set to an exposure time of 50 ms.50 and a particle tracking program provided by John C. Crocker and colleagues (http://www.physics.emory.edu/faculty/weeks//idl/). Trajectories were acquired over 1000–2000 frames. Only events with photon numbers higher than 1000 per frame were registered. Particles in adjacent frames were linked if their displacement between successive frames was less than 2.5 µm. To generate time lapse movies, stacks were analyzed using Fiji software51. Bleaching was compensated using Fiji software, and the SPIM registration plugin was used for drift compensation52.Single particle tracking was performed using a livePALM softwareSupplementary InformationSuoolementary Info File '1Supplementary FiguresSupplementary movies


Most colorectal cancer (CRC) patients are diagnosed at an advanced or metastatic stage with poor prognosis. Ubiquitin-specific protease 6 N-terminal-like protein (USP6NL) with high expression in CRC tissues regulates CRC cell proliferation via Wnt/β-catenin pathway. We hypothesized that USP6NL impacts CRC growth and inhibition of USP6NL may be a novel treatment strategy to improve CRC therapy.USP6NL level in human CRC tissues and its association with tumor growth and metastasis were examined. Its roles and potential mechanisms in regulating tumor growth were studied by genetic and pharmacological manipulation of CRC cells in vitro and in vivo.Herein, we found that USP6NL was up-regulated in tumorous tissues of CRC patients. Our data suggested that knockdown of USP6NL in human CRC cell lines (HCT116 and LOVO cells) inhibited cell proliferation, induced G0/G1 cell cycle arrest, and prevented the tumorigenicity of HCT116 cells in nude mice, and which was associated with the prevention of Wnt/β-catenin pathway. On the contrary, USP6NL overexpression in human CRC cells (SW480) showed the opposite result. Our data suggested that the promoted cell proliferation, G1/S cell cycle progression, and the enhanced expression of β-catenin Cyclin D1 and C-myc while reduced P27 induced by the overexpression of USP6NL were significantly reversed by additional treatment of XAV939, indicating that activating Wnt/β-catenin pathway was the mechanism, by which USP6NL exerted carcinogenesis in CRC in vitro. Besides, our data suggested that knockdown of USP6NL increased the ubiquitination of β-catenin, indicating that USP6NL may serve as a deubiquitinase that regulated β-catenin accumulation in this process. Furthermore, 10058-F4 down-regulated USP6NL, inhibited CRC cell proliferation and induced cell cycle arrest. The result demonstrated a possible feedback loop between USP6NL, β-catenin and C-myc in regulating CRC cell growth.USP6NL was an oncogene in CRC, and it may be a potential target for the treatment of CRC. GTPase-activating proteins (GAPs) family proteins can bind to activated G proteins and motivate their GTPase activity, impairing the binding with downstream signaling targets . GAP actWnt/β-catenin signaling is essential in carcinogenesis, embryonic development and tissue homeostasis . ActivatUbiquitin-specific proteases (USPs), acting as deubiquitinating enzymes (DUBs), is emerging as master regulator of Wnt/β-catenin signaling . We founHere, we found that USP6NL was up-regulated and functioned as an oncogene in human CRC cells. USP6NL favored β-catenin accumulation in CRC cells and regulated its downstream oncogenes . As a result, the Wnt/β-catenin signal pathway is activated and promoted pro-proliferation and normal cell cycle progression of CRC cells.https://cancergenome.nih.gov/researchhighlights/researchbriefs/ColorectalCancerInsights). Furthermore, mRNA expression data of USP6NL in 70 colorectal tissues and 12 adjacent tissues were deposited in NCBIs Gene Expression Omnibus  with accessible number GES9348. Immunohistochemistry (IHC) analysis was performed to assess USP6NL expression in 10 tumorous tissues samples and 5 precancerous tissue from patients and with CRC in the Affiliated Hospital of Jiangsu University. The use of CRC patients was permitted by the ethics committee of the Affiliated Hospital of Jiangsu University, and written informed consent of each participate were acquired.To study the role of USP6NL in CRC, mRNA expression of USP6NL in 32 pairs of tumorous colorectal tissues and adjacent non-tumorous tissue were assessed, using RT-PCT, in CRC patients recruited at the Affiliated Hospital of Jiangsu University. The researches were supported by the Independent Ethics Committee (IEC) of the Affiliated Hospital of Jiangsu University and all patients were provided written informed consent. Besides, mRNA expression data of USP6NL in 260 tumorous colorectal tissues and 41 none-tumorous adjacent tissues were downloaded from The Cancer Genome Atlas database (TCGA)  were incubated with USP6NL antibody  at 4 °C overnight followed by secondary antibodies  at 25 °C for 30 min. DAB substrate (Long Island Biotech) and hematoxylin  was used for coloration. A light microscope  with an image analysis system  were used for visualization and calculation of USP6NL-positive cell areas.2 with the confluency above 80%.HT29, SW480, LOVO, HCT116, CACO2 and FHC cells were bought from ATCC . Culture medium for HCT116, FHC and CaCO2 was DMEM  while feed medium for LOVO, SW480 and HT-29 was RPMI-1640 (Hyclone), and both of which were supplemented with penicillin , streptomycin  and heat-inactivated fetal bovine serum (FBS) ). CRC cells were cultured at 37 °C under 5% COTo verify how Wnt/β-catenin pathway played in USP6NL-induced SW480 cell growth, after USP6NL overexpression transfection, SW480 cells were treated with 10 μmol/l of XAV939  and then cultured as mentioned above. To verify the roles of C-myc in regulating USP6NL and SW480 cell growth, after transfection, cells were treated with 100 μmol/l of 10058-F4 .GAATTCATGAATTCAGACCAGGATGTAGC-3′ (forward) and 5′-CGGGATCCTCACAGCAACACTGACTCTTGG-3′ (reverse), which were implanted into pLVX-Puro vector (Clontech). Plasmids (pLVX-Puro-USP6NL), as well as psPAX2 and pMD2G (Addgen) were co-transfected into 293T cells (ATCC) using Lipofectamine™ 2000 . After 4–6 h, cells were feed in a complete medium, and lentiviruses with USP6NL expression were collected at 48 h and 72 h. 1.5 μg of lentiviruses with USP6NL expression was transfected into SW480 cells using Lipofectamine 2000 reagent. Meanwhile, cells transfected with pLVX-Puro without USP6NL expression were thought as the corresponding control.The primers of human USP6NL gene (NCBI NM_AB449916.1) were: 5′-CG® 2000 Reagent (Invitrogen). A non-specific siRNA (siRNA-NC) was used as a negative control.Three siRNAs targeting human USP6NL mRNA NM_AB449916.1) were designed in Table 6.1 were 3 cell/well in cultured medium (90 μl/well) were hatched with CCK-8 working solution (10 μl/well) at 37 °C for 1 h. Optical density (OD) value was read by a DNM-9602 microplate reader  at 450 nm.Proliferation of HCT116, LOVO and SW480 cells was assessed using CCK8. Briefly, after treatment at 0, 24, 48, 72 h, 3 × 10After knockdown of USP6NL, CRC cells were fixed in ethanol, rinsed in PBS, remove RNA using 1 mg/ml of RNase A , and then incubated with 50 μg/ml of propidium iodide solution  for 10 min in darkness at 25 °C. DNA content was monitored via flow cytometry  with FlowJo v10 .CTCGAGTATCATTTAATGGGTTCTGGAGGTC-3′ (forward with XhoI site as indicated by the underline) and 5′-CCAAGCTTATAGAGTAACTTATCTATTCCAAGATTTC-3′ , and inserted into pGL3-Enhance to construct pGL3-Enhancer-p USP6NL. SW480 cells (5 × 105 cells/well) on a 6-well plate were cultured at 37 °C overnight for adherent growth, and then transfected with plasmid pGL3-Enhancer-p USP6NL (1.5 μg) and internal control plasmid pRL-TK (0.05 μg) using Lip 2000  according to a provided instruction. Besides, pGL3-Enhancer was used as a negative control.USP6NL promoter was amplified with the primers: 5′-CC® Reporter Assay System  was used to detect the activity of firefly luciferase in plasmid pGL3-Enhancer and pGL3-Enhancer-p USP6NL, as well as the activity of renilla luciferase in internal control plasmid pRL-TK. The activity of USP6NL promoter was presented as the ratio of firefly/renilla. Each group set 3 wells, and the experiment was repeated 3 times.6 h later, SW480 cells with USP6NL overexpression were maintain in fresh culture medium with or without 10058-F4 (100 μmol/l) at 37 °C for 24 h. After fully lysis, Dual-Luciferase−∆∆Ct method according to a reported study [Total RNA from colorectal tissue, CRC cell lines , or human normal colon FHC cells was extracted by Trizol Reagent  and reverse transcribed using cDNA synthesis kit (Fermentas). The primers targeted USP6NL (NCBI NM_AB449916.1) were: 5′-TACTCAGCCTTTCAACTC-3′ (USP6NL-forward) and 5′-GCAAGTACACGTCAAATC-3′ (USP6NL-reverse); Pos: 2389-2604; size: 216 bps; primers targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NCBI NM_001256799.2) were: 5′-AATCCCATCACCATCTTC-3′ (GAPDH-forward) and 5′-AGGCTGTTG∆∆∆TCATACTTC-3′ (GAPDH-reverse). mRNA levels of USP6NL were determined using SYBR Green PCR Kit (Thermo Fisher) on an ABI7300 system (Applied Biosystem). Amplification condition was: 95 °C, 10 min  × 40; 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s; 60 °C, 15 s. USP6NL was normalized by GAPDH and quantified using the 2Colorectal tissue (20 mg) and CRC cell lines samples were lysed in RIPA . Total protein content in supernatant was determined using BCA protein assay kit . Total protein (25 μg) was separated on 15% SDS-PAGE, and electrophoretically pure were sent onto PVDF membranes  and incubated with anti-USP6NL antibody , anti-β-catenin antibody , anti-P27 antibody , anti-Cyclin D1 antibody , antibody against C-myc , and antibody against GAPDH  at 4 °C overnight followed by horseradish peroxidase-conjugated antibodies  for 1 h at 25 °C. USP6NL, β-catenin, P27, Cyclin D1 and C-myc were normalized by GAPDH and quantified using ECL system .To study whether USP6NL was associated with β-catenin, 100 μg of total protein in lysis supernatant of HCT116 cells transfected with siUSP6NL was added into protein G-Agarose beads (Roche), and then immune-precipitated with anti-USP6NL (Abcam), anti-β-catenin (Abcam) or control IgG antibody overnight at 4 °C. USP6NL and β-catenin in the immune complex were immunoblotted using anti-USP6NL (NBPI-47264), anti-β-catenin , respectively according the western blot method. Same amount of protein in each group was reserved for input control.To study the effect of USP6NL on β-catenin ubiquitination, after fully lysis of HCT116 cells transfected with siUSP6NL, 100 μg of total protein in supernatant was added into Protein A/G PLUS-Agarose , and then immune-precipitated with 1 μg of IgG  or Anti-β-catenin antibody (ab227499) overnight at 4 °C. Ubiquitination levels of β-catenin in precipitated immune-complexes were separated and quantified using Anti-ubiquitin antibody (ab7780) according the western blot.6 cells, 100 μl). Mice in siUSP6NL group were subcutaneously injected with HCT116 cells transfected with siUSP6NL . Then, mice in different groups were separately fed and the bedding was changed regularly. After tumors formation (12 day later), tumor volume (mm3) and weight (g) were calculated every 3 days from 12th day to 33th day. Tumor volume were calculated as length × (width2/2). We confirmed that the use of mice were abided by the relevant agreements of animal ethics committee in The Affiliated Hospital of Jiangsu University.12 nude mice, 4–6-week-old, were obtained from Shanghai Laboratory Animal Company, and randomly divided into siNC and siUSP6NL groups. Mice in siNC group were subcutaneously injected with HCT116 cells transfected with siNC (7 × 10In Situ Cell Death Detection Kit (Roche) was used for detection of cell apoptosis in mouse colorectal tissue. Briefly, 4–7 μm of colorectal tissue section on slides was maintained with 50 μl of TUNEL working solution  at 37 °C for 1 h, and then rinsed twice with PBS. We then dyed the cell nucleus with DBA substrate kit  followed by hematoxylin . Individual apoptotic thyroid cells were analyzed using IMS image system .t test with P value < 0.05 being statistically significance.Dates were described as mean ± standard error of the mean (SEM), which was calculated by triplicate determinations in each experiment. Comparisons between two groups were assessed by Student’s To investigate the involvement of USP6NL in human CRC, USP6NL expression in tumorous and none-tumorous colorectal tissue from CRC patients were detected. USP6NL mRNA expression data in CRC and corresponding healthy people were downloaded in TCGA and GEO database. Our results showed that USP6NL mRNA and protein expression was significantly increased in tumorous colorectal tissue when compared with none-tumorous colorectal tissues Fig. a, d, morFurthermore, mRNA expression of USP6NL in five CRC cell lines  and one human normal colon FHC cells were assessed. Our data suggested that USP6NL was obviously enhanced in CRC cell lines when compared with FHC cells, substantiating the amplification of USP6NL in CRC in vitro  or siRNA-USP6NL . Figure The proliferation of CRC cells transfected with siUSP6NL was examined using CCK-8 assay. Figure The transition from G0/G1 to S phase is regulated by the P27, Cyclin D1 and C-myc, which are targeted genes of β-catenin (the core element of Wnt/β-catenin pathway) . To inveSW480 cells were transfected with control vector or USP6NL overexpression vector. Figure To confirm that Wnt/β-catenin pathway was the mechanism, by which USP6NL regulated cell growth, SW480 cells with USP6NL overexpression were treated with XAV939 (a specific inhibitor for Wnt/β-catenin pathway). Then, cell proliferation, cell cycle, and protein levels of β-catenin, P27, Cyclin D1 and C-myc were assessed. Our data suggested that USP6NL significantly enhanced cell proliferation in a time-depended manner  from HCT116 cells, and then β-catenin (or USP6NL) was immunoblotted to analyzed the physical interaction between USP6NL and β-catenin. We found that USP6NL significantly bound β-catenin within CRC cells Fig. a, b.Fig.Without Wnt, β-catenin can be degraded through the ubiquitination/proteasome pathway . PresentTo confirm that C-myc was the mechanism, by which USP6NL regulated cell growth, control vector/USP6NL overexpression transfected SW480 cells were treated with 100 μmol/l of 10058-F4 (a specific C-myc inhibitor). Our data suggested that 10058-F4 significantly reduced USP6NL mRNA and protein levels , acting as deubiquitinating enzymes (DUBs), is emerging as the master regulator of tumor , 18–21. Previous studies have shown that the cell cycle of tumor cells is significantly different from that of normal tissue cells, and its S and G2–M phase is significantly higher than that of ordinary cells, while the percentage of G0–G1 phase representing cell division stagnation is lower . In thisWe then investigated whether and how USP6NL acts on Wnt/β-catenin to regulate CRC cell growth. By treating USP6NL overexpressing CRC cells with XAV939, a specific inhibitor of the Wnt/β-catenin pathway, the results suggest that CRC cell proliferation, G1/S cell cycle progression, and β-catenin Cyclin D1 and C-myc are promoted. Enhanced expression, while a decrease in P27 induced by overexpression of USP6NL, was significantly reversed, suggesting that activation of the Wnt/β-catenin pathway is a mechanism by which USP6NL is carcinogenic to CRC in vitro. Our data suggested that 10058-F4 (C-myc inhibitor) significantly reduced USP6NL mRNA and protein levels, and suppressed the activity of USP6NL promoter when compared with vector, demonstrating the inhibited effect of 10058-F4 on USP6NL expression.Furthermore, our data suggest that knockdown of USP6NL increases ubiquitination of β-catenin, suggesting that USP6NL may act as a deubiquitinating enzyme that regulates β-catenin accumulation during this process. In addition, 10058-F4 downregulated USP6NL, inhibited CRC cell proliferation and induced cell cycle arrest, demonstrating that USP6NL//β-catenin/C-myc may have a feedback loop in regulating CRC cell growth.Our study reveals the new highlight is that USP6NL activated Wnt/β-catenin pathway probably by the inhibition of β-catenin ubiquitination, revealing other pathways that have not been prioritized, including the Wnt/β-catenin pathway, and has filled a gap in the mechanism of USP6NL’s impact on CRC.The tumor growth rate and size of the mouse model siRNA-USP6NL group were significantly lower than that of the siRNA-NC group, which provided a strong basis for the purpose of this study, and provided a possibility for the subsequent human targeted therapy research of USP6NL.However, there are some limitations in our study. The specific mechanism of how USP6NL may activate the Wnt/beta-catenin pathway by inhibiting ubiquitination of beta-catenin is still not clear enough. The treatment of USP6NL inhibitor has not been added in animal experiments, which brings it closer to clinical trials and provides more background information for later experiments.In conclusion, USP6NL is closely related to the production of CRC, and its mechanism may be affected by intervention in the Wnt/β-catenin signaling pathway. Our data provide close evidence. However, the exact mechanism by which USP6NL affects the Wnt/β-catenin signaling pathway is unclear and needs further study. Taken together, our study not only indicates that USP6NL is up-regulated in CRC tissues, but also emphasizes its role in regulating CRC cell proliferation and metastasis in vivo and in vitro. In addition, we also found that USP6NL regulates the potential mechanism of CRC cell proliferation and metastasis by participating in the Wnt/β-catenin signaling pathway Fig. . This maUSP6NL was an oncogene in the tumorigenesis of CRC both in vitro and in vivo, and activation of Wnt/β-catenin pathway was the underlying mechanism. USP6NL activated Wnt/β-catenin pathway probably by the inhibition of β-catenin ubiquitination. Our research elucidated that targeting USP6NL may represent a novel therapeutic target in CRC treatment.


In addition, RT-qPCR and Western blot assay were applied for exploring the mRNA and protein expression levels of SIRT7, Wnt, β-catenin, and cyclin D1 after knockdown or overexpression of SIRT7. And, furthermore, Cell Counting Kit-8 assay, flow cytometry, and wound-healing assay were used to assess HAVSMCs proliferation, cell cycle, and migration. Dickkopf-1 (DKK-1), a secretory glycoprotein that can block Wnt/β-catenin pathway, was used in SIRT7 overexpression HAVSMCs; subsequently cells proliferation and migration were assessed by Cell Counting Kit-8 assay, flow cytometry analysis, and wound-healing assay. We found that knockdown of SIRT7 significantly promoted cell proliferation and migration, decreased the percentages of cells in the G1 and G2 phases, and increased those in the S phase and downregulated the protein expression levels of Wnt, β-catenin, and cyclin D1, while overexpression of SIRT7 had reverse results. After treatment with Wnt/beta-catenin pathway inhibitor DKK-1 in SIRT7 overexpression HAVSMCs, cell proliferation and migration were increased, respectively. In conclusion, SIRT7 inhibited HAVSMCs proliferation and migration via enhancing Wnt/β-catenin activation, which provided a novel therapeutic strategy for antiatherosclerosis.A huge amount of evidence indicates that sirtuin 7 (SIRT7), a key mediator of many cellular activities, plays a crucial role in the pathogenesis of various diseases. However, little is known about the role of SIRT7 in atherosclerosis. This study investigated the potential role of SIRT7 in regulating the proliferation and migration of human vascular smooth muscle cells (HAVSMCs) and its possible molecular mechanism. In this study, human vascular smooth muscle cells (HAVSMCs) were induced by oxidized low-density lipoprotein (ox-LDL) to establish atherosclerosis (AS) cell model. Immunofluorescence staining and Western blot were used to detect the level of  Atherosclerosis (AS) is a chronic degenerative disease of the arterial wall, which is well known for its high morbidity and mortality in aged people around the world . A growiThe sirtuins (SIRTs) protein family belongs to class III of histone deacetylases and consists of seven members (denoted SIRT1–7). Increasing amounts of evidence indicate that SIRTs can regulate plenty of cellular processes, such as stress responses, metabolism, cell cycle, inflammation, and senescence as well as apoptosis . Furtherβ-catenin signaling pathway [β-catenin signaling exerts a critical role in vascular pathologies. For example, several studies suggest that the β-catenin/T-cell factor (TCF) signaling pathway can regulate VSMCs proliferation and vascular calcification [β-catenin signaling participates in regulating cellular proliferation, inflammation, and cell deposition fate, which are important causes in AS progression [β-catenin signaling play roles in resisting AS and the underlying regulatory relationship between them. Therefore, Dickkopf1 (DKK-1), a secretory glycoprotein that can block the Wnt/β-catenin pathway by competitively binding to receptors on the cell membrane, was used in our present study [A recent study shows that SIRT7 is instrumental for osteogenic differentiation of bone marrow stem cells, partly by activation of the Wnt/ pathway . Accumulfication , 20. In gression –23. All nt study .β-catenin, and cyclin D1, while SIRT7 overexpression had opposite results in parallel studies. The protein expression levels of Wnt, β-catenin, and cyclin D1 were decreased after DKK-1 blocking Wnt/beta-catenin pathway. In conclusion, SIRT7 inhibited VSMCs proliferation and migration via activating Wnt/β-catenin.In this study, we demonstrated that the expression of SIRT7 was downregulated in HAVSMCs stimulated by ox-LDL. Moreover, SIRT7 knockdown enhances HAVSMCs proliferation and migration accompanied with decreasing protein expression levels of Wnt, μg/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO2 humidified incubator. HAVSMCs were seeded in 6-well plates at the density of 1 × 105 cells/well. Subsequently, they were exposed to 100 μg/ml ox-LDL  for 24 h to establish the AS cell model. Cells with passage number between 10 and 15 were used for the experiments.The human aortic vascular smooth muscle cells T/G HAVSMCs (HAVSMCs) were obtained from China Center for Type Culture Collection  and maintained in DMEM medium  supplemented with 10% fetal bovine serum , 100 6 cells/well. When grown at 40–60% confluence, HAVSMCs were transfected with 100 nM of SIRT7 siRNA or control siRNA in Opti-MEM with Lipofectamine RNAiMAX . SIRT7 siRNA (siSIRT7-1: 5′-CCUGCCGUGUGAGGCGGAA-3′ and siSIRT7-2: 5′-GCCGUGUGAGGCGGAAGCG-3′) and control siRNA (siControl: 5′-CCUUGCGUGGGAGCGCGAA-3′) were synthesized by Invitrogen . The overexpression of SIRT7 was made with the pcDNA3.1 plasmid  which was performed using Lipofectamine 2000 . Then successful transfections were determined by RT-qPCR and Western blot assay after cells were incubated for 24 h.HAVSMCs were seeded in 6-well plates at the density of 1 × 10α-SMA  overnight at 4°C. Following washing three times with PBS, cells were incubated with DyLight™ 488-conjugated secondary antibodies (Thermo Scientific) for 1 h at room temperature. Then the nuclei were subsequently stained with 4′,6-diamidino-2-phenylindole  for 5 min and then washed three times with PBS in the dark. Immunofluorescence was detected using a confocal laser scanning microscope (Olympus FV1000).HAVSMCs were seeded in 24-well plates which contained coverslips. After different treatment, cells were fixed with 4% paraformaldehyde for 15 min. Then 0.1% Triton X-100 in TBS was used to permeabilize the fixed cells for 20 min at room temperature. After being blocked in 5% bovine serum albumin for 30 min, cells were incubated with anti-3 cells/well (three replications per group) and incubated at 37°C for 6 h. The cell viability was examined by CCK-8 kit  according to the manufacturer's instruction. At the indicated time points , 10 μl CCK-8 solution was added to each well. Following incubation at 37°C for 1 h, the absorbance was measured with a plate reader at 450 nm.To explore the effect of SIRT7 knockdown or SIRT7 overexpression on the proliferation of ox-LDL-induced HAVSMCs, cells were seeded in 96-well plates at a density of 5 × 105 cells/well (three replications per group). When cells were grown at 80% confluence and merged into a monolayer, a sterile 200 μl pipette tip was used to gently generate scratch wounds at the surface of the cell layer. Then cells were rinsed twice with serum-free media to remove cell debris. An inverted microscope  was used to monitor cells at the borders of the scratches. The degree of scratch healing was observed and images were captured in each group at 0 and 24 h.For the wound-healing assay, cells were plated in 12-well plates at the density of 1 × 105 cells were treated with 70% cold ethanol at 4°C overnight to increase cell membrane penetrability. Subsequently, 100 μg/ml RNase  was used to treat cells at 37°C for 20 min. Following staining with 30 μg/ml propidium iodide  for 30 min at room temperature in the dark, cell cycle was analyzed by Gallios Flow Cytometry . The flow cytometry results were evaluated using a Cell Quest kit .The cell cycle analysis was performed using the cell cycle analysis kit . Following transfection for 48 h, 1 × 10-ΔΔCq calculation method [Total RNA was extracted using the TRIzol Reagent . RNA was reverse-transcribed to cDNA using the QuantiTect RT kit (Qiagen) following the manufacturer's instructions. Real-time PCR was performed using a System 7500 instrument  and the following PCR cycling protocol was used: 95°C for 5 min and then 40 cycles of 94°C for 15 s, 55°C for 20 s, and 72°C for 20 s, followed by 72°C for 7 min. The mRNA expression level of GAPDH was the endogenous control. The primer sequences for qPCR were as follows: SIRT7, forward 5′-ACGCCAAATACTTGGTCGTCT-3′ and reverse 5′-AGCACTAACGCTTCTCCCTTT-3′; GAPDH, forward 5′-CTCACCGGATGCACCAATGTT-3′ and reverse 5′-CGCGTTGCTCACAATGTTCAT-3′. Results were analyzed following the 2n method .α-SMA , anti-SIRT7 , anti-β-catenin , anti-cyclin D1 , and anti-GAPDH  antibodies were purchased from Cell Signaling Technology . Anti-Wnt  antibody was the product of Abcam company .VSMCs proteins were extracted with RIPA lysis buffer  and incubated for 30 min on ice. Then the supernatants were collected after centrifugation. The concentrations were detected using a BCA protein assay kit . Equal amounts of proteins were loaded onto 10% SDS-polyacrylamide gels to separate various proteins which were transferred to PVDF membranes subsequently. Then these membranes were blocked with a buffer containing 10% non-fat milk in PBS for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C, respectively. Then the membranes were incubated with the goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibodies  at room temperature for 1 h. Following washing three times with TBST, signals were detected using the enhanced chemiluminescence reagent  and the ImageJ software was provided to analyze the fold changes of protein levels. Anti- t test or one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.All statistical analyses were conducted using SPSS 14.0 software . All experimental results were expressed as mean ± SD. Statistical comparisons were made by two-tailed Student'sα-SMA in immunofluorescence imaging [α-SMA protein expression level (Figures To determine whether the successful AS cells model was made, the immunofluorescence staining and protein analysis were employed in the present study. Our data indicated that treatment with ox-LDL led to a marked increase in the level of VSMC-specific marker gene  imaging , which w Figures .α-SMA was increased in siRNA-SIRT7 group. However, the level of α-SMA was decreased after SIRT7 overexpression (In our study, the results demonstrated that the protein and mRNA expression levels of SIRT7 were reduced after HAVSMCs underwent ox-LDL stimulation Figures . To testpression . In addipression , while opression . The resWe monitored cells migration by wound-healing assay. From the results, we found that the relative distance was decreased in SIRT7 knockdown group compared with that in knockdown negative control group at the time of 24 h after scratch Figures . Importaβ-catenin signaling. After treatment with ox-LDL, the protein expression levels of Wnt, β-catenin, and cyclin D1 were decreased extremely in SIRT7 knockdown HAVSMCs (β-catenin signaling pathway in our study. The results indicated that the abilities of cells proliferation and migration were augmented after DKK-1 was employed in SIRT7 overexpression HAVSMCs, which were exhibited in Figures β-catenin signaling pathway.To further explore the molecular mechanism underlying the SIRT7-mediated protective effects on ox-LDL-induced proliferation and migration of HAVSMCs, the following study investigated the relative variation in Wnt/ HAVSMCs ; however HAVSMCs . Furtherβ-catenin signaling pathway.It is well known that AS is a chronic degenerative disease and has become a predominant factor of a plenty of cardiovascular diseases, which has high morbidity and mortality. However, the specific pathogenesis mechanisms of AS are still not fully elucidated. In this study, we found that SIRT7 regulates the VSMCs proliferation and migration induced by ox-LDL via Wnt/Aberrantly proliferation and migration of VSMCs play a considerable role in the progression of AS . It has β-catenin signaling is an evolutionarily conserved pathway and activation of this signaling regulates the expression of downstream target genes including cyclin D1, subsequently resulting in many variations of pathophysiological behaviors, such as cell differentiation, proliferation, migration, and polarity [β-catenin signaling has been shown to be involved in SMA and calponin expression which are marker genes during the progression of AS [β-catenin signaling pathway [β-catenin signaling intrigues our interest because of its crucial role in regulating essential aspects of cell proliferation and migration [β-catenin signaling was affected by the knockdown or overexpression of SIRT7 in HAVSMCs. In our present research, we found that the expression levels of β-catenin and cyclin D1 were notably decreased in SIRT7 silencing HAVSMCs, while an increased result was detected in SIRT7 overexpression condition. Taken together, these findings indicated that there was a regulatory relationship between SIRT7 and Wnt/β-catenin signaling in HAVSMCs.The Wnt/polarity , 31. Accon of AS , 33. In  pathway . To inveigration , 35. Theβ-catenin signaling, DKK-1, a classical inhibitor of canonical Wnt/β-catenin signaling, was employed in our study [β-catenin signaling pathway.To further elucidate the underlying regulation between SIRT7 and Wnt/ur study . The resβ-catenin and cyclin D1, while SIRT7 overexpression had reverse results. These findings indicated that SIRT7 plays a protective role in regulating proliferation and migration of AS via Wnt/β-catenin signaling pathways.According to our results, the present study demonstrated that SIRT7 was significantly downregulated in ox-LDL-stimulated HAVSMCs. SIRT7 knockdown notably promoted cell proliferation and migration and downregulated the expression of


RAS were significantly increased and correlated in tissues of 756 gastric cancer (GC) patients and tissues of primary- and acquired-resistance patient-derived xenograft tumors treated with 5-fluorouracil and oxaliplatin modulated with leucovorin (FOLFOX). On the basis of our previous studies, where small molecules to suppress colorectal cancer (CRC) via degrading both β-catenin and RAS were developed, we tested the effectiveness of KYA1797K, a representative compound functioning by binding axin, in the growth of GC cells. The efficacy test of the drugs using gastric tumor organoids of 1638NApc mice showed that the CD44 and ALDH1A3 cancer stem cell markers were induced by FOLFOX, but not by KYA1797K. KYA1797K also efficiently suppressed tumors generated by re-engrafting the FOLFOX-resistant patient-derived xenograft (PDX) tumors, which also showed resistance to paclitaxel. Overall, the small-molecule approach degrading both β-catenin and RAS has potential as a therapeutic strategy for treating GC patients resistant to current standard chemotherapies.Treatment of advanced gastric cancer patients with current standard chemotherapeutic agents frequently results in resistance, leading to poor overall survival. However, there has been no success in developing strategies to overcome it. We showed the expression levels of both β-catenin and  Helicobacter pylori (H. pylori) infection, smoking, diet, and genetics are major causes of its occurrence [Gastric cancer (GC) is one of the leading causes of cancer-related death globally, and currence ,2,3,4. Ccurrence ,6. Althocurrence .H. pylori infection, a major known risk factor for GC [adenomatosis polyposis coli (APC), which are present in approximately 70% of patient with intestinal-type gastric adenoma [RAS and more frequently its upstream receptors such as ERBB2 and EGFR, frequently occurs in GC patients and plays roles in tumorigenesis [RAS mutation is rare, its protein level was commonly found to be significantly increased via focal high-level amplification of its gene in tissues of GC patients [KRAS-amplified tumor had poorer survival compared to those without KRAS amplification [r for GC ,9, and m adenoma ,10, are igenesis ,12. Althpatients ,14,15. AAPC and KRAS, which critically enhanced the occurrence and progression of tumors [APC and KRAS mutations were attributed to increase in the levels of both β-catenin and mutant KRAS; these increases were caused by their stabilizations via APC loss, which inactivates GSK3β involved in phosphorylation-dependent β-catenin and Ras degradation [The Wnt/β-catenin and RAS-ERK pathways interact in colorectal cancer (CRC) tumorigenesis by gene mutations of the components in these two pathways such as the f tumors ,17,18. Tradation . In the Apc1638N mouse gastric tumors. KYA1797K, but not FOLFOX, inhibited the enrichment of CSC markers with the critical inhibition of β-catenin and Ras, although both FOLFOX and KYA1797K reduced the size of the tumoroids. The effectiveness of KYA1797K on the suppression of gastric tumors, particularly those resistant to chemotherapy, was further confirmed by establishing PDX derived from acquired-resistant gastric tumor [We recently identified small molecules that degrade both β-catenin and Ras proteins by the screening of libraries of small molecules that inhibit the Wnt/β-catenin pathway ,21. KYA1ic tumor  against ic tumor . An approach of the suppression of CSCs via the degradation of both β-catenin and RAS by targeting the Wnt/β-catenin pathway has a potential as a therapeutic strategy to overcome the current limitations of resistance and recurrence after adjuvant chemotherapies in the clinical management of GC.P < 0.05). The majority of AGC patient tumors (586 of 693 cases) was characterized by high expression levels of activated β-catenin and pan-Ras (P < 0.05).To identify the involvement of the levels of β-catenin and RAS in the pathogenesis of GC, we analyzed tissue microarrays retaining tumor tissues of 756 AGC patients by immunohistochemical (IHC) analyses of β-catenin and pan-RAS. The correlations between β-catenin or pan-RAS expression and clinicopathological features of AGC are summarized in https://www.oncomine.org). The expression of the Wnt/β-catenin pathway target genes, particularly those related to cancer, such as CD44, S100A4, MMP7, and LBH, was significantly increased in GC tumors compared with that in normal tissue  10.0 pathway annotation [fragments per kilobase of transcript per million mapped reads (FPKM) [CD44, S100A4, MMP7, and LBH genes in FOLFOX AR-PDX tumors   values oX tumors E. KYA1797K dose-dependently reduced levels of both β-catenin and RAS with the inactivation of ERK, downstream kinases of RAS, in GC cell lines . Growth Treatment with KYA1797K also effectively suppressed the FOLFOX-induced increments of both β-catenin and RAS as well as the activities of ERK, respectively, in NCI-N87 cells . IntriguAPC1638N mice, and tested the effects of these drugs. The growth of tumoroids was significantly suppressed by treatment with both FOLFOX and KYA1797K although no synergistic effects were observed when both of these drugs were administered together /ERK pathways ,33,34. TConsidering the roles of β-catenin and RAS in the resistance and recurrence of GC after chemotherapy, we investigated the effectiveness of a compound that destabilize both β-catenin and RAS proteins via the activation of GSK3β through the specific binding of axin, a negative regulator of the Wnt/β-catenin pathway , on the 1638NApc mice, which reduced their size upon FOLFOX treatment. The CSC induction by FOLFOX treatment indicates that the recurrence of tumors after initial response to FOLFOX treatment may be related to the activation of CSCs. Although both FOLFOX and KYA1797K similarly suppressed the growth of tumoroids, KYA1797K, which degrades β-catenin and RAS proteins, suppressed CSC activation of the tumoroids. Moreover, the induction of CSC markers by FOLFOX did not occur in the tumoroids co-treated with KYA1797K and FOLFOX.A potential contribution of chemotherapy to the activation of CSCs was shown by the enrichment of CD44 and ALDH1A3 in the tumoroids derived from gastric tumor tissues from The specific inhibition of both Wnt/β-catenin and Ras-ERK pathways on the suppression of CSCs involving the resistance and regrowth of GC was further confirmed using PDX tumors derived from a tumor with AR to FOLFOX. The second-line PDX tumors generated from first-line PDX tumors resistant to FOLFOX were not significantly suppressed by paclitaxel ,36. HoweIn this study, we identified increased levels of both β-catenin and RAS in GC and revealed their roles in CSC activation involving the resistance to chemotherapy and recurrence after an initial response to this treatment. Furthermore, the effectiveness of a compound that destabilizes both β-catenin and RAS for suppressing of GCs, especially those resistant to chemotherapies due to CSCs, was proven using PDX models. Overall, the therapeutic approach degrading both β-catenin and RAS could be a potential approach for suppressing GCs, particularly those showing resistance and reoccurrence after initial treatment with standard chemotherapies.The present study included 756 GC patients who underwent surgical therapy at Yonsei University College of Medicine during the period from 2000–2003. Tissue samples of primary GC were obtained from these patients with written informed consent for it to be used for research purposes. Clinical data were retrieved from each patient’s medical records. Ethical approval to perform this study was obtained from the Institutional Review Board of Severance Hospital, Seoul, South Korea (4-2015-0616). This study did not include any sensitive information regarding patient privacy or identifiable information. Thus, a patient permission form was not required.3 fragments; then, these fragments were subcutaneously implanted into their flanks. The drug treatment was initiated when the mean tumor volume reached between 100–150 mm3, and the mice were randomly assigned into specific treatment groups. The FOLFOX regimen was delivered with the following schedule: leucovorin (5 mg/kg/day) was injected intraperitoneally, followed by 5-FU (15 mg/kg/day) after 20 min once a day. Oxaliplatin (5 mg/kg/day) was injected intraperitoneally once a week. KYA1797K was injected intraperitoneally at a dose of 25 mg/mL/day daily, and paclitaxel was injected at 10 mg/mL once a week during 30 days. The sizes of the implanted tumors were measured three times a week using Vernier calipers, and the tumor volume was calculated as follows: (length × width2)/2. The mice were sacrificed, and the tumors were isolated, weighed, sliced, and fixed in formalin or liquid nitrogen for further analyses.The detailed protocol for PDX establishment was as described previously . Brieflyhttps://en.wikipedia.org/wiki/FASTQ_format) data selected only the human genome. The paired-end reads were aligned to a human genome reference (UCSC hg19) using TopHat2 version 2.0.12 (https://ccb.jhu.edu/software/tophat/index.shtml) [http://cole-trapnell-lab.github.io/cufflinks/) [q-value < 0.05 and |log2 fold change (FC)| > 1. The statistical overrepresentation test module in PANTHER version 10.0 [p-value < 0.05 were considered statistically significant. RNA-seq data for FOLFOX-treated PDX model (accession number: GSE128967) have been deposited in NCBI Gene Expression Omnibus database.We used Xenome software tools to classify Xenograft RNA-seq human data and mouse (mm9) genome reference . The clax.shtml) . The aliflinks/) , and Cufion 10.0  was used2 at 37 °C. All chemicals were dissolved in dimethyl sulfoxide  for the in vitro studies, except for 5-FU  and oxaliplatin , which were dissolved in phosphate-buffered saline (PBS). Unless otherwise indicated, cells were treated with KYA1797K at a concentration of 25 µM for 24 h.Human GC cells (MKN74 and NCI-N87) were purchased from the Korea Cell Line Bank. MKN74, and NCI-N87 cells were cultured in RPMI  containing 10% fetal bovine serum , 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco), and 5% CO1638NApc mice were trypsinized for single-cell culture, and mixed with Matrigel , and plated in 48-well plates. After polymerization of Matrigel, gastric culture medium (Advanced DMEM/F12 supplemented with B-27 Supplement (50×), N2, 1 mM N-acetylcysteine , and gastrin  containing growth factors ) was overlain. For the first two days after seeding, the medium was also supplemented with 10 mM ROCK inhibitor Y-27632 (Sigma Aldrich) to avoid anoikis. Growth factors were added every second day, and the entire medium was changed every two days. For passage, gastric organoids were removed from the Matrigel, mechanically dissociated, and transferred to fresh Matrigel. Passage was performed every week with a 1:5–1:8 split ratio.Isolated gastric tumors from 1638NApc mice were obtained from Jackson Laboratory . Mice were housed in a micro-ventilation cage system with a computerized environmental control system . Room temperature was maintained at 24 °C with a relative humidity of 40–70%. For mouse genotyping, the tail was used to extract genomic DNA; male mice were used for further animal experiments. For tissue preparation, stomach tissues from the mice were removed immediately after sacrificing the animals. The abdomens of the mice were cut open longitudinally and cleaned by flushing with PBS. After washing vigorously, resected tissues were fixed with 10% neutral formaldehyde (Sigma-Aldrich) for 24–48 h at 4 °C. Fixed tissues were embedded in paraffin according to standard procedures. The animal studies were approved and performed in accordance with the guidelines of the Institute of Health Guidelines for the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine .C57BL/6JCells were washed in ice-cold PBS  and lysed using radio-immunoprecipitation assay (RIPA) buffer containing 20 mM NaF, 1 mM sodium vanadate, 10 mM Tris-HCl at pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM PMSF, and protease inhibitor cocktail . The tissues were homogenized and dissolved in RIPA buffer, as described previously, and stored in a liquid nitrogen tank. Proteins were separated on a 6–12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose membrane . Immunoblotting was performed with the following primary antibodies: anti-pan-Ras monoclonal , anti-β-catenin , anti-p-ERK , anti-ERK , anti-p-Akt , and anti-β-actin . Horseradish peroxidase-conjugated anti-mouse  or anti-rabbit  secondary antibodies were used. Immunoblots were detected by enhanced chemiluminescence  using a luminescent image analyzer .2O2  was applied to sections to block endogenous peroxidase activity for 15 min. Before incubating sections with mouse primary antibody, mouse imunoglobuline G (IgG) was blocked using an M.O.M. Mouse IgG blocking kit (Vector Laboratories). Sections were incubated with anti-β-catenin, anti-pan-Ras, anti-ALDH1A3 or anti-CD44 primary antibody overnight at 4 °C, followed by incubation with biotinylated anti-mouse  or biotinylated anti-rabbit  secondary antibodies for 1 h at room temperature. The samples were then incubated in avidin biotin complex solutions (Vector Laboratories), stained with 3,3-diaminobenzidine  for maximum 5 min, and counterstained with Mayer’s hematoxylin . All incubations were performed in humid chambers. Signals were analyzed using a bright-field microscope . For IHC staining, 4-µm paraffin-embedded tissue sections were treated with citrate buffer (pH 6.0) and autoclaved for 15 min. The sections were then blocked with 5% bovine serum albumin and 1% normal goat serum  in PBS for 1 h for human and mouse tumor samples. For fluorescent IHC, sections were incubated with primary antibody , anti-pan-Ras monoclonal , anti-ALDH1A3 , or anti-CD44 ) overnight at 4 °C, followed by incubation with anti-mouse Alexa Fluor 488  or anti-rabbit Alex Fluor 555  secondary antibodies for 1 h at room temperature. The sections were counterstained with 4,6-diamidino-2-phenylindole  and mounted in Gel/Mount media . All processes were conducted in dark, humid chambers. A confocal microscope  was used to visualize the fluorescence signal at excitation wavelength of 488 nm (Alexa Fluor 488), 543 nm (Alexa Fluor 555), or 405 nm (DAPI). At least three fields per section were analyzed. Mean fluorescence intensities of the markers were measured at least three different tissue samples using NIS-Elements AR 3.1. . For peroxidase IHC analysis, 3.45% H3 cells/well, and NCI-N87 cells were seeded at a density of 1 × 104 cells/well in a 24-well plate. The cells were then treated with 5 or 25 µM KYA1797K or with control (DMSO) for 72 h. Next, 3--2-5-diphenyltetrazolium bromide  reagent was added to each well at a concentration of 0.25 mg/mL. After incubation for 1 h at 37 °C, insoluble purple formazan was obtained by incubating in 500 μL (24-well) or 200 μL (96-well) of DMSO for 20 min. The absorbance of the formazan product was determined at 590 nm every 24 h. For colony formation assays, cells were seeded in 12-well plates (500 cells/well for NCI-N87 and MKN74 cells). Control (DMSO) or KYA1797K was added to the cells with medium changes every three days until visible colonies formed. At the end of the experiment, cells were fixed in 4% paraformaldehyde (PFA) for 30 min and stained with 0.5% crystal violet in 20% ethanol for 30 min.To assay cell proliferation, MKN74 cells were plated at a density of 8 × 10t-tests, and statistically significant p-values are presented as follows: * P < 0.05; ** P < 0.005; and *** P < 0.0005.Statistical analyses were performed using Microsoft Excel or GraphPad Prism 5 software . All data are represented as the mean ± standard deviation. For the TMA analysis of the AGC patients, statistical analysis was also performed using GraphPad Prism 5 software. The associations between β-catenin and pan-RAS staining were evaluated by the chi-squared test . The staChemotherapy resistance and disease recurrence remains major causes of gastric cancer  patient


Biobased nanofillers, such as cellulose nanofibrils (CNFs), have been widely used as reinforcing fillers for various polymers due to their high mechanical properties and potential for sustainable production. In this study, CNF-based composites with a commercial biobased epoxy resin were prepared and characterized to determine the morphology, mechanical, thermal, and barrier properties. The addition of 18–23 wt % of CNFs to epoxy significantly increased the modulus, strength and strain of the resulting composites. The addition of fibrils led to an overall increase in strain energy density or modulus of toughness by almost 184 times for the composites compared to the neat epoxy. The addition of CNFs did not affect the high thermal stability of epoxy. The presence of nanofibrils had a strong reinforcing effect in both glassy and glass transition region of the composites. A significant decrease in intensity in tan δ peak for the epoxy matrix occurred with the addition of CNFs, indicating a high interaction between fibrils and epoxy during the phase transition. The presence of highly crystalline and high aspect ratio CNFs (23 wt %) decreased the water vapour permeability of the neat epoxy resin by more than 50%. Epoxy resins are a class of polymers that exhibit high strength, stiffness, and creep resistance, as well as dimensional and thermal stability. They have been widely used in coatings, as well as in composites, construction, adhesives, electronics, and electrical applications ,2. Most Despite all the good properties of biobased epoxy systems, they are inherently brittle. Epoxy resins fail under less vigorous conditions when compared to other engineering polymers due to its high crosslink density ,5. As a Biobased nanofillers, such as cellulose nanofibrils (CNFs) have widths in nano-dimensions and high aspect ratio (length-to-width ratio). In plant fibers, several cellulose molecules are bundled together by strong hydrogen bonding to form elementary fibrils or microfibrils. These microfibrils or aggregates of microfibrils with diameters less than 100 nm are called as CNFs ,18. InteSeveral studies have reported the use of CNFs to enhance the mechanical properties of synthetic or petroleum based thermoset resin ,24,28,29The epoxy investigated is a commercial biobased epoxy resin, BioPoxy 36, supplied by Ecopoxy Inc. . A medium hardener was the curing agent obtained from the same manufacturer. Cellulose nanofibril suspensions were obtained from the Center for Biocomposites and Biomaterials Processing, University of Toronto, Toronto, Canada. The cellulose content for the CNFs were 83% (based on dry weight), and the average fibril diameter was approximately 10–25 nm. The degree of polymerization and crystallinity for the CNFs were 1200 and 71%, respectively. All other chemicals were obtained from Caledon labs .CNF suspension at 0.1 wt % solid consistency was vacuum filtered through a membrane  in a Buchner funnel. The wet films obtained after dewatering the suspensions along with the membranes were placed between blotting papers and pressed at 345 kPa for 6 min each. Some of the films were removed and dried at 50 °C under a pressure of approximately 23 kg for two days. A separate batch of wet films was then solvent exchanged to acetone by soaking in acetone for 48 h. The dried as well as solvent exchanged films were cut into dog-bone shape tensile specimens according to ASTMD 638-Type V specifications.The cellulose nanofibril network obtained by vacuum filtration was solvent exchanged with acetone before impregnating with epoxy. The solvent exchange is done to prevent aggregation of fibrils. The epoxy resin consisted of epoxy/curing agent mixed in a ratio of 4.29:1 by weight. To reduce the viscosity of resin, the epoxy resin was diluted with acetone in three different ratios: higher viscosity (epoxy and acetone at a weight ratio of 3:1); medium viscosity (1:1 ratio); and lower viscosity (1:3 ratio). Solvent exchanged CNF sheet specimens were immersed in each of these epoxy resin solutions. The impregnated specimens were removed from the solutions and suspended using fine metal wires in an oven for two hours prior to the curing. In addition to impregnated CNF specimens, neat epoxy films were solvent casted in aluminum pans. The specimens were then cured in the oven for 2 h at 120 °C. The cured samples were analyzed for various physical properties.−1 were used. Dynamic mechanical analysis (DMA) were carried out using a TA Instruments Q800 analyzer  in tension mode. Rectangular test specimens were 6–7 mm wide and the span was 15 mm. The frequency and amplitude of oscillation were set at 1 Hz and 15 μm, respectively. The temperature varied from room temperature to 180 °C at a heating rate of 3 °C/min.The dried CNF films, neat epoxy films and composites were tested according to ASTM D 638-Type V specifications. Tensile tests were performed using an Instron machine  equipped with a 2 kN load cell, in which a gauge length of 2.5 cm and a cross head speed of 10 mm minonset for the sample was defined as the temperature at which significant weight loss begins and determined using the tangent method. The Tmax, defined as the temperature at which the maximum weight loss rate occurred, corresponds to the maximum value of the derivative weight curve (dm/dTmax).The thermal stabilities of the CNF films, neat epoxy films, and composites were obtained using a TGA Q500  equipped with platinum crucibles. Samples (8−10 mg) were heated at a constant rate of 10 °C/min from room temperature to 800 °C in nitrogen atmosphere. The TWVT was determined according to ASTM E-96-95 water method. The test conditions were set to 23 °C and 50 % relative humidity (RH). The CNF films, neat epoxy and composite films were cut into 6.4 cm diameter specimens and conditioned to test conditions. The specimens were used to seal a test dish containing 50 mL of distilled water. The weight change of the test dish was recorded periodically and WVT was determined from the slope of weight change versus time, using the following equation:Here, ΔG is the weight change of dish, Δt is the time during which the change occurred, and A is the test area (dish mouth area).−1 at a resolution of 4 cm−1 after 64 scans. Transmittance spectra of the CNF, epoxy and composite films were obtained in the visible region (300–800 nm) using UV–VIS spectrophotometer .Scanning electron microscopy (SEM) images of the cross-sectional fracture surfaces of the CNF, epoxy and composite films were obtained using a JEOL 6610LV  operated at 15 kV. Samples were mounted on an aluminum stub using carbon tapes and sputtered with gold to provide adequate conductivity. Attenuated total reflectance−Fourier transform infrared spectroscopy (ATR-FTIR) was used to obtain spectra of CNF film, neat epoxy, and composites . The sample spectra were collected from 600 to 4000 cmTwelve to fifteen specimens were tensile tested to obtain the average mechanical properties of pure CNF, neat epoxy and composite films. A graphical representation of the change in mechanical properties of composites with varying CNF weight percent will be obtained. Three specimens from each group  will be analyzed for TGA, DMA, WVT, FTIR, and UV–VIS transmission spectra.A series of composites were prepared by impregnating the pure CNF films in different epoxy/acetone solution ratios. The average thickness of CNF film ranged from 55–60 µm a. The imThe medium viscosity led to good impregnation and resulted in composites with different CNF contents ranging from 18 to 23 wt %. Furthermore, the experimental data were compared with the predicted values using the rules of mixture (ROM). The predicted values were obtained using the following equations:composite and σcomposite are the predicted modulus and tensile strength for the composites, respectively. Ef, Em, σf, σm is the tensile modulus of CNF, tensile modulus of matrix, tensile strength of CNF, and tensile strength of matrix, respectively. Vf denotes the volume fraction of CNF. The tensile property of pure CNF film , neat epoxy  obtained from f = 13.7%) were 84.08 MPa and 3.02 GPa, respectively. The predicted strength and modulus values for 23 wt % loading (Vf = 17.77%) were 90.49 MPa and 3.24 GPa, respectively. The experimental values (composite = 3.31 GPa and σcomposite = 97.11) and 23 wt % (Ecomposite = 3.79 GPa and σcomposite = 118.7 MPa) were higher than the predicted values. Here, El values  for 18 wCellulose molecules show high polarity and, therefore, need chemical functionalization of the CNF to have a significant reinforcement effect on the epoxy matrix . A good −1 (O-H stretching), 1161 cm−1 (C-O-C asymmetric stretching), 990 cm−1 and 1056 cm−1 (C-O stretching), 1369 cm−1 (symmetric C-H deformation), 1425 cm−1 (asymmetric C-H deformation) associated with various polysaccharides (cellulose and hemicellulose) [−1 (O-H stretching), 1607 cm−1 (C=C stretching in phenyl groups), 1036 cm−1 (C-O symmetric stretching in ether groups), 1334 cm−1 (C-H deformation in epoxide groups), 1295 cm−1 (C-H vibration in epoxide groups), and 915 cm−1 (in-plane asymmetric deformation of epoxide ring) [−1, related to the epoxide/oxirane rings in the resin. The CNF has high surface area with enormous amount of hydroxyl groups, which reacts with epoxides forming stable ether linkages [llulose) ,36. The de ring) ,37,38. Fde ring) ,35. Complinkages . In addilinkages . Figure linkages .onset and Tmax of 286 and 321 °C, respectively. Despite being abundant and sustainable, the CNFs are still limited in composite industry mainly due to its low thermal stability [onset and Tmax of 323 and 355 °C, respectively. The composites with 23 wt % CNF loading showed a Tonset and Tmax of 308 and 345 °C, respectively. The decrease in Tonset and Tmax was only 5% and 3%, respectively, in the composite compared to the pure epoxy. The results are impressive, given the fact that we have added high amount of cellulose nanofibrils in the composite. The high thermal stability for the composite can be attributed to effective interaction between fibrils and epoxy [g) at 91 °C did not show any significant difference between the epoxy and the composite. However, a significant decrease in intensity in tan δ peak for the epoxy matrix occurred with the addition of CNFs. The results imply that there was high interaction between fibrils and epoxy during the phase transition [tability . Howeveransition .A strong reinforcement of CNFs on the epoxy polymer matrix was quite evident from different loading of CNFs (18–23 wt %). The average tensile modulus increased from 2.28 to 3.56 MPa, the tensile strength increased from 62.5 to 108.25 MPa, and the strain increased from 2.92% to 4.81%, which led to an overall increase in modulus of toughness by 184 times for the composites compared to the neat epoxy. The addition of fibrils also decreased the water vapor permeability of the neat epoxy, increasing the water vapour barrier efficiency of resulting composites by more than fifty percent. All these results indicate an effective interaction between the CNFs and epoxy used in this study. Also, the addition of substantial amount of cellulose fibrils (23 wt %) in the resulting composites did not affect the high thermal stability of the epoxy matrix. With improved mechanical properties, the commercial biobased epoxy resin reinforced with CNFs obtained in our study has great potential for various structural applications. The use of biobased epoxy and the addition of CNFs also highlight their environmental suitability. The low water vapour permeability obtained by the addition of CNFs in the epoxy are favorable attributes and can be extremely desirable for other applications such as coating or adhesives.