Alleviation of liver injury by hucMSC-Ex was determined. We further explored the production of oxidative stress and apoptosis in the development of liver injury and compared the antioxidant effects of hucMSC-Ex with frequently used hepatic protectant, bifendate (DDB) in liver injury. hucMSC-Ex alleviated CCl4-induced acute liver injury and liver fibrosis and restrained the growth of liver tumors. Decreased oxidative stress and apoptosis were found in hucMSC-Ex-treated mouse models and liver cells. Compared to bifendate (DDB) treatment, hucMSC-Ex presented more distinct antioxidant and hepatoprotective effects. hucMSC-Ex may suppress CCl4-induced liver injury development via antioxidant potentials and could be a more effective antioxidant than DDB in CCl4-induced liver tumor development.Mesenchymal stem cells (MSCs) have been increasingly applied into clinical therapy. Exosomes are small (30–100 nm in diameter) membrane vesicles released by different cell types and possess the similar functions with their derived cells. Human umbilical cord MSC-derived exosomes (hucMSC-Ex) play important roles in liver repair. However, the effects and mechanisms of hucMSC-Ex on liver injury development remain elusive. Mouse models of acute and chronic liver injury and liver tumor were induced by carbon tetrachloride (CCl The imbalance between oxidant and antioxidant results in oxidative stress. Most chronic liver diseases, such as alcoholic liver disease, nonalcoholic fatty liver disease, liver fibrosis, and viral hepatitis, possess the increased oxidant stress . Even inMesenchymal stem cells (MSCs), with multilineage differentiation potential and self-renew ability, have given rise to interests in the potentials of repairing tissues. Increasing researches have taken advantage of MSCs to cell-based clinical trials for numerous diseases including liver diseases . It is rExosomes (30–100 nm) are small membrane-bound vesicles derived from multivesicular bodies, which can be secreted by a wide variety of cells and contain proteins, mRNAs, and noncoding RNAs as cargos being transferred to other cells , 16. Exo4-induced acute and chronic liver injury and liver tumor growth in mice. Furthermore, we compared the antioxidative and antiapoptotic effects of hucMSC-Ex with that of bifendate (DDB) in CCl4-induced acute liver injury.Bifendate, a synthetic intermediate of schisandrin C, was found to protect against drug-induced liver injury in animals and is now used clinically for the treatment of hepatitis . In this2. Human normal hepatic L02 cells (Chinese Academy of Science) were maintained in RPMI 1640 containing 10% FBS  at 37°C with 5% CO2.Fresh umbilical cords were harvested from informed, consenting mothers and processed within 6 h according to the experiment protocols approved by Jiangsu University 2012258) as previously described [ as previ2O cushion and ultracentrifuged at 100000 ×g for 2 h at 4°C. Exosomes were gathered from the bottom of the tube and washed with PBS for three times by centrifugation at 1000 ×g for 30 min using a 100 kDa MWCO . Exosomes were finally filtrated on a 0.22 μm pore filter  and stored at −80°C. Concentration of concentrated exosomes was determined by nanoparticle tracking analysis (NTA) . Exosomes were also identified by transmission electron microscopy  for the morphology and the size and ImageStreamX Imaging Flow Cytometer  for exosomal markers, CD9 and CD63.Exosomes were isolated and purified as described previously . Cell-co4 dissolved in mineral oil, and the final dose was 0.3 ml CCl4/kg body weight. The normal group without any treatment was used as control (n = 6). Mice with liver tumor were induced with CCl4 every 3 days for 8 months and then treated with PBS (n = 6) or hucMSC-Ex (n = 6) via the tail vein. At 1 month after treatment, livers were harvested from sacrificed mice for further analysis. Mice for establishing liver fibrosis were treated with CCl4 every 3 days for 5 months and randomized into 2 groups for treating with PBS (n = 10) or hucMSC-Ex (n = 10). The dose of hucMSC-Ex was 6.4 × 109 particles per mouse diluted in 330 μl PBS. One month later, mice were sacrificed to collect livers. Mice for acute liver injury were injected with CCl4 twice for an interval of 3 days. For the analysis of hucMSC-Ex in acute liver injury, mice were randomized into 2 groups for treating with PBS (n = 10) or hucMSC-Ex  by tail vein administration. For the comparison of antioxidative ability between hucMSC-Ex and DDB, mice were randomized into 2 groups for treating with hucMSC-Ex or DDB by intragastric administration at 24 h post-CCl4 injection. In each group, mice were divided into 3 groups randomly with the doses of hucMSC-Ex at 6 × 1010 particles/kg (n = 10), 1.2 × 1011 particles/kg (n = 10), and 2.4 × 1011 particles/kg (n = 10) or DDB at 8 mg/kg (n = 10), 16 mg/kg (n = 10), and 32 mg/kg (n = 10). At another 24 h, mice were sacrificed for further analysis.BALB/c female mice aged 4-5 weeks were purchased from the Laboratory Animal Center , and all experiment procedures were in accordance with the Chinese legislation regarding experiment animals. All the models were induced by intraperitoneal injection with 10% CCl5 cells/well and were cultured with medium containing 0.1 mM CCl4/hucMSC-Ex  for 24 hr. After hucMSC-Ex treatment, L02 cells were collected for further dectection.L02 cells were seeded in six-well plates at 1 × 104-induced mice via the tail vein. After injection for 24 h, mice were imaged using a Maestro in Vivo Imaging System (CRI) to observe the distribution of exosomes.According to the manufacturer's instructions, exosomes were incubated with the cross-linkable membrane dye, CM-DiR , for 30 min at 37°C in the dark. After washing with PBS, the labeled exosomes were concentrated with a 100 kDa MWCO  at 1000 × g for 30 min at 4°C to remove nonbinding dye. Then CM-DiR-labeled exosomes were injected into CClμm sections. The sections were stained with hematoxylin and eosin, Sirius red , and Masson trichrome (MT)  in accordance with standard protocols. To analyze the extent of liver fibrosis, randomly picked fields of MT sections were captured from each animal.Liver tissues were fixed in 4% formaldehyde solution at room temperature overnight, embedded in paraffin, and cut into 4 Following deparaffinization and rehydration, the liver slides were steamed in citrate buffer  for 30 min for antigen retrieval and exposed to 3% hydrogen peroxide for 30 min for inhibiting endogenous peroxidase activity. Slides were then blocked in 5% bovine serum albumin for 1 h and incubated with the primary antibodies against SOX9 , 8-OHdG (Japan Institute for control of aging), activated caspase 3, Bax, and PCNA  overnight and then incubated with secondary antibody for 30 min at 37°C. Finally, slides were visualized with 3,3′-diaminobenzidine and counterstained with hematoxylin for microscopy examination (200x).w/v) skim milk for 1 h and incubated with the primary antibodies against CD9, CD63, Bcl2 , cleaved Casp3 , and GAPDH  at 4°C overnight and then incubated with HRP-conjugated goat anti-rabbit antibody for 1 h at 37°C. The signals were detected with a luminata™ crescendo Western HRP substrate  quantitated by a Molecular Dynamic Densitometer (Sage Creation Science) with LANE 1D software.hucMSC-Ex were lysed in RIPA buffer. Equal amount of protein was loaded and separated on a 12% SDS-PAGE gel. After electrophoresis, protein was transferred to PVDF membranes. The transferred membranes were blocked in 5%  and total protein concentration with a BCA assay kit . MDA levels were normalized to milligrams of protein.β levels in fibrotic liver tissues treated with PBS or hucMSC-Ex were determined using an ELISA kit  according to the manufacturer's instructions.TGF-The apoptotic cells in liver slides were measured by using an in situ cell apoptosis kit  according to the manufacturer's instructions.Total RNA of mouse livers was extracted with the Trizol reagent according to the manufacturer's instructions . The cDNA was synthesized using Super Scrispt™ RT kit according to the manufacturer's instructions . The sequences of primers are shown in Cellular reactive oxygen species (ROS) of L02 cells was measured with a 2′-7′-dichlorofluorescein diacetate (DCF-DA)  staining according to the instruction. Percentage and fluorescence intensity of DCF-positive cells were detected with the ImageStreamX Imaging Flow Cytometer  and Olympus Fluorescent Microscope, respectively .t-test (two-tailed) using Prism software . P value < 0.05 was considered significant.Data are expressed as the means ± standard deviation (SD). Statistical significance was assessed by Student's To characterize hucMSC-Ex, primary hucMSCs were cultured in exosome-free media . The exo4-induced mouse liver tumor models were established and hucMSC-Ex was infused into the mouse intravenously. In vivo fluorescent imaging showed that CM-DiR labeled hucMSC-Ex mainly located at the liver at 24 h postinjection . Hematoxylin and eosin (H&E) staining confirmed reduced areas of inflammation infiltration after hucMSC-Ex treatment .Liver tumors develop in the context of chronic liver diseases such as CClcontrols . In the controls . Moreoveβ in livers were also reduced in the hucMSC-Ex group . Thus, hucMSC-Ex may have potentials of oxidation resistance and antiapoptosis in liver fibrosis.To confirm the abilities of hucMSC-Ex against oxidative stress and apoptosis in liver fibrosis, 8-OHdG and activated caspase 3 were detected by immunohistochemistry. Compared to PBS, hucMSC-Ex significantly inhibited activated caspase 3 and 8-OHdG production in mouse liver fibrosis models . The lev4. The decrease of 8-OHdG production in an injured liver was found at 24 h after treatment with hucMSC-Ex . Activated caspase 3 and BAX have been shown to be involved in cell apoptosis. Then we explored the expression of activated caspase 3 and BAX. Results of Bax and activated caspase 3 staining showed cell apoptosis-associated gene expression was decreased after hucMSC-Ex treatment . TUNEL staining also revealed that hucMSC-Ex treatment could inhibit the apoptosis in liver injury . Therefore, hucMSC-Ex can reverse oxidative stress-induced apoptosis in CCl4-induced acute liver injury.To further assess the antioxidative and antiapoptotic effects of hucMSC-Ex, they were injected intravenously into mouse models with acute liver injury induced by CCl4. To identify the antioxidant ability of hucMSC-Ex, different doses of hucMSC-Ex  and DDB  were given 24 h after administration of CCl4. We observed more integrated hepatic tissue structure and less hepatic lobule destruction in the hucMSC-Ex group at 1.2 × 1011 particles/kg compared with the DDB group at 16 mg/kg. Expression of 8-OHdG and activated caspase 3 was dose-dependently decreased in both the hucMSC-Ex group and the DDB group . Compared with the PBS group, hucMSC-Ex (2.4 × 1011 particles/kg) exerted a more distinct inhibition effect on the expression of 8-OHdG and activated caspase 3 .Bifendate (DDB) is frequently used in clinical therapy and can alleviate liver damage induced by CClin vitro, ROS level of the L02 cells were detected by using a DCF-DA probe. Results of imaging flow cytometry and fluorescence microscope showed that the percentage and fluorescence intensity of DCF-positive hepatocytes decreased after hucMSC-Ex treatment . hucMSC-Ex treatment also promoted the expression of proliferating cell nuclear antigen (PCNA)   and inhin (PCNA) . These fd injury .The capacities of MSCs including differentiation, immunomodulation, and bioactive molecule release determine the potentials to treat organ diseases induced by tissue injury or degeneration. A series of clinical trials was performed on patients with diseases such as liver cirrhosis and acute-on-chronic liver failure, which have demonstrated that hucMSC improved liver function and increased survival rates , 28. Inc4 is a hepatotoxic chemical and is frequently used to induce liver injury. After 8-month injection of CCl4, we successfully established a mouse model of liver tumor. Our data showed that after administration of hucMSC, the average volume of liver tumors was distinctly diminished. Although doses of 250 μg exosomes were enough to ameliorate liver fibrosis and 200 μg exosomes to improve wound healing, we still chose the high dose of hucMSC-Ex to transplant to observe the tumor-suppressive function with the consideration of severity of injured tissues in liver tumor.CClβ levels which are always induced in damaged livers and trigger hepatocyte destruction and hepatic stellate activation [Approximately 90% of liver tumors were developed in liver cirrhosis accompanied with chronic inflammation in which oxidative stress plays an influential role . So far,tivation . With retivation . In in vIn spite of our understanding about the hepatoprotective effects of hucMSC-Ex, we still did not know well these potencies compared with other medicines. As is known to all, DDB, a synthetic intermediate of schisandrin C, is used to treat hepatitis with minimal side effects and is always regarded as positive control when exploring hepatoprotective actions , 38. In In conclusion, we unraveled that hucMSC-Ex presented hepatoprotective activities through antioxidant defenses in the disease progression from initial liver injury to fibrosis and even to liver tumor. During the pathogenesis and regression of liver diseases, nevertheless, there are multifarious hepatotropic networks concerning tissue regulation and homeostasis , and henhucMSC-Ex may suppress liver injury development via antioxidant potentials and could be a more effective antioxidant than DDB in liver injury.


Impregnation of expandable graphite (EG) after thermal treatment with an epoxy resin containing surface-active agents (SAAs) enhanced the intercalation of epoxy monomer between EG layers and led to further exfoliation of the graphite, resulting in stacks of few graphene layers, so-called “stacked” graphene (SG). This process enabled electrical conductivity of cured epoxy/SG composites at lower percolation thresholds, and improved thermo-mechanical properties were measured with either Kevlar, carbon or glass-fiber-reinforced composites. Several compositions with SAA-modified SG led to higher dynamic moduli especially at high temperatures, reflecting the better wetting ability of the modified nanoparticles. The hydrophilic/hydrophobic nature of the SAA dictates the surface energy balance. More hydrophilic SAAs promoted localization of the SG at the Kevlar/epoxy interface, and morphology seems to be driven by thermodynamics, rather than the kinetic effect of viscosity. This effect was less obvious with carbon or glass fibers, due to the lower surface energy of the carbon fibers or some incompatibility with the glass-fiber sizing. Proper choice of the surfactant and fine-tuning of the crosslink density at the interphase may provide further enhancements in thermo-mechanical behavior. Carbon nanotubes (CNTs) have been suggested as an efficient conductive filler because of the outstanding electrical properties and the high aspect ratio. CNT-modified carbon epoxy composites have been studied, where the CNTs are either dispersed in the matrix and/or grafted on the carbon fibers –2. Howev3/g. For instance, the increase to about 200 cm3/g caused a thickness increase of about 80–100 times by a thermal shock at 600 °C [Graphite nanoplatelets (GNPs) or stacked graphene (SG) have been developed as a low-cost conductive alternative . GNPs ca3/g at 900 °C was obtained by exfoliating graphite treated by a mixture of H2SO4/HNO3/KMnO4 at a ratio of 1:9:3:0.44 over an immersing time of 150 min in formic acid [3 followed by hydrolysis resulted in the the formation of graphite nitrate with negligible damage to the sp2 graphite lattice. An interlayer distance of 0.336 nm was measured. Nevertheless, numerous multi-pores ranging from 2 to 10 nm were also detected. The acid and hydroxy groups on the multi-pore walls promote the interaction of EG with organic compounds, more specifically, with monomers before curing and with polymer segments after curing, thus contributing to the mechanical properties and percolation threshold reduction of conductive polymer/graphite nanocomposites [Further size reduction of these large  structures produces fine particles consisting of a few layers of graphene loosely joined together, i.e., SG or GNP, also called few layered graphene (FLG). The exfoliation volume is governed by the structure of the starting expandable graphite, exfoliation temperature and heat rate . The larmic acid . Intercamposites –12.Graphite intercalation compounds (GIC) may provide a plausible high-yield source for polymer nanocomposites . HoweverLow percolation thresholds have been demonstrated in polymer compositions with nanosized fillers. The high aspect ratio and large surface area of graphene, along with the high electrical conductivity, promote percolation thresholds much lower than with metallic powders, carbon fibers or carbon black .Epoxy resins are used as a matrix in high-performance composite materials for aerospace structures, coatings and adhesives for a variety of applications. Epoxy systems combined with reinforcing fibers provide composites with high strength and stiffness, ease of molding complex shapes and environmental resistance at low densities. The properties of epoxy systems can be varied as a function of the molecular weight or the functionality of the hardener constituent, by variations in processing conditions or by changing the ratio between hardener and monomer. Incorporation of functional silanes can improve the dispersibility of fillers in an epoxy system and increase the mechanical properties of the cured resin. The combination of SG and epoxy fiber composites was hardly studied. Nevertheless, it may offer special properties by reinforcing the epoxy matrix and providing higher electrical conductivities depending on the localization of the SG. Consequently, the present investigation deals with epoxy-based conductive compositions containing treated stacked graphene and continuous fabric reinforcement based on carbon fibers, Aramid and glass fibers.MW = 4000, hydroxyl number: 28 mg KOH/g, (Grade 4200 from Bayer)), octylphenol ethoxylate , as well as 3-methacryloxypropyltrimethoxysilane (MEMO) and (3-glycidyloxypropyl)trimethoxysilane (GLYMO) (Sigma-Aldrich); fabrics used: 3k carbon fiber of plain weave, 200 g/m2 (Primetex ZB type 43199 from Hexcel), Kevlar of plain weave, 450 g/m2 (Type 745) and fiberglass fabric of plain weave, 300 g/m2.Diglycidyl ether of bisphenol A (DGEBA)  with epoxy equivalent weight (EEW) 182–192; triethylenetetramine (TETA) hardener, amine equivalent weight 24 (LEUNA-Harze GmbH); graphite intercalated compound (GIC) of 30–50 mesh, 3.1% sulfur ; surface-active agents (SAAs) for SG treatment used in this study: polyether polyol  was subjected to thermal shock at 600–620 °C in a preheated muffle furnace in air, with further heating of the expanded material for 15–20 min to perform homogeneous exfoliation of the graphite sheets into stacked graphene (SG) consisting of layered graphene structures.The properties of epoxy-based compositions with various concentrations of EG incorporated by different techniques were investigated. The principle method comprised the following steps: impregnation at elevated temperatures of EG in epoxy resin premixed with SAAs, further size reduction of EG worm-like particles using intensive mixing, and degassing of the paste-like epoxy/SG compositions in vacuum to remove the entrapped air. Two nonionic surfactants (Triton X100 and X-15) with different hydrophilic/lyophilic balance (HLB) were used in order to evaluate the thermodynamic effect on the localization of the SG. Hardener TETA was added to the mixture before specimens casting.The basic formulation (by weight) was as follows: epoxy: 100 parts; SAA: 1 pph; expanded graphite (EG): 2.5 pph (or 2.1 wt %); hardener TETA: 14.1 pph.Composite laminates were prepared using carbon fiber (CF) fabric, Kevlar fabric alone or a combination of CF/Kevlar combinations, and fiberglass (FG) fabrics. CF was used as received, and Kevlar was cleaned by dipping in isopropyl alcohol (IPA) with further drying.FG was used as received or modified by silanes and surfactants. FG specimens (150 × 150 mm) were dipped in solutions of MEMO and GLYMO silanes in IPA/water. The silane/IPA/water ratio was 2:90:10. Acetic acid (2 g) was added to adjust the pH value of the solution to 5.0. Wet specimens were rinsed with IPA, dried at room temperature and finally cured at 120 °C for 30 min.Specimens of FG fabrics were heat-treated at 600 °C for 2 h to burn out the sizing material and dried at 150 °C just before dipping into a variety of treatment solutions. Solutions of (3-aminopropyl)triethoxysilane (AMEO), Triton X-15 and TETA in IPA were used as modifiers of the FG surface.Laminates were fabricated by the hand lay-up technique using brush and roller to apply the matrix composition on the fabric plies. The samples were designed to provide a thickness of 2.5–3.0 mm. The fiber volume fraction was controlled by applying pressure to the laminates to a predetermined thickness. The samples were cured under pressure for two days at ambient temperature, and after that post-cured at 80 °C for 4 h and at 120 °C for 2 h.The thermo-mechanical properties of the compositions were measured at 1 Hz according to ASTM D 4065 using a DMA-Dynamic Mechanical Analysis (Q800 TA Instruments). Flexural testing followed ASTM D790 (3 point bending) at 1.3 mm/min. Electrical resistivity, both surface and volume, was determined with an electrometer (Keithley 6517D). Typically, the characterization comprised five specimens with standard deviations of 5–8%. Morphology of the composite materials was characterized with high-resolution scanning electron microscopy (Zeiss). Specimens were cut from samples frozen in liquid nitrogen.The electrical, mechanical and thermo-mechanical properties of composite materials prepared with Kevlar fabrics are exhibited in Table S1 . LaminatFor compositions in which epoxy resin was modified by hydrophobic Triton X-15 or GLYMO the fiber coverage is less pronounced. Some uncovered sections of Kevlar filaments can be observed in SEM images of composite materials based on carbon fabrics in All laminated composites manifested increased DMA storage moduli. Composites with modified epoxy/SG matrices demonstrated a significant increase of storage moduli at elevated temperatures, and higher Young’s moduli 3,asymmetric structure: (0/90 CF)2/(0/90 Kev)1/(0/90 CF)1/(0/90 Kev)1/(0/90 CF)2.symmetric structure: (0/90 CF)Tg evaluated from the tan δ values of the DMA showed some scatter but remained at roughly the same level as the unmodified resin system.The properties of the composite materials are presented in Table S3 and Table S4 . LaminatThe properties of laminated composite materials prepared with the second configuration are demonstrated in Table S4 . The intSurface resistivities of the asymmetric laminate in Table S3 demonstrate the ability to control one-sided conductivity or gradient conductivities of up to three orders of magnitude in a composite structure. Surface resistivities for both symmetric and asymmetric laminates are expectedly similar to the all-carbon laminates.Table S4 indicates that, again, Kevlar plies interleaved between carbon plies do not prevent volume conductivity, especially with Triton X-15 and MEMO surfactants where resistivity is only one order of magnitude higher than the all-carbon laminates.SEM images of FG/epoxy-laminated composites indicated no marked differences in the morphology of composite materials with the investigated SG modified matrices. The epoxy based compositions do not provide sufficient wetting and coverage of fiberglass filaments, even when surfactants were added .The properties of laminated composite materials based on FG fabrics are summarized in Table S5 . As can Experiments aimed at modification of the FG surface, using silanes with either acrylic or epoxy functionalities, in order to provide better wetting of filaments, were carried out, as explained in detail in the Experimental section. Examination of morphology and mechanical properties of laminated materials did not reveal any substantial changes in the performance of the laminates . It can Further FG treatments were carried out with (3-aminopropyl)triethoxysilane (AMEO), triethylenetetramine (TETA), and the non-ionic SAAs Triton X-100 and Triton X-15 dissolved in IPA at various concentrations. Results of electrical and thermo-mechanical tests are shown in Table S6 . As can Interesting results were observed with the non-ionic hydrophobic SAA Triton X-15. Addition of this SAA to the epoxy/SG matrix blend, as well as the modification of FG with 1% solution of Triton X-15 led to improved mechanical properties. The SAA concentration must be balanced an excess of this SAA during FG modification (2% solution) had a negative effect on the stress at yield.Improved thermo-mechanical properties of laminated FG composites were observed also with moderate concentrations of Triton X-100 applied for FG modification and incorporated into the matrix composition. Here, an excess of SAA in the FG fabric also results in a decrease of the stress at yield value. Increase in DMA storage moduli and stress at yield was noticed when FG was treated with TETA hardener. Further crosslinking at the interphase may take place when testing at high temperatures, explaining the higher modulus at 100 °C. Fine tuning of the epoxy composition at the interphase may turn out to be an interesting way to improve its properties, and there is some potential in this practice to improve properties.SEM images of composite materials based on modified FG are shown in Figures 5–7. Morphology of the composite materials in The unknown composition of the sizing material used for the FG filaments could be the reason for FG/matrix composition incompatibility. Hence, experiments were carried out to remove the original treatment in order to reveal an influence of selected FG impregnating material on the properties of laminated composites. Subsequently, composites based on heat-treated FG fabrics that were further modified with SAAs  demonstrated higher DMA storage moduli, especially at elevated temperatures as shown in Table S7 . FG modiThe exfoliation of commercially available expandable graphite (EG) is enhanced by impregnation in an epoxy/surfactant mixture, allowing on to downsize the nanoplatelets to "stacked" graphene (SG) or few graphene layers (FGL) platelets. The effect of such exfoliation on continuous-fiber-reinforced epoxy composites was studied in terms of electrical conductivity and thermo-mechanical properties at low loadings. Modification of epoxy resin by SAAs prior to EG impregnation enhanced intercalation of epoxy monomer between EG layers and further exfoliation. Morphology and compositions are reflected by the mechanical properties of composite laminates. Electrically conductive laminated composites, based on Kevlar or glass fibers, can be obtained by introducing treated stacked graphene at low concentrations.The morphology of composite materials based on Kevlar fabrics varies depending on the composition of the matrix. Laminates prepared with neat epoxy or unmodified epoxy/SG exhibited bare surfaces of Kevlar fibers. Modification of epoxy resin with Triton X-100 and methacrylate-functionalized silane (MEMO) resulted in improved wetting of the Kevlar filaments with the nanocomposite matrix. Enhanced rigidity in three-point bending and increased DMA storage moduli were achieved for composites where EG was impregnated with epoxy resin modified by Triton X-100, Triton X-15 and MEMO. All Kevlar-based laminates demonstrated improved electrical properties, indicating the possibility to obtain electrically conductive composite materials on the basis of electrically insulating fibers using binder matrices with low content of inexpensive SG. Both surface and volume resistivity dropped by 7–8 orders of magnitude.Laminated composites based on carbon fabric showed increased DMA storage moduli, improved thermal stability and increased Young's moduli. SEM images of SAA-modified epoxy/SG/carbon fabric composites revealed no distinctive effect of the epoxy composition on the morphology of laminates, yet the compounds demonstrated significant improvement in thermal stability at elevated temperatures, especially with a polyol surfactant. A reduction of electrical surface resistivity was registered, while volume resistivity remained at the level determined for the neat epoxy composite.Composite materials consisting of Kevlar/carbon fabric combinations with epoxy/SAA/SG manifested increase in DMA storage moduli and enhanced Young's moduli. Asymmetric layering of the Kevlar/carbon layers provided the possibility to form hybrid composite materials with regulated surface resistivity for the different external plies of the laminate.For fiberglass-based composites with various nanocomposite matrices no marked differences in the morphology were noticed. Epoxy-based compositions, even modified with SAA, do not provide sufficient wetting and coating of fiberglass filaments. DMA storage moduli and Young's moduli remained at the level of the neat epoxy matrix, or were even reduced slightly. Treatment of fiberglass fabrics using moderate concentrations (1–2%) of non-ionic SAA (Triton X-15 and Triton X-100), or TETA hardener in IPA allowed for improvement of mechanical and thermo-mechanical properties of FG composites. Electrical properties manifested significant improvement as surface and volume resistivities dropped by 7–9 orders magnitude.Thermomechanical properties of FG-reinforced laminated composite materials can be improved by removal of the commercial sizing and modification of the fiberglass surface using moderate concentrations (about 1%) of either AMEO, non-ionic Triton X-15 or even TETA hardener. We suggest to develop strategies to fine-tune the crosslink density at the interphase. A graded crosslink density transition from the fiber to the matrix may prove beneficial.Supporting Information contains all tables with the measured thermo-mechanical and electrical properties of all samples.File 1Thermo-mechanical and electrical properties.


Wincr1 and Wincr2. These two lncRNAs are highly expressed in mouse embryonic skin and perinatal dermal fibroblasts. Furthermore, we found that Wincr1 expression levels in perinatal dermal fibroblasts affects the expression of key markers of fibrosis , enhances collagen contraction, and attenuates collective cell migration. Our results show that β-catenin signaling-responsive lncRNAs may modulate dermal fibroblast behavior and collagen accumulation in dermal fibrosis, providing new mechanistic insights and nodes for therapeutic intervention.Wnt/β-catenin signaling is required for embryonic dermal fibroblast cell fate, and dysregulation of this pathway is sufficient to promote fibrosis in adult tissue. The downstream modulators of Wnt/β-catenin signaling required for controlling cell fate and dermal fibrosis remain poorly understood. The discovery of regulatory long non-coding RNAs (lncRNAs) and their pivotal roles as key modulators of gene expression downstream of signaling cascades in various contexts prompted us to investigate their roles in Wnt/β-catenin signaling. Here, we have identified lncRNAs and protein-coding RNAs that are induced by β-catenin activity in mouse dermal fibroblasts using next generation RNA-sequencing. The differentially expressed protein-coding mRNAs are enriched for extracellular matrix proteins, glycoproteins, and cell adhesion, and many are also dysregulated in human fibrotic tissues. We identified 111 lncRNAs that are differentially expressed in response to activation of Wnt/β-catenin signaling. To further characterize the role of mouse lncRNAs in this pathway, we validated two novel Wnt signaling- Induced Non-Coding RNA (Wincr) transcripts referred to as  Wnt/β-catenin signaling has a diverse role in both embryonic mouse skin development and in human skin diseases such as pilomatricomas, dermal hypoplasia, and fibrosis . β-catenCol7a1, Ccn3/Nov, Biglycan, and Matrix Metalloproteinase 16 (Mmp16), a subset of which are also up-regulated in human skin fibrosis and tumor stroma  Nagy (R26rtTA)Gt(ROSA)26Sor Jax labs stock: 005670 .b1Δex3/+ , Engrail(En1Cre) , Gt mice and minced . Dermal fibroblasts were infected with Adenovirus-Cre (366-500 MOI) or Adenovirus-GFP (366 MOI) (Adenovirus purchased from University of Iowa). After 48 h, recombination of Ctnnb1 Exon 3 was confirmed with site specific PCR under standard conditions. Primers Neo PMR (5′ AGACTGCCTTGGGAAAAGCG 3′) and Cat-AS5 (5′ ACGTGTGGCAAGTTCCGCGTCATCC 3′) were used to identify the targeted allele with a ∼500 bp amplicon and GF2 (5′GGTAGGTGAAGCTCAGCGCAGAGC 3′) and Cat-AS5 identified the recombined allele with an expected amplicon of ∼700 bp  per gram of body weight by intraperitoneal injection at embryonic day 12.5 (E12.5) and embryonic cranial and dorsal dermal skin were harvested at E13.5. In vitro stabilized β-catenin expression was induced by treating Engrailed1Cre/+; Rosa26rtTA/+; Teto-deltaN89β-catenin/+ P4 ventral dermal fibroblasts cells with 2 μg/mL doxycycline in complete growth media for 4 days. For induction-reversal experiments, duplicate cultures were then switched to complete growth media without doxycycline for 48 h prior to RNA isolation.For in vivo and dermal fibroblasts in vitro using TRIzol reagent (Thermo Fisher Cat. No. 15596026). RNA was isolated using the RNeasy MinElute kit (Qiagen Cat. No. 74204) with DNAse1 (Qiagen Cat. No. 79254) treatment, following the manufacturer’s protocol. RNA concentration and quality were measured using the NanoDrop 8000 UV-Vis Spectrophotometer.Total RNA was extracted from embryonic tissues P-value less than 0.05. Normalized mapped reads are available on Gene Expression Omnibus (GSE103870) using the private token from the editor.Libraries were prepared by the CWRU Genomics Sequencing Core, using the True-Seq Stranded Kit (Illumina). Paired-end sequencing was carried out on the Illumina HiSeq 2500 platform. Resulting 100 bp reads were mapped to the mm10 mouse genome release using TopHat and Cufflinks. Mapped raw reads were counted, normalized to total mapped reads, and used for differential gene expression (Cuffdiff and Seqmonk standard settings). Differential expression was defined as an absolute fold change greater than 2, and Benjamini–Hochberg adjusted Ct or ΔΔCt method where applicable  or HPRT (mm1545399_m1). Wincr1 isoform1  and Wincr2  quantities were measured relative to reference gene RPL32  using custom-designed primers (Invitrogen) and SyBr Green reagents(Invitrogen Cat. No. 4367659). Mmp10 and Col1a1 quantities  were measured relative to reference gene ActB expression, using TaqMan Gene Expression Master Mix (Thermo Fisher Cat. No. 4369016). Non-coding gene primers were designed using Primer3Plus1. Primer sequences for RPL32 and Mmp10 were acquired through the MGH Primer Bank Site2. Primer sequences for Col1a1 were identified previously . Relative mRNA quantities of select genes were determined using the Applied Biosystems StepOnePlus Real-Time PCR System (Life Technologies Cat. No. 4376600) and the Δplicable . In all eviously .t-test was used. In all other instances, an unpaired t-test was used. Significance for all purposes was defined as ∗P-value ≤ 0.05, ∗∗P-value ≤ 0.01, ∗∗∗P-value ≤ 0.001, ∗∗∗∗P-value ≤ 0.0001. Paired samples are shown as dots connected by a line where applicable. Expression across different tissues are shown as mRNA levels relative to reference gene (2-ΔCt). In all other plots, individual relative quantities (2-ΔΔCt) are shown along with mean and standard error of the mean.Statistics were performed using GraphPad Prism 7. For experiments in which cells from the same animal were used in control and experimental conditions, a paired P-value < 0.05] between GOF and control samples. Color was assigned to each sample on a basis of Z-score of fragments per kilobase mapped (FPKM) compared to other samples’ FPKM of the same gene (row Z-score). Heatmaps were clustered hierarchically based on the aggregate differential gene expression profiles using the DESeq2 package in R.Heatmaps were generated using all mRNA or lncRNA genes considered significantly differentially expressed . Gene lists were entered as Gene Symbols. All settings were default. The top five ranked clusters are shown, with clusters enriched among up-regulated genes shown as positive and clusters enriched among down-regulated genes shown as negative.DAVID Functional Annotation Clustering4 (Z-score and a Fisher score for overrepresentation. TFBS were then plotted according to these scores using GraphPad Prism 7. Thresholds were determined as follows: Z-score - Mean + (2 × Standard Deviation), Fisher score -Mean + Standard Deviation as previously described 4 . Gene liescribed .gene_set permutations were used. All other settings remained default.Gene Set Enrichment Analysis (GSEA) was used to identify known gene expression signatures similar to the differential expression of genes in GOF dermal fibroblasts . GSEA wa5. The intersection of this list with others was performed in Microsoft Excel.The human matrisome gene list was accessed through the MIT Matrisome Project at MatrisomeDBP-value < 0.05 and fold change > 2) and inclusion in the Matrisome gene list (see above). An FPKM table was constructed in Microsoft Excel consisting of these candidate genes as well as Axin2, Wincr1, and Wincr2. This table was loaded into Cytoscape as an unassigned table. The plugin “Expression Correlation” was used (with a Low Cutoff of -1 and a High Cutoff of 0.6) to generate a correlation network. The built-in tool “Network Analyzer” was used to further analyze the network for node degree. A custom style was created to visualize the network, in which edge color correlates with strength of correlation (red:high CC). Node size correlated with number of first neighbors (degree).Coexpression networks were constructed using Cytoscape version 3.4.0 . CandidaP-value < 0.05 were considered significantly differentially expressed.Microarray data from nine human studies of various fibrotic conditions were analyzed using Gene Expression Omnibus (GEO). Data were analyzed using the GEO2R tool. Genes with average fold change > 1.5 and adjusted Wincr1 was assembled from synthetic oligonucleotides and PCR products and subcloned into pMA-T . Wincr1 lentivirus was generated by subcloning Wincr1 from pMA-T into the Nco1-EcoRV region of pENTR4 (Thermo Fisher Cat. No. A10465) vector and then swapping it into the pGK destination vector (Addgene Cat. No. 19068). Lentivirus was generated in 293T (f-variant) cells (ATCC Cat. No. 3216) by simultaneously transfecting pGK Destination Vector containing Wincr1 (4.5 μg), PMD2G helper plasmid (1.6 μg) (Addgene Cat. No. 12259), and pCMV-dR874 plasmid (3.2 μg), using Lipofectamine 3000 reagent (18 μL) (Invitrogen Cat. No. L30000015) in 1.5 mL Opti-MEM (Thermo Fisher Cat. No. 31985062) media in a 6 cm dish. Cells were selected in Puromycin (2 μg/mL) (Sigma Cat. No. P8833). Virus was collected from 293T conditioned cell media at 24 and 52 h after transfection, clarified with 45 μm pore filter  and frozen. Lentivirus conditioned media was titered on 293T cells at 3.25–50% dilutions. After 3 days, infected cells were selected by treatment with Puromycin (2 μg/mL) for 3 days. Stable expressing P4 mouse ventral dermal fibroblast cells lines were propagated for further experiments. Wincr1 overexpression was confirmed by qRT-PCR as described.Full-length isoform 1 of Wincr1 knockdown was achieved with custom LNA-GapmeRs designed and synthesized by Exiqon6. Sequences targeting Wincr1 (GACTAGGATGATAGAT) and a negative (scrambled) control (AACACGTCTATACGC) were acquired. LNA GapmeRs were reconstituted to 50 μM in tissue culture-grade water as per manufacturer’s instruction. Transfection was carried out using Lipofectamine 3000 Reagent in Opti-MEM. Dermal fibroblasts were seeded in a 12-well cell culture treated plate and transfected in 500 μL of Opti-MEM containing 2 μL Lipofectamine 3000 and a final LNA GapmeR concentration of 50 nM. Active transfection was carried out for 6 h, at which time the Lipofectamine 3000 containing media was replaced with complete growth media. Additional LNA GapmeRs were added for a final concentration of 100 nM. After 48 h of unassisted transfection, cells were harvested and RNA was extracted and analyzed for gene expression changes as described above. The knockdown of Wincr1 expression was confirmed by qRT-PCR.To measure cell proliferation, a standard growth curve assay was performed on P4 ventral dermal fibroblasts between passage 4–6 as described above. 30,000 cells were plated in duplicate into a 12 well plate, with cell number being assessed using Trypan blue (Invitrogen/Gibco Cat. No. 1520061) exclusion on the Cell Countess (Invitrogen Cat. No. C10281). Collective cell migration was assessed using a qualitative scratch assay . BrieflyCtnnb1Δex3/+ with Adenovirus Cre (Ad-Cre) . Recombination of Exon 3 of Ctnnb1 produces a stabilized form of β-catenin protein, referred to as Gain of Function (GOF), which lacks the phosphorylation site for degradation and constitutively activates the canonical Wnt signaling pathway. Using site-specific primers, we confirm the consistent and effective Ad-Cre-mediated excision of Ctnnb1 Exon 3 by 48 h post-infection, compared to fibroblasts carrying the same transgene and transduced with Adenovirus GFP   further demonstrating successful excision of Ctnnb1 Exon 3 and activation of the pathway.To identify coding and non-coding RNAs downstream of Wnt/β-catenin signaling, we infected neonatal dermal fibroblasts carrying gure 1B) . Axin2, -catenin , was up-n = 3) by whole-genome RNA-sequencing at 72 h post Ad-Cre infection . We quantified gene expression using FPKM  and further verified GOF status by examining FPKM values of several well-known Wnt/β-catenin signaling targets in dermal fibroblasts in vivo, such as Wnt5a, Wnt11, Apcdd1, and Twist2 . Within expressed mRNAs and lncRNAs (FPKM > 1), GOF samples independently clustered together via Pearson correlation against the controls . In total, we identified 1,661 mRNAs and 111 lncRNAs that were differentially expressed across all GOF samples  . These findings demonstrate that Wnt signaling differentially regulates lncRNAs as well as mRNAs in dermal fibroblasts.Next, we isolated total RNA and performed expression profiling on control and GOF neonatal dermal fibroblasts . The highest scoring functional clusters enriched in up-regulated genes included Glycoprotein, Membrane, Cell junction, and Wnt signaling . Functions including Extracellular Matrix and Secreted/Glycoprotein were also highly enriched among those genes that were down-regulated in GOF dermal fibroblasts .We performed Gene Ontology (GO) Analysis for all differentially expressed mRNAs. These analyses identified key functional groups affected by Wnt/β-catenin signaling in dermal fibroblasts. Through DAVID, the differentially expressed mRNAs were grouped by functional clusters, with positive enrichment scores indicating functional clusters comprised of up-regulated genes and negative scores indicating clusters of down-regulated genes . Of the 258 differentially expressed matrisome genes, 59 were annotated as ECM glycoproteins, 16 are Collagen proteins, 10 are Proteoglycans and 34 are ECM affiliated genes, while the rest serve ECM regulatory functions. Within the various functions of differentially expressed genes, the ECM-encoding and matrisome-related genes are of note due to their function in fibrogenesis.Since DAVID functional clustering analysis highlighted a strong overrepresentation of ECM genes, we used an existing gene set consisting of matrix encoding and related genes or “matrisome” , to furtP-value < 0.05) expressed in our control and GOF fibroblasts, 2371 of which corresponded to human genes with microarray identifiers in the GSEA database . The top four gene sets enriched in GOF samples were targets of Suz12, EED, and exhibited histone modification of H3K4Me2/H3K27Me3. They are all related to the function of the Polycomb Repressive Complex 2 (PRC2), a well-characterized epigenetic mechanism of gene repression . A recent study found functional links between lncRNAs, Wnt/β-catenin signaling, and components of PRC2 in liver cancer stem cells  to identify biological signatures enriched in GOF samples . We inpuem cells . The ontFigure 2C). Our analysis shows that the expression changes in GOF dermal fibroblasts in vitro after 48 h of stabilization of β-catenin is consistent with findings from profiling in vivo fibrotic mouse dermis in that Wnt/β-catenin signaling may regulate the expression of matrisome genes and contribute to fibrotic conditions , desmoid tumors, idiopathic lung fibrosis, and tumor stroma . Of the nditions . TherefoWincr1 and Wincr2) were highly differentially expressed . Wincr1 isoform 1(ENSMUST00000146678.1) (GM12603) was expressed at 37 FPKM in control samples and at 551 FPKM in the GOF samples (average 14x fold-change). Wincr2 (GM12606) was expressed at 3.24 FPKM in control samples and 28.27 FPKM in GOF samples, marking over an 8x fold change between conditions. Therefore, we validated increase of Wincr1 with isoform 1 specific PCR primers and Wincr2 expression by qRT-PCR in four independent biological replicates of both GOF and controls in P4 ventral dermal fibroblasts in vitro .We next focused on the 111 differentially expressed lncRNAs in the GOF samples as compared to controls. Two top novel candidate Wnt Induced Non-Coding RNAs  and dorsal skin, as compared to expression in the embryonic liver, heart, and gut (n = 2) . Wincr1’s enhanced presence in embryonic cranial and dorsal skin vs. other tissues suggests that our lncRNA candidate could play a role in fibroblast biology. Wincr2 followed a similar tissue-specific trend in embryonic tissues .To investigate the tissue-specific expression profile of itiation . We assaWincr1 with doxycycline-inducible stabilized β-catenin (β-catistab) in E13.5 Engrailed1Cre/+; Rosa26rtTA/+; Teto-deltaN89β-catenin/+ cranial and dorsal dermal fibroblasts in vitro . In response to β-catistab, Wincr1 was significantly higher in embryonic dorsal and cranial dermal fibroblasts . In the same in vitro GOF samples assayed for Wincr1 expression, an increase in Wincr2 expression was observed but did not reach statistical significance .We further tested β-catenin-responsive expression of in vitro . RelativWincr1 in doxycycline inducible-reversible levels of β-catistabin vitro in P4 ventral dermal fibroblasts. Addition of doxycycline and subsequent withdrawal in culture media allowed us to induce and reverse expression of stabilized β-catenin as shown by Axin2 mRNA levels in Engrailed1Cre/+; Rosa26rtTA/+; Teto-deltaN89β-catenin/+ P4 dermal fibroblasts . Similarly to the previous result, Wincr1 responded dynamically to the induction and reversal of β-catistab levels , further establishing Wincr1 as a Wnt/β-catenin signaling-responsive lncRNA in embryonic and perinatal dermal fibroblasts obtained from cranial and trunk skin.We tested for dynamic β-catenin-responsive expression of Wincr1. It has been shown that correlation of expression between two genes across samples can imply a common regulatory factor (Axin2 (97 genes), and those that do not correlate with Axin2 (6 genes). By excluding genes whose expression correlated with that of Axin2, a known direct target of β-catenin, we identified a small number of matrisome genes that might be indirect targets of β-catenin regulation. Relevant fibrosis-related marker genes were selected as possessing some or all of the following traits:We next utilized co-expression network construction to identify and separate putative regulatory targets of both β-catenin and y factor . Using F(a)-catistab dermal fibroblasts by comparison of FPKM Up-regulated in β(b)Figure 2B)Inclusion in the Matrisome gene set ((c)Figure 2C)Overexpressed in human fibrotic tissue microarray ((d)Axin2 cluster of the correlation network Exclusion from the Matrix Metalloproteinase-10 (Mmp10) was selected for functional investigation. Furthermore, there was no significant enrichment of predicted Tcf/Lef binding motifs within 5 kb of the transcriptional start site of Mmp10 and other differentially expressed mRNAs .Of the genes that passed all four criteria, Wincr1 function in gene regulation and dermal fibroblast biology, we either overexpressed or knocked down Wincr1 in P4 ventral dermal fibroblasts in vitro. For overexpression, we generated a lentivirus construct to overexpress Wincr1 (LV-Wincr1). For knockdowns of Wincr1, we utilized LNA GapmeRs (Wincr1 GapmeRs). We confirmed increase in Wincr1 RNA expression levels after infection of LV-Wincr1  and reduction after transient transfection with Wincr1 GapmeR . Dermal fibroblasts with either over- or knocked down expression of Wincr1 and the parent control cells were then used to investigate changes in the expression of dermal identity and fibrosis-related gene candidates.To elucidate Mmp10 mRNA level was highly correlated with levels of Wincr1 in dermal fibroblasts . After infection with LV-Wincr1, expression of Mmp10 increased dramatically compared to the control . Conversely, knockdown of Wincr1 via GapmeRs resulted in a decrease in Mmp10 expression by ∼30% of basal levels . Also, the expression of Col1a1, a key fibrotic marker, correlated negatively with overexpression of Wincr1 . However, we did not observe a significant difference in Col1a1 mRNA levels upon knockdown of Wincr1 . The mRNA levels of markers for fibroblasts identity Platelet derived growth factor receptor alpha (Pdgfra) and β-catenin responsive genes and fibrotic markers such as Loxl4, Col5a1, which were up-regulated in GOF, did not correlate with Wincr1 mRNA level, indicating that Wincr1 does not regulate their expression in our system . Co-expression analysis also identified Has2 and Methylthioadenosine phosphorylase (Mtap), the latter of which shares a locus with Wincr1. However, the relative mRNA levels of Has2 and Mtap did not correlate with changes in Wincr1 level . Thus, our findings demonstrate that Mmp10 is a key target of Wincr1.We found Wincr1 and β-catenin have synergistic effects on gene expression, we infected Engrailed1Cre/+; R26rTA/+; Teto-β-catenin (β-catistab) P4 ventral dermal fibroblast with LV-Wincr1. We validated induction of Axin2 in β-catistab condition and confirmed comparable overexpression of Wincr1 in lentivirus infected β-catistab condition . Mmp10 and Col1a1 mRNA levels in LV-Wincr1+ β-catistab were not significantly altered from LV-Wincr1 only . These results suggest that Wnt/β-catenin signaling and Wincr1 do not synergize to regulate Mmp10 and Col1a1 mRNA and likely function in a linear pathway.To test if Wincr1 on Mmp10 expression, we sought to determine functional effects of Wincr1 on cellular behavior of dermal fibroblasts. In order to prevent discrepancies, we used the same stably infected LV-Wincr1 lines in all functional assays . Two different dermal fibroblast lines with over-expression of Wincr1 had comparable rates of proliferation to controls over 7 days . Next, we used an in vitro scratch assay to study the rate of collective cell migration of dermal fibroblasts in a wound healing setting. This multistep process is relevant in many biological processes such as embryonic development and wound healing  and serum-free conditions within 14–22 h .Given the gene regulatory effect of  healing . Comparein vitro model for fibrotic process and matrix turnover . The difference in collagen gel shrinkage by 14 h was significant and consistent across the two LV-Wincr1 cell lines. Thus, gain of function Wincr1 positively correlated with collagen gel contraction, thereby linking Wincr1 to a key fibroblast functionality.Collagen type1 gel embedded with fibroblasts is considered an turnover . We deteWincr1, is a potentially new regulator of dermal fibroblast behavior and ECM-related gene expression.In summary, we have identified key mRNAs and lncRNAs that are responsive to Wnt/β-catenin signaling levels and we have demonstrated that a lncRNA responsive to Wnt/β-catenin activity, Wincr1, that act as putative regulators of key genes such as Mmp10 and Col1a1 in ECM biology. Wincr1 also affects complex cellular behaviors such as collective cell migration and collagen processing and contraction.Long non-coding RNAs have emerged as key regulators of many cellular processes, and their dysregulation has been observed in many human diseases . StudiesOur expression profiling study allowed us to identify genes that were under the influence of Wnt/β-catenin signaling in dermal fibroblasts. Consistent with our previous gene expression analysis from mouse fibrotic dermis after 21 days of sustained Wnt/β-catenin signaling , gene onAnalysis of the gene expression signature induced by β-catenin stabilization reveals enrichment for PRC2 targets, signifying epigenetic regulation. Unbiased gene set enrichment analysis of genes expressed in dermal fibroblasts shows an enrichment of PRC2 targets. This epigenetic regulatory repressive complex has been shown to interact with lncRNAs, suggesting that a portion of the β-catenin-dependent expression signature controlling ECM and fibrosis-related genes may be regulated by PRC2 or other mechanisms . While fWincr1 was identified as a Wnt/β-catenin signaling induced lncRNA candidate due to its consistent response to β-catenin activity levels in multiple contexts in mouse dermal fibroblasts. Further context-specific functional roles of Wincr1 will be elucidated in future in vivo studies with fibroblast-restricted mutants. Our in vitro modulations of Wincr1 expression levels in dermal fibroblasts demonstrated that this lncRNA has a robust gene regulatory effect on distinct genes. Identification of other Wincr1 targets will require extensive expression profiling in dermal fibroblasts and other cell types. In addition, our studies reveal that β-catenin stabilization and overexpression of Wincr1 do not synergize to increase Mmp10 levels, suggesting that β-catenin and Wincr1 may function linearly in a uncharacterized genetic or epigenetic pathway to modulate key genes in matrix production and turnover. Our current data show that while Wincr1 levels are increased after stabilizing β-catenin, Wincr1 can also independently regulate expression of protein coding genes. Identifying other Wincr1 targets will allow us to further refine our understanding of how Wincr1 intersects with Wnt/β-catenin signaling for gene regulation and fibroblast cell behavior. In our current study, we elaborate on the role of Wincr1 in fibroblast gene regulation and cellular behavior, given that this is a context in which Wnt/β-catenin signaling is known to be important.Using sustained and inducible-reversible systems of Wnt/β-catenin signaling activation, Wincr1 has a restricted gene regulatory role. Specifically, Mmp10 expression was consistently responsive to Wincr1 levels, suggesting it is a putative regulatory target of this novel lncRNA. Wincr1’s gene regulatory role does not appear to be pan-ECM, but instead can regulate a defined set of genes. Further mechanistic-based experiments are required to gain insight into how Wincr1 regulates Mmp10, and such studies would be helpful in predicting other targets through binding motifs or other means. Mmp10/Stromelysin-2 has emerged as a key player in fibrosis and “degradomics” (-Wincr1 dermal fibroblasts. Mmp10 is not a collagenase, but it has the ability to regulate expression of other MMPs, such as Mmp13, that have collagenolytic function and promote the resolution of matrix in wound healing. It is not clear if Mmp10 has a similar role in mitigating ECM accumulation in chronic fibrosis settings that are the result of excessive ECM production and inadequate resolution (Wincr1 participates as a negative feedback to the pro-fibrotic Wnt/β-catenin pathway in dermal fibroblasts. Future functional studies with Wincr1 in fibrosis models will be needed to demonstrate if it has a fibroprotective function.Our query of several fibrotic genes suggests that adomics” . MMPs caadomics” , and we solution . It is tWincr1 to Wnt/β-catenin signaling indicates the possibility of this and other lncRNAs as targets for therapeutic intervention to treat fibrosis, but also as circulating diagnostic biomarkers (in vivo functions of Wincr1 and other β-catenin responsive lncRNAs. Also, mechanistic understanding into Wincr1 may lead to novel insights into the Wnt signaling pathway and how it regulates key genetic networks throughout embryonic development and adult diseases.Wnt/β-catenin signaling has diverse roles in skin development, tissue homeostasis, and disease. Thus, identifying new genetic and epigenetic regulatory mechanisms will provide novel insight in our understanding of this important pathway in dermal fibroblasts. Discovery of this lncRNA and its preliminary regulatory links to β-catenin signaling, skin development, and known fibrotic protein-coding gene expression indicate that lncRNAs may play an important regulatory role in directing Wnt/β-catenin signaling. This is of particular interest in the context of fibrosis, a condition that can be caused by activation of this pathway, but with gene regulatory intermediates that will require further characterization in order to be of clinical value. The robust responsiveness of omarkers . FurtherNKM, NVM, RA, and AK contributed to experimental design, experiment collection, analysis, writing, and figure preparation. EH-S contributed to experimental design and writing of the manuscript. BI contributed to analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Epoxidized natural rubber fibers (ERFs) are developed through one-step electrospinning and directly deposited into epoxy resins without collecting and distributing of fibers. The shape of ERFs shows rough surface due to different evaporation rate of solvent mixture consisting of chloroform and dichloromethane and the average diameter of ERFs is 6.2 µm. The increase of ERFs loading from 0 to 20 wt % into the epoxy resin increases the fracture strain significantly from 1.2% to 13% and toughness from 0.3 MPa to 1.9 MPa by a factor of 7. However, the tensile strength and Young’s modulus decrease about 34% from 58 MPa to 34 MPa and from 1.4 GPa to 0.9 GPa, respectively. Due to the crosslinking reactions between oxirane groups of ERFs and amine groups in the resin, surface roughness and the high aspect ratio of ERFs, ERFs result in more effective toughening effect with the minimum loss of tensile properties in epoxy resins.  GIC) of 158%, 261% and 281% at 1, 3 and 5 wt % of fiber loadings, respectively [Electrospinning as an efficient and versatile technology has been developed for generating ultrafine fibers in a variety of materials, such as polymers, inorganic materials, and hybrid materials . These fectively . The epoectively . Electroectively . Nanofibectively . Many studies have investigated improving toughness of epoxy resin using synthetic copolymers and metal derivatives as toughening agents such as carboxyl-terminated acrylonitrile butadiene, synthetic ductile polymers such as the acrylic rubber, hydroxy-terminated butadiene or acrylonitrile butadiene, epoxy-terminated acrylonitrile butadiensbse, epoxy-terminated butadiene and amine-terminated acrylonitrile butadiene ,15,16,17−2, 1.5 times that of neat epoxy resin even metal conductivity of 0.85 W m−1 K−1 [However, little research has been conducted on eco-friendly toughening agents to improve brittle epoxy resins. For example, epoxy resins with 60% functionalized green halloysite nanotubes resulted in increased impact strength to about 5.8 kJ m m−1 K−1 . After a m−1 K−1 . In anot m−1 K−1 .The problem in ductile toughening agents is that most resins experience a decline in their tensile properties with the addition of ductile materials. Hence, good interfacial bonding via chemical reactions between the matrix and the toughening agent has been an active research area in order to improve the toughness without loss of tensile properties in the matrix. For example, as 0.5 wt % dodecane functionalized tannic acid incorporated into epoxy resin as the toughening agent increased the impact strength by up to 196% compared to that of neat epoxy . This teThe primary objectives of this study were to develop micro-sized green elastic ENR fibers (ERFs) with surface modification through one-step electrospinning. bT) and volatility rate because chloroform (bT: 61 °C) has higher boiling temperature than dichloromethane (bT: 39 °C) [: 39 °C) . The internal structures of ERFs are shown in −1 (epoxy C–O–C ring deformation) [−1 range (C–N stretching), 1375 cm−1 and 1494 cm−1 (CH and CH2 deformation), 1528 cm−1 (NH stretching) and 2862–2937 cm−1 range (CH and CH3 asymmetric stretching) [−1 corresponds to NH2 bending of excess amine group from the curing agent in the epoxy resin. Neat ERFs transmittance peaks at 751 cm−1, 852 cm−1 and 1246 cm−1 corresponding to the oxirane groups appeared in the spectra [rmation) , 1020–12etching) . The bro−1 range to indicate the presence of hydroxyl groups (–OH) produced by crosslinking reaction between oxirane groups of epoxy resin or ENFs and amine of the curing agent, as shown in −1 range corresponding to C–N linkages [−1 range), C–O–C (ether stretching vibration at 945 cm−1) and –OH (3000–3600 cm−1), as shown in The E-ERF resins show additional peak at 3000–3600 cmlinkages . The incTg1 (glass transition temperature of ENR) at −25 °C, whereas neat epoxy matrix shows Tg2 (glass transition temperature of epoxy resin) at 55 °C. E-ERF resins, however, show little change in Tg1 from −28 to −25 °C but significant change in Tg2 from 55 to 65 °C as ERFs loading increases from 0 to 20 wt %. At 5 wt % loading of ERFs in epoxy resin, there is little increase in Tg2. However, with the 10% addition of ERFs, Tg2 increases by 7 °C. This increasing trend continues with ERFs loading of 15 and 20 wt % in epoxy resins to 63 and 65 °C, respectively. This trend is attributed to cross-linking reactions between oxirane group of ERFs and excess amine or hydroxyl group in epoxy resins to create a 3D cross-linking structure. These cross-links increase the chain length and interactions between chains, resulting in limited freedom to rotate about the bonds and consequently, increases the Tg2 temperature of E-ERF resins [Tg with increasing loading concentrations of liquid rubber [F resins ,37. The F resins  for epoxd rubber . They exd rubber . E′) of E-ERF resins is plotted as a function of temperature in Tg1 of ENR. The E′ for neat ENR resin shows the final storage modulus close to 0 MPa at 60 °C, whereas E-ERF resins show E′ values above 4 GPa at 60 °C. δ) plots for E-ERF resins are shown in Tg because ENR is at elastic state whereas rigid epoxy resin stays at glassy state. The tan δ peak at around −25 °C represents relaxation of the neat ENR. As ERFs loading increases from 0 to 20 wt %, the Tg1 values of E-ERF resins decrease slightly from −28 to −25 °C; however, Tg2 values of E-ERF resins increase from 51 to 65 °C. The reason is that the chemical crosslinking reactions between oxirane groups of ENR and excess crosslinking agent in epoxy resin occur and result in long molecular chain and offer higher than intermolecular hydrogen bonding. Therefore, free motion of epoxy molecular chain segments are restrained, which results in an increase of Tg2 [The mechanical loss factor  was purchased from Huntsman Co. . Aradur® 956-2  was purchased from Krayden Inc. . Epoxidized natural rubber  was donated by Malaysian Rubber Board. Adirondack green 520 fluorescent dye, chloroform, dichloromethane were purchased from Sigma Aldrich Chemical Co. .Aralditev/v) of dichloromethane and chloroform and then stirred for 12 h at room temperature (RT). The solution was put into a 10 mL syringe, and electrospun into fibers (ERFs) with a high voltage of 12 kV. The syringe feeding rate used was 30 µL min−1 and the distance between the syringe needle tip (diameter: 1 mm) and the collector was 15 cm. Precured epoxy resin was placed above the bath containing metal collector and heated to 40 °C with stirred at 50 rpm. ERFs were directly deposited into the epoxy resin. ENR (1 wt %) was dissolved in the mixture of 1:2  and stirred at 50 rpm for 30 min. Electrospun ERFs using 1 wt % ENR polymer concentration were directly deposited into the epoxy resin. Then the epoxy resin containing ERFs at different loadings was poured into a Teflon® mold (127 × 127 mm) and precured for 3 h at 40 °C. The epoxy matrix containing ERFs was cast in the form of sheets and the epoxy sheets were then cured in the oven at 1 h for 100 °C. Afterwards, all specimens were conditioned at 21 °C and 65% RH for 72 h prior to their characterization.The curing agent, 35 wt % Aradur−1 from −50 to 200 °C to evaluate the changes in their thermal properties under a nitrogen flow rate of 100 mL min−1.All epoxy resins containing ERFs at 5, 10, 15 and 20 wt %  were tested their thermal properties. E-ERF resins were scanned on differential scanning calorimeter  at a heating rate of 5 °C min−1 and gauge length of 30 mm. All tests were performed on Instron universal tensile tester  using at least ten specimens [All E-ERF resins were cut by a laser cutter to obtain the dimensions of 50 mm × 10 mm and were characterized for their tensile properties according to ASTM D882-02 standard at a strain rate of 0.6 mm minpecimens .−1. All tests were performed at 5 µm amplitude and sinusoidal tensile stress , and conducted at least three times [Dynamic mechanical properties of E-ERF resins were analyzed using DMA  in 3-point bending mode. Resins were cut into the final specimen dimensions of 30 mm × 10 mm and scanned from −50 to 100 °C at a ramp rate of 5 °C minee times . −1 wavenumbers with a total of 300 scans and a resolution of 2 cm−1. ENR solution was stained with 0.5% Adirondack green 520 fluorescent dye of ENR before electrospinning. Then, it was electrospun directly into epoxy matrix. The resins were analyzed using a confocal laser scanning microscope  and 520 nm excitation filter with 25× objective lens.Microstructures of the tensile fracture surfaces of all E-ERF resins were analyzed using Tescan Mira3 FESEM. ATR-FTIR  was used to confirm crosslinking reactions of E-ERF resins. Neat ENR, neat epoxy matrix and all E-ERF resins were scanned from 700 to 4000 cmA convenient one-step electrospinning was developed for green elastic natural rubber fibers. ERFs were directly deposited to epoxy resins without collecting or redepositing of fibers. The surface of ERFs displayed bumpy (spines-like) and rough edges due to different volatility rate of solvents. The range of diameter of ERFs varied from 1.6 µm to 13 µm and the average diameter was approximately 6.2 µm. The increase of ERFs loading from 0 to 20 wt % into epoxy resins increased the fracture strain significantly from 1.2% to 12.8% and toughness at break from 0.3 MPa to 1.9 MPa, an increase by a factor of 7. However, Young’s modulus decreased minimally from 1.4 GPa to 0.9 GPa. E-ERF resins showed the presence of strong interfacial bonding between the ERFs and the epoxy resin without breakage or pullout of fibers. Inherent rubber toughness and high aspect ratio of fiber form of ERFs could contribute to efficient energy dissipation, since effective crosslinking reactions between the oxirane groups and amine or hydroxyl groups in epoxy resin allow the 3D structure to serve as an effective toughening agent. Green elastic ERFs could be useful in many applications such as construction materials, biomaterials, smart textiles, automotive and aerospace, therefore replacing petroleum-derived elastic fibers.


Hippo/YAP pathway is known to be important for development, growth and organogenesis, and dysregulation of this pathway leads to tumor progression.We and others find that YAP is up-regulated in human gliomas and associated with worse prognosis of patients. However, the role and mechanism of YAP in glioma progression is largely unknown.The expression of YAP in glioma tissues was detected by quantitative polymerase chain reaction (qPCR) and immunoblotting. The effect of YAP on glioma progression was examined using cell growth assays and intracranial glioma model. The effect of YAP on β-catenin protein level, subcellular location and transcription activity was examined by immunoblotting, immunofluorescence and RT-PCR.Firstly, knockdown of YAP inhibited glioma cell proliferation in vitro and tumor growth in vivo. In addition, YAP modulated the protein level, subcellular location and transcription activity of β-catenin via regulating the activity of GSK3β. Lastly, β-catenin partially mediated the effect of YAP on glioma cell proliferation.Our findings identify that YAP promotes human glioma growth through enhancing Wnt/β-catenin signaling. In addition, this study provides a new crosstalk mechanism between Hippo/YAP and Wnt/β-catenin pathways, which suggests a new strategy for human glioma treatment.The online version of this article (10.1186/s13046-017-0606-1) contains supplementary material, which is available to authorized users. Malignant glioma is the most common brain cancer. Affected patients are usually treated with a combined approach of surgery, chemotherapy and radiation therapy, but the median survival time is only 12–15 months [Recent studies have identified the Hippo/YAP signaling as a key mechanism that controls organ size by imping on cell growth and proliferation , 3. The The canonical Wnt pathway regulates many biological processes, including cell proliferation, cell fate decision, axis formation and organ development during embryonic development and tissue homeostasis , 11. TheSeveral studies have shown that the Hippo/YAP pathway genetically and functionally interacts with Wnt/β-catenin signaling. For instance, Varelas et al. have shown that cytoplasmic TAZ binds to dishevelled and inhibits Wnt signaling . MoreoveWhether and how YAP regulates β-catenin to promote glioma cell proliferation remains poorly understood. In this study, we demonstrated that knockdown of YAP expression inhibited glioma cell proliferation in vitro and tumor growth in vivo. YAP modulated the protein level, subcellular location and transcription activity of β-catenin via regulating the activity of GSK3β. At last, β-catenin mediated the effect of YAP on glioma cell proliferation. These results provide a novel mechanism that YAP promotes human glioma cell growth via β-catenin activation.A total of 26 human glioma samples and 13 nontumor brain tissues (decompressive surgery) were obtained from Affiliated Hospital of Xuzhou Medical University. All of the glioma samples used in this study were astrocytomas, which were histologically diagnosed according to the World Health Organization grading system . All the glioma and nontumor brain tissues had been collected immediately after surgical resection and stored in −80 °C. Written informed consent was obtained from the patients and the study was approved by the Ethic Committee of the hospital.The U87 and U251 glioma cells were purchased from Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Science. Cells were cultured in DMEM/F-12 (Gibco) media, supplemented with 10% fetal bovine serum .(Ser33/37/Thr41), p-β-catenin and GAPDH were obtained from Cell Signaling Technology . Antibody against Ki67 was purchased from Thermo. β-cateninCA and β-cateninWT plasmids were kindly donated by Prof. Zhen-Ge Luo at the Institute of Neuroscience and Key Laboratory of Neurobiology, Chinese Academy of Sciences. YAP wild type and YAP S94A plasmids were kindly gifted by Prof. Bin Zhao at the Life Sciences Institute of Zhejiang University.Antibodies against YAP1 were purchased from Abcam . Antibodies specific for GSK3β, p-GSK3β, β-catenin, Non-phospho active β-catenin To establish YAP down-regulation glioma cells, short hairpin RNA (shRNA) targeting human YAP1 was inserted into lentiviral pLL3.7 backbone at HpaI and XhoI sites. For over-expression of YAP, the YAP2 cDNA was inserted into the pWPXLd backbone with GFP tag using PacI and MluI sites. The sequences (Sangon Biotech Shanghai) of shYAP were: F: 5′-TGCAGCAGAATATGATGAACTTCAAGAGAGTTCATCATATTCTGCTGCTTTTTTC-3′; R: 5′-TCGAGAAAAAAGCAGCAGAATATGATGAACTCTCTTGAAGTTCATCATATT CTGCTGCA-3′. Primers sequences for YAP2 were: F: 5′-CCTTAATTAAATGGATCCCGGGCAGCAG-3′; R: 5′-CGACGCGTCCCTATAACCATGTAAGAAAGC-3′.RNA was extracted from the human glioma cell lines U87 and U251 with YAP down-regulation or over-expression and the cDNA was synthesized using reverse transcription reagents  according to the manufacturer’s protocol [Cellular fractionation was conducted by using Membrane and Cytosol Protein Extraction Kit (Biovision), according to the instruction of manufacturer. Equal amount of protein lysates were subjected to 10% SDS-PAGE and then transferred to 0.45 μm pore size PVDF membrane (Millipore). After blocking with 5% non-fat milk, the membrane was probed with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. Bound antibodies were detected by the Pierce ECL Plus Western Blotting Substrate  and exposed to X-ray films. Band densities were quantified by ImageJ Software . The relative amount of proteins was determined by normalizing the densitometry value of interest to that of the loading control .3 cells per well. Twenty-four hours later, the cells were exposed to 50 μM of 5-ethynyl-20-deoxyuridine  for additional 2 h at 37 °C. Then, the cells were fixed with 4% paraformaldehyde for 20 min and treated with 0.5% Triton-X-100 for another 20 min at room temperature. After being washed with PBS for five times, the cells were reacted with 100 μL of 1× Apollo® reaction cocktail for 30 min. Thereafter, the DNA contents of cells were stained with 100 μL of Hoechst 33,342 (5 μg/mL) for 20 min and visualized under a fluorescent microscope . Data were obtained from three independent assays performed in triplicate [Cells were seeded into 96-well plates at 7 × 10iplicate .Cell growth curves were obtained by detecting the cell viability with the Cell Counting Kit-8  assay every 24 h according to the manufacturer’s instruction.Three days after lentivirus infection, cells were seeded into 6-well plate (300 cells per well). The medium was changed at three days intervals. After 14 days of culture at 37 °C, the colonies were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The colonies were then stained with 0.05% crystal violet for 10 min, washed with water and air-dried. The total number of colonies with more than 50 cells was counted.n = 6 per group) and were anesthetized by intraperitoneal injection of 3% chloral hydrate (0.01 mL/body weight (g)). According to the coordinates, a small burr hole, 2 mm diameter, was drilled with 1.4 mm away from the midline at the right side of the cranium. The head of syringe was held in a horizontal position and shYAP or scramble U87 cells (1 × 106) were injected into right striatum at a depth of 2.6 mm using a small animal stereotactic apparatus. The cells were injected slowly within 10 min at a rate of 0.5 μL/s and the syringe stayed for 10 min before withdrawing. The burr hole was sealed with bone wax, strict aseptic conditions were applied in the whole experiments.Intracranial model of glioma in nude mice was performed according to our previous study . All the2 with ‘a’ representing the longest diameter and ‘b’ representing the shortest diameter.To obtain the survival curve, the mice were sacrificed when they appeared hemiplegia, listlessness, cachexia and other neurological symptoms or without neurological symptoms at 50 days after transplantation. The cryosections of brain were subjected to H.E. staining and the tumor volume was calculated according to the formula V = 1/2 abImmunofluorescence was performed according to our previous study . The brat test or ANOVA for multiple comparisons. In all analysis, quantitative data were obtained from at least three independent experiments and expressed as mean ± SEM. P values less than 0.05 were considered statistically significant .Data were analyzed by using Student’s In order to study the effect of YAP on glioma progression, we firstly examined the mRNA and protein level of YAP in human glioma tissues by real-time PCR and immunoblotting respectively. As shown in Fig. Therefore, we down-regulated YAP with shRNA lentivirus to examine the effect of YAP on glioma cell proliferation. As shown in supporting Fig. To study the effect of YAP on intracranial glioma growth in vivo, YAP down-regulation U87 cells were transplanted into the right striatum of nude mice. We found that the tumors derived from YAP down-regulation cells were significantly smaller than those of the scramble mice Fig. . In addiNext, we address how YAP regulates glioma progression. Hippo/YAP pathway is closely related to the Wnt/β-catenin signaling , 25. In , we therefore wonder whether YAP modulates the protein level of β-catenin via regulating the activity of GSK3β in destruction complex. As shown in Fig. As the protein level of β-catenin is mainly modulated via phosphorylation of GSK3β in AXIN/APC/GSK3β destruction complex , 11, we Next, we examined whether YAP affects the subcellular location and activity of β-catenin. As shown in Fig. As β-catenin functions as a transcriptional co-activator to promote cell proliferation after being transported into the nucleus , 11, we WT and β-cateninCA, the active form of β-catenin, to address this question. β-cateninCA is the mutated form of β-catenin with four serine/threonine residues  changed to alanine, which cannot be phosphorylated by GSK3β and exhibits constitutive stabilization [WT and its active form β-cateninCA in U251 and U87 glioma cells promoted glioma cell proliferation and β-cateninCA exhibited a stronger promotion effect than β-cateninWT. In line with our previous results shown in Figs. WT and β-cateninCA were over-expressed in the cells with YAP depletion, the reduced proliferation capacity of glioma cells induced by YAP down-regulation could be partially rescued. Especially, β-cateninCA exhibited a stronger rescue effect than that of β-cateninWT -expressing cells, activated YAP enhances IGF signaling by phosphorylating AKT and GSK-3β, leading to GSK-3β activity inhibition. Consistently, the level of β-catenin and the active non-phosphorylated β-catenin increased in YAPS112A-expressing cardiomyocytes [We found that the protein level of p-GSK-3β increased after YAP over-expression, leading to β-catenin and active-β-catenin increase. In addition, β-cateninmyocytes . In 2014We observed that, after YAP over-expression, the increased β-catenin and active-β-catenin translocated into the nucleus, leading to c-myc and cyclin D1 transcription. As nuclear accumulation of β-catenin participates in malignant progression of gliomas and implicates poor prognosis , we deduWe found that knockdown of YAP inhibited glioma cell proliferation in vitro and tumor growth in vivo. Our results identify a novel mechanism that YAP modulates the protein level, subcellular location and transcription activity of β-catenin via regulating the activity of GSK3β in destruction complex. β-catenin mediates the promoting effect of YAP on glioma cell proliferation, which provides a new crosstalk mechanism between Hippo/YAP and Wnt/β-catenin pathways and suggests a new strategy for human gliomas treatment.The use of human tissues was approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University (No: xyfylw2014002). Written informed consent was obtained from each patients. All animal experiments were performed according to the guidelines for the care and use of laboratory animals and were approved by IACUC of Xuzhou Medical University .Additional file 1: Figure S1.(A) The infection efficiency of scramble and shYAP lentivirus in U251 and U87 cells. U87 and U251 glioma cells were infected with viral supernatant and the infection efficacy was evaluated by GFP positive cells 72 h later. Scale bar, 100 μm. PH: Phase contrast. (B) Representative immunoblots of protein extraction from shYAP infected U251 and U87 glioma cells (up panel)and YAP protein levels were quantified (bottom panel). *** P < 0.001. (PDF 358 kb)Generation of YAP down-regulation U87 and U251 cells. Additional file 2: Figure S2.(A) The infection efficiency of vector and YAP lentivirus in U87 cells.U87 glioma cells were infected with viral supernatant and the infection efficacy was evaluated by GFP positive cells 72 h later.Scale bar, 100 μm. PH: Phase contrast. (B) Expression analysis of YAP protein levels by Western blotting in YAP over-expression U87 glioma cells. (PDF 90 kb)Generation of YAP over-expression U87 cells. Additional file 3: Figure S3.(A&B) Representative immunoblots of GSK-3β, p-GSK-3β, β-catenin, active-β-catenin and p-β-catenin after over-expression of YAP or YAP S94A in U251 (A) and U87 (B) glioma cells.The results indicate that YAP S94A recapitulated the effect of YAP wild type on GSK3β and β-catenin phosphorylation. (PDF 153 kb)The effect of YAP on GSK-3β and β-catenin phosphorylation.


The Wnt/beta catenin pathway has been highlighted as an important player of brain tumors aggressiveness and resistance to therapies. Increasing knowledges of the regulation of beta-catenin transactivation point out its hub position in different pathophysiological outcomes in glioma such as survival and migration. Crosstalks between integrins and beta-catenin pathways have been suggested in several tumor tissues. As we demonstrated earlier that α5β1 integrin may be considered as a therapeutic target in high grade glioma through its contribution to glioma cell migration and resistance to chemotherapy, we addressed here the potential relationship between α5β1 integrin and beta-catenin activation in glioma cells. We demonstrated that overexpression and activation by fibronectin of α5β1 integrin allowed the transactivation of beta-catenin gene targets included in an EMT-like program that induced an increase in cell migration. Hampering of beta catenin activation and cell migration could be similarly achieved by a specific integrin antagonist. In addition we showed that α5β1 integrin/AKT axis is mainly involved in these processes. However, blockade of beta-catenin by XAV939 (tankyrase inhibitor leading to beta-catenin degradation) did not synergize with p53 activation aiming to cell apoptosis as was the case with integrin antagonists. We therefore propose a dual implication of α5β1 integrin/AKT axis in glioma cell resistance to therapies and migration each supported by different signaling pathways. Our data thus suggest that α5β1 integrin may be added to the growing list of beta-catenin modulators and provide new evidences to assign this integrin as a valuable target to fight high grade glioma. Since several years association of the canonical Wnt/beta-catenin signaling pathways with tumorigenesis and tumor aggressiveness has garnered significant attention in different solid tumors thus leading to the development of potential inhibitors –3. Its aDue to the complexity of the networks involving beta-catenin and its major contribution to the pathology, the search for unknown drivers remains an important topic in the field of glioma. We and others have previously characterized the α5β1 integrin, also known as the fibronectin receptor, as playing a key role in the pathobiology of glioma , 17. IntTo date, very few data concerning an interconnection between α5β1 integrin and beta-catenin activity are available  and, to We first determined whether β-catenin activation could be linked to α5β1 expression in U87MG and U373MG glioma cell lines. Measure of the beta-catenin basal level of activation was achieved with cells deprived of serum for 90 min as described in Material and Methods. Activation of beta-catenin was recorded with a specific antibody which recognizes a non-phosphorylated Ser37/Thr41 N-terminal beta-catenin epitope known to be generated upon Wnt signaling or GSK3β pharmacological inhibition . As showWe then analyzed if α5β1 integrin activation through binding to fibronectin may enhance beta-catenin activation. For this purpose, U87MG-α5 high cells were plated on fibronectin pre-coated wells. The effects of other ECM components  were compared to those obtained on non-coated or with poly-L-lysine (a non ECM component) coated wells. As compared to uncoated wells, poly-L-lysine and laminin did not improve the active beta-catenin fraction in U87MG-α5 high cells Figure  thus rulIn the former assays, beta-catenin activation was determined by mean of protein level with a specific anti-active beta-catenin antibody . ActivatWe already demonstrated the implication of α5 integrin in glioma chemoresistance and tumor cell migration. Our recent data implicated the integrin-dependent PI3K/AKT pathway activation in resistance to p53 activators. Inactivation of AKT via integrin antagonist K34c was required to sensitize U87MG-α5 high cells to p53 activator Nutlin-3a . As inacIn addition to its role in proliferation or survival, beta-catenin also impacts on cell migration . We alreTaken together, data show that α5 integrin activation triggers AKT and beta-catenin stimulation, inducing an EMT-like program and cell migration.We present here data suggesting that α5β1 integrin is to be considered as a new important driver of beta-catenin activation leading mostly to tumor cell migration through EMT-like processes. Aberrant activation of the Wnt/beta-catenin pathway has been largely documented in brain tumors where it contributes to the maintenance of stem-like cells, resistance to therapies and to an invasive phenotype . Unlike Expression level (mRNA and protein) of beta-catenin is considered as a negative prognostic marker in glioma with an enhanced expression in glioblastoma , 38. Intin vitro motility but not proliferation. Interestingly, these authors also showed that nuclear (active) beta-catenin was predominantly found in the infiltrative zone of glioblastoma supporting its role in cell migration in vivo.Among the hallmarks of glioblastoma, resistance to apoptosis and invasion into the normal brain are both implicated in their propensity to resist standard therapies. As reviewed in Schaffner et al.  and BlanIn solid tumors, acquisition of a migratory phenotype is often the result of an epithelial to mesenchymal transition (EMT). EMT-like processes in glioma  have recently been described  and a “mU87MG and U373MG cells were obtained from ATCC and ECACC respectively several years ago. Cells were manipulated genetically to overexpress (U87MG- and U373MG-α5 high cells) or repress (U87MG- and U373MG-α5 low cells) the α5 integrin subunit as described earlier , 21. Bri2 at 37°C.To measure basal levels of signaling pathways (i.e. in conditions where integrins are not exogenously activated by serum components), trypsinized cells were washed 3 times in serum free medium by centrifugation and then plated  in the presence of drugs or controls (DMSO 0.2%). When required, cells were plated on substrate-coated dishes  and incubated for 90 minutes  or 6 hours  in a humidified incubator containing 5% CO2 incubator for 3 hours. The coated and uncoated chambers were then placed in the 24-well plates filled with respective culture medium supplemented with the drugs as required. Cells (1 × 104 cells/chamber) in culture medium containing or not the required drugs were seeded into the upper part of the chamber and the plates were transferred in a humidied incubator containing 5% CO2 at 37°C. After 24 h of incubation, cells that reached the bottom layer were fixed with paraformaldehyde (4%) and stained with DAPI. Fluorescent images were acquired under a Zeiss Axio microscope under 10× magnification (6 views field) and cell number quantified with a home-made macro using the ImageJ software.Cell migration was analyzed by a Boyden Chamber assay using an 8 μm pore size Transwell permeable support . The bottom part of the Transwell membrane was either uncoated or coated with fibronectin. Fibronectin coating  was achieved at 37°C in a COProteins were separated on precast gradient 4–20% SDS PAGE gels (Biorad) and transferred to PVDF membrane (Amersham Bioscience) under established protocol settings. After blocking, membranes were probed with primary antibodies (1/1000) against α5 integrin , β1 integrin , fibronectin , (non-phospho Ser33/37/Thr41) active β-catenin , β-catenin (Millipore. #06-734), GAPDH , AKT , pSer473-AKT , vimentin , cleaved caspase 3  and PARP  antibodies. Immunological complexes were revealed with HRP-conjugated secondary antibodies (Promega) at 1/10 000 dilution using chemoluminescence  and visualized with Las4000 image analyser . Quantification of non-saturated images was performed with ImageJ software. GAPDH was used as the loading control for all samples.RNA extracted with RNeasy minikit (Qiagen), according to the manufacturer protocol, was transcribed into cDNA using the high capacity cDNA kit (Applied Biosystem). Real time quantitative PCR was performed using the Fast SYBR Green Master Mix and the StepOne Plus Real time PCR system (Applied Biosystem)  according to the manufacturer's instructions. Luciferase activity was monitored with a Multidetection Microplate Reader (TriStar2 LB 942 − Berthold Technologies).Cells plated were transiently transfected using Lipofectamine 2000 (Invitrogen) with a mixture of an inducible transcription factor responsive firefly luciferase reporter vector or a non inducible vector and a constitutively expressing Renilla construct to normalize for transfection efficiency . Three independent transfections were performed. Following transfection, cells in serum free medium were plated on uncoated or fibronectin-coated wells during 3, 6, 12, 24 or 48 hours. Lysates were harvested, and luciferase activities were recorded with a Dual-LuciferaseSpheroids were formed and analysed as described in Blandin et al. . Brieflyt test with the GraphPad Prism program. P < 0.05 was considered significant.Data are represented as the mean ± SEM, and n is the number of independent experiments. Statistical analyses were conducted using the Student Taken together, our results show for the first time that the α5β1 integrin modulates the beta-catenin pathway in glioma to sustain an EMT-like based migratory program. In addition with our previous results, the interplay we describe here highlights the potential pivotal role of the α5β1 integrin in hallmarks of brain tumors, i.e. resistance to apoptosis and invasion in the normal brain. Antagonists of this specific integrin may thus be proposed as new options for helping to block both processes in subpopulations of high grade glioma.