APC−/− organoids. Furthermore, xStAx-VHLL could effectively restrain tumor formation in BALB/C nude mice, and diminish the existing tumors in APCmin/+ mice. More importantly, xStAx-VHLL could potently inhibit the survival of colorectal cancer patient-derived organoids. These findings suggest that xStAx-VHLL exhibits the ability of cancer prevention and cure, highlighting the potential of β-catenin degrader PROTACs as a new class of promising anticancer agent.Aberrant activation of Wnt/β-catenin signaling has been associated with the onset and progression of many types of tumors and thus β-catenin represents one attractive intracellular target for cancer therapy. Based on the Axin-derived peptide that binds to β-catenin, two stapled peptides SAHPA1 and xStAx were reported to enhance or impair Wnt/β-catenin signaling, respectively. In this study, we designed PROTACs (proteolysis targeting chimeras) by coupling SAHPA1 or xStAx with the VHL ligand to achieve efficient β-catenin degradation. The obtained xStAx-VHLL sustained β-catenin degradation and manifested strong inhibition of Wnt signaling in cancer cells and in  The components of canonical Wnt signaling consist of extracellular Wnt ligands, transmembrane receptors Frizzled/LRP5/6 (low-density lipoprotein receptor-related protein), the intracellular destruction complex  and β-catenin3. As the canonical Wnt signal transducer, the stability of β-catenin is tightly controlled by the interplay between Wnt ligands, receptors and the destruction complex2. Once mutations occur to Wnt signaling components, excessive β-catenin accumulates in the cytoplasm and translocates into the nucleus to promote cell proliferation4. Aberrant activation of Wnt signaling is highly implicated in various types of cancer, including colorectal cancer, in human, for example, hotspot mutations of APC, Axin, and β-catenin have been found to drive tumorigenesis2. Hence, inhibition of the Wnt/β-catenin pathway has already become an important approach for cancer therapy, and β-catenin is the most attractive intracellular target5.The Wnt/β-catenin signaling pathway plays a pivotal role in cell proliferation, differentiation, and survival6. However, none of them has been approved for the clinical use. As β-catenin is the central player of the canonical Wnt signaling and is frequently mutated in cancers, it is the most attractive target for cancer therapy. For instance, PRI-724, which can disrupt the interaction between β-catenin and its co-activators, has been tested for clinical use7. One of the caveats of this type of inhibitors could be insufficient to disturb all the protein–protein interactions due to the large binding interface and complicated binding partners in heterogeneous tumor tissue. Therefore, it is ideal to design inhibitors that can induce β-catenin degradation8. The small-molecule MSAB is a such example9, although its specific binding site and the working mechanism are still unclear.Many small-molecule inhibitors of Wnt/β-catenin signaling have been identified10. Peptide stapling chemistry developed by Verdine and coworkers, takes advantage of ruthenium-catalyzed metathesis to crosslink the side chains of paired anchoring residues at suitable positions, and has been successfully applied to design various peptide inhibitors with improved proteolytic stability, membrane permeability, and thus biological activity12. Modeled after the same Axin-derived peptide motif, we and Verdine lab independently designed and prepared two different stapled helical peptides, termed as SAHPA1 and xStAx, respectively14. Both SAHPA1 and xStAx are capable of penetrating the cell membrane and binding with β-catenin. Interestingly, SAHPA1 was shown to activate Wnt/β-catenin signaling, while xStAx inhibited it14. The opposite effect of these peptides could be due to their different working mechanisms: xStAx blocks the β-catenin-TCF interaction in the nucleus, while SAHPA1 enhances the dissociation of β-catenin from Axin in the presence of Wnt. Nevertheless, it still remains challenging to drive xStAx efficiently inhibit Wnt-dependent tumor growth in cell culture, and importantly, in vivo, due in part to the rapid accumulation of β-catenin in cancer. To overcome these limitations of β-catenin inhibitors, new strategies are urgently in need to achieve the long-lasting degradation of β-catenin and thus tumor regression.Peptides are superior as therapeutics to small molecules in interacting with β-catenin but still suffer from the poor membrane permeability and protease stability17. PROTACs consist of two ligands connected by a linker, with one ligand specifically binding to the protein of interest and the other ligand recruiting an E3 ligase. When PROTACs interact with the target protein and an E3 ligase concurrently, the target protein will be poly-ubiquitinated and then undergo proteasomal degradation. Compared with traditional inhibitors that need a 1:1 (inhibitor:target) activity ratio, PROTACs can continuously function and show sub-stoichiometric property19. For instance, a stabilized peptide-based PROTAC has been successfully developed to target estrogen receptor α for the efficient degradation20.In recent years, proteolysis-targeting chimaeras (PROTACs) has emerged as a useful technology to efficiently degrade protein of interest in vivoAPC–/– mice. xStAx-VHLL also restrained tumor formation in xenograft mouse models and reduced intestinal tumors in APCmin/+ mice. Besides, xStAx-VHLL potently inhibited the survival of the CRC patient-derived organoids. This study is the first attempt to block Wnt signaling via PROTAC-mediated β-catenin degradation, highlighting the potential of PROTAC peptides as a new class of promising agents against the diseases caused by overactivation of Wnt/β-catenin signaling.In this study, to achieve efficient elimination of β-catenin, we designed novel PROTAC β-catenin degraders by coupling SAHPA1 or xStAx with VHL (von Hippel-Lindau protein) ligand via an Ahx chemical linker, termed as SAHPA1-VHLL and xStAx-VHLL, respectively. We found that xStAx-VHLL manifested strong inhibition of Wnt signaling and sustained degradation of β-catenin in cancer cells and the intestinal organoids derived from wild-type and 21, we and Verdine group designed stapled helical peptides that specifically targets β-catenin to modulate Wnt signaling14. Although two of the stapled peptides (SAHPA1 and xStAx) shared high similarity in sequences, SAHPA1 acted as an agonist for Wnt signaling while xStAx functioned to inhibit the pathway.Based on the observation that the β-catenin binding domain of Axin (amino acid 469–482) forms a stable continuous α-helix and fits into a shallow groove of β-catenin16, is employed as a ligand for the VHL E3 ligase recruitment. SAHPA1 or xStAx is conjugated to the VHL ligand via amide bonds with a simple 6-Aminocaproic acid as the linker, generating the two PROTAC peptides: SAHPA1-VHLL and xStAx-VHLL  and pulldown assay, stapled peptides and the corresponding PROTACs displayed comparative binding potency with β-catenin  or with vehicle DMSO treatment  significantly impeded the growth of APC−/− organoids, and the inhibitory effect was observed at day 3 . The exon sequencing indicated the mutations of Wnt/β-catenin pathway components in all tumor tissue , TCF7L2, KRAS, TP53, SMAD4, PI3KCA, and others. For example, xStAx-VHLL manifested a strong inhibition function on C-08 and C-05 compared with the DMSO control  was purchased from Tianjin Nankai Hecheng Science & Technology Co. Ltd.2O = 95/2.5/2.5, v/v/v) for 2 h at room temperature. After completion of the cleavage reaction, TFA was evaporated by blowing with Argon. The crude peptides were obtained by precipitation with 40 mL of cold diethyl ether and centrifugation at 3500 r/min for 3 mins (three times). The supernatant diethyl ether was decanted from the centrifuge tube, the crude peptides were allowed to air dry and purified by RP-HPLC to give the final products.As a typical example, 400 mg Rink Amide MBHA resin was swelled with DCM (5 mL) for 20 min. Then the resin was treated with 20% piperidine/DMF/0.1 mol/L Oxyma pure twice (5 min × 2), followed by washing with DMF (five times), DCM (five times) and DMF (five times) at 35 °C. For coupling of the natural amino acid, Fmoc-AA-OH (1 mmol), DIC (1 mmol), Oxyma pure (1 mmol) and NMP (6 mL) were mixed for 1 min and then added to the resin at 60 °C. After 15 min, the resin was washed with DMF (five times), DCM (five times), and DMF (five times). The peptide couplings of S5 and R8 were carried out over a single 2 h coupling cycle using 2 eq. of the Fmoc protected amino acids at 60 °C. The deprotection, washing, coupling and washing steps were repeated until all the amino acid residues were assembled reagent. The peptide-bound resin was treated with 20% piperidine/DMF/0.1 mol/L Oxyma pure to remove the Fmoc group from the N-terminus. After the resin was washed, it was treated with 5 mL solution of acetic anhydride and pyridine (1:1) for 20 min. Then the resin was washed with DMF (five times), DCM (five times), and DMF (five times). The ring-closing metathesis reaction was carried out in 1,2-dichloroethane (DCE) at room temperature using Grubbs’ first-generation catalyst. The resin was washed with DCM (three times), and DCE (three times) and then treated with 10 mM solution of Grubbs’ first-generation catalyst in DCE. After the first round of the 2 h metathesis, we repeated the same procedure for a second round of catalyst treatment with fresh catalyst solution. Then the peptide-resin was washed with DMF (five times) and DCM (five times). Peptides were cleaved from their resin by treatment with cocktail trifluoroacetic acid (TFA) (TFA/Triisopropylsilane (TIPS)/HAfter elongation of the peptide and metathesis reaction were conducted on the resin according to a general protocol. Then, the Fmoc group was removed and the resin was treated with Fmoc-Ahx-OH (4 eq.), DIC (4 eq.), Oxyma pure (4 eq.) and NMP (6 mL) for 15 min at 60 °C. The Fmoc group was removed and the resin was treated with fluorescein isothiocyanate  and DIPEA (2 eq.) in DMF (6 mL) at room temperature overnight. The biotinylated labeling was carried out as routine amino acid. Cleavage was conducted following a general protocol and crude peptide was purified by RP- HPLC to give the final products.Antibodies used in this study were as following: β-catenin and caspase3 were purchased from Santa Cruz; GAPDH, cleaved caspase3, Axin1, Dvl2, GSK3β; TCF4 from Cell Signaling Technology and E-cadherin from BD Biosciences. XAV939 and VH-298 was purchased from MCE, MSAB from Sigma, PRI-724 and PNU-74654 from Selleck. Peptides used in this study, including SAHPA1, SAHPA1-VHLL, xStAx and xStAx-VHLL, were dissolved in DMSO for 10 mM stock solution.2. HCT116 cells were cultured in McCoy’s 5 A medium supplemented with 10% FBS (Hyclone) in a 37 °C humidified incubator containing 5% CO2. LoVo cells were cultured in DMEM medium supplemented with 10% FBS (Hyclone) in a 37 °C humidified incubator containing 5% CO2.Colorectal cancer cell lines SW480, HCT116, and LoVo were obtained from ATCC. SW480 cells were cultured in L15 medium supplemented with 10% FBS (Hyclone) in a 37 °C humidified incubator without CODual luciferase reporter assay system (Promega) was used. HEK293T cells, cultured in 24-well plates, were transfected with 200 ng Topflash plasmid and 30 ng Renilla plasmid for 3-well cells. At 12 h post-transfection, cells were treated with certain concentrations of peptides with or without Wnt3a conditioned medium (CM) for another 24 h. Then harvested the cells for luciferase activity measurement.g centrifugation for 10 min, the lysates were immunoprecipitated with specific antibody and protein A-Sepharose beads and then washed the beads with TNE buffer for three times before immunoblotting.For immunoprecipitation, the cells were lysed in TNE lysis buffer . For the β-catenin ubiquitination detection experiment, the raw cell lysates were heated to 95 °C for 5 min and sonicated to completely denature the proteins. After 18,000×36. Briefly, mouse intestine was cut longitudinally and washed three times with cold PBS. Villi were carefully scraped away and small pieces (5 mm) of intestine were incubated in 0.5 M EDTA in PBS for 40 min on ice. These pieces were then vigorously suspended in cold PBS and the mixture was passed through 70 μm cell strainer (BD Biosciences). The crypt fraction was enriched through centrifugation (3 min at 300–400 g). Then the crypts were embedded in Matrigel (BD Biosciences) and seeded on 48-well or 24-well plates. The intestinal organoid culture medium consists of Advanced DMEM/F12 supplemented with Penicillin/Streptomycin, GlutaMAX-I, N2, B27 and N-acetylcysteine (Sigma-Aldrich) and combination of growth factors including EGF , Noggin  and R-spondin1 (500 ng/mL). The organoids were treated with proper dilution of peptides in the medium.Mouse intestinal crypts isolation and organoid culture were performed as previously described37. All samples were obtained with informed consent, and the study was approved by the Peking University Third Hospital Medical Science Research Ethics Committee (M2018083). All relevant ethical regulations of Peking University Third Hospital Medical Science Research Ethics Committee were followed.Intestine tumor tissues were surgically resected and sampled, from 12 patients who had been diagnosed with intestine tumors at Peking University Third Hospital, Beijing, China, as referred beforeCRC patient tumor tissues were washed with cold HBSS. After removing the muscle tissue, the epithelial tumor tissues were cut into small pieces and vigorously suspended. Then the tissue suspension was centrifuged (3 min at 300–400 g) and tumor fraction was enriched at the bottom. Next, the tumor fraction was embedded in Matrigel (BD Biosciences) and seeded on 24-well plates with the culture medium, which includes Advanced DMEM/F12 supplemented with Penicillin/Streptomycin, GlutaMAX-I, N2, B27, N-acetylcysteine (Sigma-Aldrich), CHIR-99021 , A-83–01 , SB202190 , gastrin , Y27632 , PEG2 , nicotinamide , EGF , Noggin  and R-spondin1 (500 ng/mL).Lgr5, 5′-CGGGACCTTGAAGATTTCCT-3′ and 5′-GATTCGGATCAGCCAGCTAC-3′; Aixn2, 5′-GCTCCAGAAGATCACAAAGAGC-3′ and 5′-AGCTTTGAGCCTTCAGCATC-3′; Ccnd1, 5′-GCCATCCAAACTGAGGAAAA-3′ and 5′-GATCCTGGGAGTCATCGGTA-3′; Myc, 5′- GCTGTTTGAAGGCTGGATTTC-3′ and 5′-GATGAAATAGGGCTGTACGGAG-3′; and Gapdh, 5′-AAGAAGGTGGTGAAGCAG-3′ and 5′-TCATACCAGGAAATGAGC-3′.Total RNA was extracted with TRIzol (Invitrogen) and cDNA was prepared using Revertra Ace (Toyobo). Real-time PCR reactions were performed in triplicates on a LightCycler 480 (Roche). Relative target genes expression values were normalized to GAPDH expression. Data were analyzed according to the ΔCT method. The following primers were used: Equal number of cells  were seeded in the 24-well plates and then subjected to different treatments. The cell numbers were counted every 24 h with the CCK-8 cell counting kit (Dojindo Molecular Technologies) according to manufacturer’s instructions.6 cells. Three weeks later, the nude mice were sacrificed and dissected for tumors. Tumor weight was quantified.LoVo cells cultured were pretreated with DMSO pr peptides (50 μM) for 24 h. Then the cells were digested with trypsin and centrifuge (3 min at 300–400 g). Cell pellets were washed with cold PBS three times and the cell number was counted. The BALB/C nude mice were subcutaneously injected with 3 × 10APCmin/+ mice and fixed in 4% paraformaldehyde at room temperature overnight. Paraffin sections (5 μm) were cut . The sections were de-paraffinized in xylene and graded alcohols, followed by antigen retrieval in 95 °C water bath for 25 min, followed by permeabilization with 0.1% Triton X-100 for 15 min at 4 °C and block for 1 h at room temperature in 5% BSA. After incubation overnight at 4 °C with the primary antibody , the fluorescein-labeled secondary antibody  was added for 1 h at room temperature. The sections were scanned using Olympus FV3000 Laser Scanning Microscope.The small intestines were isolated from Axin2LacZ reporter mice, washed with PBS and fixed in 4 % paraformaldehyde for 1 h at room temperature, then cut into small pieces (1 cm). The tissue pieces were stained with in situ β-galactosidase staining kit (Beyotime) according to the manufacturer’s instruction. The stained tissues were transferred to tissue cassettes and paraffin blocks prepared using standard methods. The tissues were sliced into 5 μm thick sections with paraffin slicing machine  and subjected to hematoxylin staining.The small intestines were isolated from APCmin/+ mice and relative Axin2 expression level, statistical analysis was performed with one-way ANOVA. Results were presented as mean ± SD. P < 0.05 was considered statistically significant: *P < 0.05, **P < 0.01, and ***P < 0.001.All experiments were carried out with at least three biological replicates. GraphPad Prism 6 software was employed for statistical analysis. For reporter assays, qRT-PCR, cell proliferation assays, immunofluorescence marker intensity and organoid experiment, statistical analysis was performed with two-way ANOVA. For immunoblot band intensity, tumor weight of nude mice, tumor number of Supplementary Figures


The epoxy compound samples were seasoned for the following period of time . The environment was tap water and the acidic environment had three different concentrations of acetic acid . Strength tests of the epoxy compound samples were carried out in accordance with the ISO 604 standard. In the case of the modified composition, it is noted that the samples immersed in tap water were characterized by a higher strength than in acidic environments. A similar tendency was observed for unmodified compositions, although the differences were smaller than for the modified compositions. It was also noticed that the increase in the pH of the acidic solution in many analyzed cases contributed to the decrease in mechanical properties, although the immersion time in the acidic solution is important.The aim of this work was to determine the impact of the acidic environment on the mechanical properties of two epoxy compounds in different conditions. The samples were made from the epoxy compounds composed of the epoxy resin (based on Bisphenol A), triethylenetetramine curing agent (unmodified compound), and calcium carbonate (CaCO A wide variety of epoxy resins have become the most popular polymers since the mid 20th century ,2. TheseThe important and typical characteristics for the epoxy compounds are high mechanical resistance, good adhesion to different types of materials, and resistance to weather and environmental effects as well as the possibility of modification to obtain specific properties ,9,10,11.Works dealing with the analysis of the degradation process of epoxy compositions in various aqueous environments have previously been published ,30,31,323, and the issues related to the modification of epoxy resins with this additive are presented in several papers [3, as a modifying agent, is considered to be a suitable substitute for rubbery elastomer in the epoxy resin matrix due to the fact that it is characterized by high rigidity and may influence the behavior of the rubber elastomer [3 particles [3 particle size and the ratio of kenaf to CaCO3 content on the impact strength of the hybrid composite based on the epoxy resin [3 reinforced epoxy resin composite based on the values of the input concentration and sustained stress at impact was tested by Nwoye et al. [3 particles and different polyvinyl chloride (PVC) content on the mechanical and electrical properties of epoxy composition [3 content on the mechanical and thermal properties of epoxy/nano-CaCO3 composites was tested by He et al. [3 [3 and Al2O3 fillers on the mechanical properties of glass/epoxy composites was studied by Sai Sravani et al. [3 powder was investigated by Al-Zubaidi et al. [3 powder to the epoxy resin matrix produced a harder, less ductile, and better heat insulator product.The processes occurring in epoxy compounds, epoxy compositions during aging, or interaction in various environments, based on the environmental factors, are also affected by the type of epoxy compound itself as well as the type of additive/modifying agent. One such additive is CaCOl papers ,39,40,41lastomer . Li et aarticles . Sosiatixy resin . The pree et al. . Abd preposition . The effe et al. . He et at al. [3 . The effi et al. . Mechanii et al.  and the 3 can have a negative impact on the mechanical performance of polymers. Sai Sravani et al. [3 in the epoxy matrix (in cured state) can increase the thermal stability and mechanical properties of the modified epoxy material. On the other hand, with regard to the exploitation process in the aquatic environment, He et al. [3 particles. The effect of nano-CaCO3 on the mechanical properties was found to be positive, which is promising for the application of modified epoxy resin in high-humidity environments. However, different environments and exposure time can affect the properties of the modified epoxy compositions.Some authors  have shi et al.  also indi et al.  demonstre et al.  presenteThe analysis of the compressive strength in the work answers the question of what load should be applied to damage the structure of the insulating material. The higher the compressive strength value, the greater the load on the material can be. This relationship is widely used in engineering because it allows the application of a specific material  where the load acting on building structures is mainly snow, wind, earth, and other materials including water .3) and an unmodified one.The main focus of the present article was on examining the impact of tap water and acidic environments of three different concentrations on the resistance of two types of epoxy compounds based on the epoxy resin (based on bisphenol A), taking into consideration different periods of exploitation. This article presents the research concerning the impact of one of the exposure variants, which was an acidic environment in the form of an aqueous solution of acetic acid, on the mechanical properties of two epoxy compounds: a modified calcium carbonate powder (CaCO3 powder (in the case of the modified compound) mixed in appropriate weight proportions were used to make test samples. The quantitative proportion of the constituents used is presented in Two types of epoxy compounds containing epoxy resin and curing agent (in the case of the unmodified compound) as well as CaCOEpoxy resin is a styrene-modified epoxy resin based on bisphenol A . The curing agent is an amine curing agent . The properties of the used epoxy resin and curing agent were presented in ,36. Stre3, in the form of a powder (micro-filler) with a molecular weight of 100.09 g/mol, which is an inorganic chemical compound-a salt of carbonic acid and calcium, was used as a modifier. CaCO3 is commercialized by Carl Roth GmbH + Co KG, Karlsruhe, Germany, with the trade name E170. According to the literature [3 as a modifying additive for resins or polymer composites is in the range of 2–8 parts by weight. Yang et al. [3 added, but decreased when the filler content reached 2%. He at al. [3 per 100 g of epoxy resin was used in the study.CaCOterature ,45, the e at al.  mentioneThe tests included two types of the epoxy compound samples whose characteristics were presented in The samples were made of two types of epoxy compound. Seventy-two samples were made of each type of epoxy compound , which is a transparent and scentless preparation used, among others, in rubber and polymer processing. Covering the forms with the aforementioned agent enabled us to create a thin layer of the silicone oil that made it possible to take the cured epoxy compound samples out later on.3) were measured with the use of electronic scales. The weight proportions used to prepare the specific epoxy compounds are presented in 3, this modifier was added to the epoxy resin first. The curing agent was added to the ingredients mixed beforehand. The mixing time of the three-ingredient compound (modified) was 2 min, which allowed us to distribute the CaCO3 in the epoxy compound evenly.The next phase was to prepare the epoxy compound. Taking into consideration the appropriate weight proportions, the particular ingredients of the specific epoxy compound , triethylenetetramine curing agent (Z-1), and CaCOThe conditions of preparing and curing the samples for both the strength and the microscopic tests were as follows: air temperature of 20 ± 2 °C and relative air humidity of 25 ± 2%. Time of curing in the forms was seven days. After that, the cured epoxy compound samples were taken out of the forms. They were then subject to mechanical processing in order to equalize their length.The epoxy compound (modified and unmodified) samples prepared in such a way were exposed to environmental factors (specific environment and seasoning time) . The unaThe operating environments presented in After the assumed seasoning time in the acidic environments , the epoCompression tests of the epoxy compound samples were carried out with accordance to the norm ISO 604 on the testing machine Zwick/Roell Z150 . The parameters used during the strength tests of the epoxy compound samples were the following: initial force of 10 N, compression modulus speed of 2 mm/min, crosshead speed of 10 mm/min, force cut-off point of 80% Fmax, and maximum unit shortening of 10%. Six samples were tested in each run of the strength tests.Microscopic tests were carried out with the use of the DiGi ViTiny UM06 microscope . The main objective of the microscopic tests was to observe the surface of the samples in order to determine the signs of influence of the exploitation environments, the exposure time, and presumable material discontinuity .The results of the compression tests  of the unmodified epoxy compound (Epidian 53/Z-1) seasoned in four water environments  for the When comparing the compressive strength of the unmodified compound’s samples with the same seasoning time, but different water environments a, it wasThe comparison of the results presented in In the shortest seasoning period of time (one week), the highest compressive strain was observed for the samples seasoned in the 9% acetic acid solution and was 7.1% c. The veThe microscopic test images conducted on the samples of the unmodified epoxy compound seasoned in exploitation environments are presented in 3 can react with acetic acid in some cases and thus would produce a porous structure. The most visible impact on the unmodified epoxy compound’s structure was exerted by the 3% acetic acid solution, whose samples were seasoned for three months. The sample’s surface was much rougher, the air bubbles that appeared on the surface were much bigger than in the case of the samples seasoned for a shorter period of time. The 6% acetic acid had the same impact on the samples seasoned for one month.The microscopic tests disclosed the tested epoxy compounds’ microstructure development, structure changes, and defects that are not distinguishable to the unaided eye. It was observed that as the seasoning time passed by, the samples seasoned in all the exploitation environments  changed their color . In addiThe samples of epoxy compounds were subject to the ideal axial and centric compression. During the compression of the epoxy compound samples, the following deformations were observed: buckling, spalling, and cracking. Several samples were completely damaged. 3) seasoned in four water environments  of the modified epoxy compound , the strength of the refrain samples was higher than that of the samples of the compositions seasoned in these environments, regardless of the seasoning time.With respect to the results of the compression modulus b, the fo3 seasoned in four water environments for the following periods of time: one week, one month, and three months  seasoned in the exploitation environments changed color as time passed by. A special change was observed for the sample seasoned for three months in the 6% acetic acid solution and 9% acetic acid solution (Like in the case of the samples made from the unmodified epoxy compound (Epidian 53/Z-1), the samples of modified epoxy compound  in many analyzed cases contributed to the decrease in mechanical properties, although the immersion time in the acidic solution was important. Mahmoud and Tantawi  underlin3, it can be noted that the unaged samples of the unmodified epoxy compound (Epidian 53/Z-1) had a higher strength than the unaged samples of the modified epoxy compound (Epidian 53/Z-1/CaCO3). This conclusion can be applied to all environments (water and acid solutions) and times used in the experiments, although in some of the compared cases, the differences were not very significant. Li et al. underlined that micron-sized CaCO3 may have a negative impact on the mechanical performance of polymers [3, however, showed a higher strength after seasoning. Higher values of the compressive strength after seasoning in different environments were observed for the modified epoxy compound 53/Z-1/CaCO3. The highest compressive strength was 15.2 MPa, where in the case of the unmodified epoxy compound Epidian 53/Z-1, the highest value was 9.6 MPa. The compressive strain at the compressive strength for both compounds was at a similar level. According to He et al., nano-CaCO3 particles have a large surface area and they may act as efficient barriers against moisture transport through the polymers [3 content can be explained by the growth of void content and interphase layer content [3 particles. Among all the samples made from the unmodified epoxy compound Epidian 53/Z-1, the highest compressive strength was observed for the samples seasoned in the 9% acetic acid solution for three months. The lowest compressive strength, in turn, was observed for the samples seasoned for three months in tap water (1.5 MPa). In the case of the modified epoxy compound 53/Z-1/CaCO3, the samples seasoned for one month in the 6% acetic acid solution showed the highest compressive strength (15.2 MPa). The least strong samples, in turn, were those seasoned for one month in the 9% acetic acid solution (2.7 MPa).Regarding the modification of the epoxy compositions with CaCOpolymers . Some repolymers , Calvez polymers , Bowditcpolymers , and othpolymers ,16,23,24polymers . In addipolymers . The tra content . MoreoveThe samples designed for the microscopic tests were also subjected to tests. After analyzing the microscopic images of the epoxy compounds, it was observed that after being seasoned in different exploitation environments and periods of time, all samples changed their color. One of the mechanisms of destruction of the surface layers of polymer composites during the exposure to water is a bubble formation, which at a further stage may cause degradation of the polymer composite. In the conducted research, the appearance of bubbles was noticed in the microscopic images in the case of a longer seasoning in water, which can be one of the causes of polymer degradation. The effect of bubble based degradation on the physical and structural properties of single wall carbon nanotube (SWCNT)/epoxy resin composite samples was investigated by Hashemi and Mousavi . Additio3. The tests concerned the mechanical properties  of the epoxy compounds that were subject to seasoning for different periods of time  in various exploitation environments . The obtained results showed the following:The aim of this work was to determine the impact of the acidic environment on the mechanical properties of two epoxy compounds in different conditions. The subjects of the present research were two types of epoxy compound made of epoxy resin based on bisphenol A, the triethylenetetramine curing agent, and a modifier in the form of powdered CaCONo significant effect of exposure time in selected operating environments on the compressive strength of epoxy compounds was observed.(i)In many cases, the unaged samples (not exposed to any water environment) of the unmodified epoxy compound had an insignificant higher compressive strength than the unaged samples of the modified epoxy compound, regardless of the seasoning variant, although there were different differences depending on the environment and immersion time.(ii)In the case of the modified composition, it was noted that the samples immersed in tap water were characterized by a higher strength than in acidic environments. A similar tendency was observed for the unmodified compositions, although the differences were smaller than for the modified compositions.(iii)The increase in the pH of the acidic solution in many analyzed cases contributed to the decrease (to a slight but noticeable degree) in the mechanical properties, although the immersion time in the acidic solution is important.3.It can be assumed that during the exploitation in acidic environments, some kind of chemical reaction is triggered between the modifier and the acetic acid aqueous solution in the epoxy compound modified with CaCO


APC loss, in CRC.Most cancer cells employ the Warburg effect to support anabolic growth and tumorigenesis. Here, we discovered a key link between Warburg effect and aberrantly activated Wnt/β-catenin signalling, especially by pathologically significant APC-loss or Wnt ligand on the identified enzymes, PKM2 and LDHA, as well as Warburg effects were investigated. A linkage between activation of Wnt/β-catenin signalling and cancer metabolism was analysed in tumour of Apcmin/+ mice and CRC patients. The roles of PKM2 in cancer metabolism, which depends on Wnt/β-catenin signalling, were assessed in xenograft-tumours.Proteomic analyses were performed to evaluate the global effects of KYA1797K, Wnt/β-catenin signalling inhibitor, on cellular proteins in CRC. The effects of APC-loss caused the increased expression of metabolic genes including PKM2 and LDHA, and increased glucose consumption and lactate secretion. Pathological significance of this linkage was indicated by increased expression of glycolytic genes with Wnt target genes in tumour of Apcmin/+ mice and CRC patients. Warburg effect and growth of xenografted tumours-induced by APC-mutated-CRC cells were suppressed by PKM2-depletion.By proteomic analysis, PKM2 and LDHA were identified as key molecules regulated by Wnt/β-catenin signalling. APC loss activates the Warburg effect in CRC.The β-catenin-PKM2 regulatory axis induced by  However, the mechanistic basis by which cancer metabolism is controlled by oncogenes and tumour suppressor genes varies in different tumour types, and in most cases, the molecular mechanisms that induce the alterations in the expression of the metabolic enzymes are poorly understood. Furthermore, although cellular metabolism reprogramming is considered as a critical event during tumorigenesis, the regulatory mechanisms and signalling pathways that initiate and control it remain elusive.Cancer cells, in contrast with normal cells, exhibit an altered metabolism, increased aerobic glycolysis, and decreased oxidative phosphorylation.12 Aberrant activation of Wnt/β-catenin signalling has been frequently found in various cancers. Colorectal cancer (CRC) is the most notable cancer type associated with activation of Wnt/β-catenin signalling because loss of function mutations of adenomatous polyposis coli (APC) occur in up to 90% of human CRC patients.13 APC plays a role as a gatekeeper in colorectal tumorigenesis, and APC loss initiates the pathogenesis via stabilisation and subsequent nuclear translocation of β-catenin for transcriptional activation of target genes involving cell proliferation and transformation.13 CRC development caused by this aberrant Wnt/β-catenin signalling is regarded to most likely result from the inappropriate activation of genes.14 Recently, speculative links between Wnt/β-catenin signalling and cancer metabolism have been poised in several cancers.18 However, regulation of cancer metabolism by Wnt/β-catenin signalling, especially APC mutations, implying the major pathological significance of activation of Wnt/β-catenin signalling and initiation of CRC, is poorly characterised.The Wnt/β-catenin signalling pathway is a major pathway that regulates important biological processes including normal development and oncogenesis.19 The Wnt/β-catenin-signalling-dependent regulation of the metabolic enzyme expression was confirmed by various in vitro studies. The positive relationships between the expression of metabolic enzymes and β-catenin were further confirmed in tumour tissues of CRC patients, indicating the pathological significance of the linkage. The APC mutation-induced Wnt/β-catenin signalling activation increased the PKM2 and LDHA along with the Warburg effect in CRC cells. The induction of PKM2, a key enzyme mediating the Warburg effect,21 by APC loss occurs via β-catenin/Tcf4-mediated transcription. The role of Wnt/β-catenin signalling in the pathogenesis of CRC related to the Warburg effect is shown by elevation of PKM2 and subsequent increment of the glycolytic genes including LDHA and glucose transporter (GLUT1) due to APC loss. In addition, the critical role of PKM2 in tumour growth induced by APC-mutated CRC cells was confirmed in vivo by xenograft mouse model.In this study, through systematic proteomic analysis, we identified significant decreases in the levels of glycolytic enzymes such as pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) in DLD1 cells treated with KYA1797K, a small molecule that inhibits Wnt/β-catenin signalling by degradation of β-catenin.APC loss, and the Warburg effect in CRC. In addition, these results suggest that PKM2 could be a potential therapeutic target for CRC caused by aberrant Wnt/β-catenin signalling activation.These findings provide a key link between aberrantly activated Wnt/β-catenin signalling, especially due to Human CRC cells , U87 cells, and human embryonic kidney (HEK) cell line 293T cells were obtained from the American Type Culture Collection . DLD1, SW48, SW480, and HCT15 cells were cultured in in RPMI 1640 medium (Gibco) supplemented with 10% foetal bovine serum , and RKO, WiDr, U87, and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium  containing 10% FBS. HCT116 cells were maintained in McCoy’s 5a Medium (Gibco) containing 10% FBS. Mycoplasma contamination tests were performed for all cells used in this study. KYA1797K was dissolved in dimethyl sulfoxide  for the in vitro studies. Recombinant wnt3a, EGF and bFGF were purchased from PeproTech.6 DLD1 cells per well were seeded in 100 cm2 plates and treated with DMSO or 25 μM KYA1797K for 3 h. The cells were harvested and lysed in the sample buffer ) complemented with complete protease inhibitors (Roche) on ice for 30 min by adding DNase I. The lysates were applied to immobilised pH 3–10 nonlinear gradient strips (Amersham Biosciences) and isoelectric focusing was performed at 80,000 Vh. The second-dimensional separation was performed in 9–16% linear gradient polyacrylamide gels at a constant 40 mA per gel for ∼5 h. After protein fixation in 40% methanol and 5% phosphoric acid for 1 h, the gels were stained with Coomassie brilliant blue G-250 for 12 h. Stained gels were scanned using a GS-710 imaging densitometer (Bio-Rad) and analysed with an Image Master 2-DE Platinum image analysis program (Amersham Biosciences). Expression levels of the spots were determined by the relative spot volume of proteins compared with the volume of a single spot in the gel using the Melanie II program (GenBio).22A total of 3 × 1023 Aliquots of the peptide mixtures obtained from digestion were applied onto a target disk and allowed to air-dry. Spectra were obtained using a Voyager DE PRO MALDI-TOF spectrometer (Applied Biosystems). Protein database searching was performed with MS-Fit24 using monoisotopic peaks. A mass tolerance was first allowed within 50 ppm, and then recalibration was performed at 20 ppm after obtaining the protein lists.For mass spectrometry fingerprinting, protein spots were cut out of the gel and digested using trypsin (Promega), as previously described.4 cells per well were seeded in 12-well plates. Forty-eight hours after plating, the medium was collected, and the glucose and lactate levels were examined using a glucose colorimetric/fluorometric assay kit (Biovision) and lactate colorimetric/fluorometric assay kit (Biovision), respectively, according to manufacturer’s instruction. Glucose and lactate were calorimetrically measured at 590 nm using a FLUOstar OPTIMA (BMG LABTECH), and the glucose consumption and lactate production were normalised to cell number.A total of 5–7 × 10PKM2 was subcloned into the pGL3-Basic vector  to obtain a pGL3-PKM2 promoter-LUC plasmid. The pGL3-PKM2 promoter-LUC plasmid was co-transfected with internal control pCMV-β-galactosidase  reporter plasmid (Clontech). When treating Wnt3a proteins, the cells were treated with 50 ng/ml of Wnt3a with fresh media for 24 h after 24 h of transfection. Forty-eight hours after transfection, cells were harvested and lysed in Reporter Lysis Buffer (Promega) according to the manufacturer’s instructions. Luminescence and β-galactosidase activity were measured with a FLUOstar OPTIMA (BMG LABTECH). Luciferase activity was normalised to the β-galactosidase activity. Relative luciferase activity was normalised to the control for each experiment.The promoter of ApcMin/+ (Apcmin/+) mice were obtained from Jackson Laboratory . Mice were housed in micro-ventilation cage system (MVCS) cages with a computerised environmental control system (Threeshine Inc.). The temperature was maintained at 24 °C with a relative humidity of 45–55%. Mice were euthanised by carbon dioxide. To generate Apcmin/+ mice, Apcmin/+ mice were crossed with C57BL/6J+/+ (WT) mice. Mouse genotyping was performed using genomic DNA extracted from the tail, and we used male mice for further animal experiments. To control genetic background effects, sex-matched littermates were always used. To investigate the expression level of glycolytic enzymes and β-catenin in WT and Apcmin/+ mice, 12-week-old mice were used and were euthanised by carbon dioxide . For the study of in vivo efficacy of KYA1797K, 5-week-old Apcmin/+ mice were randomly assigned to 2 groups receiving either vehicle or KYA1797K. The mice were injected i.p. with vehicle  or KYA1797K  dissolved in the vehicle 5 days per week for 7 weeks. At that time, the mice were euthanised by carbon dioxide and sacrificed. Immediately after sacrifice, the abdomen of each mouse was cut open longitudinally and cleaned by flushing with PBS. Proximal regions of the small intestine were dissected, stained with 0.025% methylene blue (Sigma-Aldrich) for 2 min, and fixed with 4% paraformaldehyde (PFA). Gross images of tumour tissues were captured, and the tissues were resected and embedded in paraffin according to standard procedures. The tumours were classified according to standard World Health Organization histopathological criteria. For histopathologic analyses, a subset of freshly isolated tissues was snap frozen in liquid nitrogen and stored at −80 °C.All animal experiments were performed in accordance with the Korean Food and Drug Administration guidelines. Protocols were reviewed and approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine. C57BL/6J-athymic BalbC nu/nu mice were purchased from Joongabio Inc. Mice were housed in micro-ventilation cage system (MVCS) cages with a computerised environmental control system (Threeshine Inc.). The temperature was maintained at 24 °C with a relative humidity of 45–55%. After acclimatisation for 1 week, sixteen mice were randomly divided into two groups and injected subcutaneously in the dorsal flank with 2 × 107 of SW480 stably expressing control shRNA or shRNA targeting PKM2 (shRNA #1) in 100 µl of PBS:Matrigel  (n = 8 per each group) after anesthetised by i.p. injection of sterile avertin (tribromoethanol: 250 mg/Kg). The vital signs of mice were observed each week after injection, and no death was observed in the all groups. Tumours were measured using Vernier callipers, and tumour volume was calculated according to the following formula: π/6 × length × width × height. The mice were euthanised by carbon dioxide when the tumour volume exceeded 1500 mm3. Eighty-six days after injection, the mice were sacrificed, and the tumours were excised, weighed, and fixed in 4% PFA or snap frozen in liquid nitrogen for further analysis.All animal experiments were performed in accordance with the Korean Food and Drug Administration guidelines. Protocols were reviewed and approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine. Four-week-old male 25 All signals were analysed in a double-blind manner.Tissue microarrays (TMA) for normal and cancer tissues, colon disease spectrum tissue array , were purchased from US Biomax. IHC was performed with antibodies against PKM2, LDHA, or β-catenin. From the TMA, 30 normal tissue samples and 104 CRC adenocarcinoma tissue samples were used for further analyses. Signals of the TMA slides were analysed using a bright field microscope (Nikon TE-2000U). For quantitative analysis, the intensity of each staining was determined by IHC Profiler plugin.PKM2, two DNA fragments ; fragment 2, from −230 of intron 1 to 5′flanking region of exon2 (21 bp)) were obtained by PCR. The following primers were used: fragment 1, forward 5′-CCTACTATGCACCTAATGTGAGC-3′ and reverse 5′- ACACTTACTGAGTGTGCCACATCC-3′ (including EcoRV restriction site); fragment 2, forward 5′-GTCTAGGTAGATGTCAGTCAGCC-3′ (including SmaI restriction site) and reverse 5′-TTCGAGATGGCTGCTGAGGTCCTGG-3′. The two DNA fragments were ligated and inserted into the pGL3-Basic (pGL3) vector. The cloned plasmid was verified by DNA sequencing (Cosmogenetech). Myc-TCF4-pcDNA3.0 and Myc-ΔN-TCF4E-pcDNA3.0 (dnTCF4) vectors were obtained from Eric R. Fearon of the University of Michigan26 and pMD2G and psPAX2 were a gift from Dr. KunLiang Guan, University of California.27 β-gal reporter plasmid and pLKO.1puro-β-catenin (β-catenin shRNA) plasmids were purchased from Clontech and Addgene, respectively.To generate luciferase reporter plasmid containing the promoter of APC knockout (KO) RKO or WiDr cell lines were generated using CRISPR/Cas9 methodology28. Briefly, annealed oligonucleotides , targeting the APC exon 15 were cloned into the lentiCRISPRv2 (Addgene). The sequences of sgRNA were as follows: sgRNA-1 5′-CACCGTCGCTCTTCATGGATTTTTA-3′; sgRNA-2 5′-AAACTAAAAATCCATGAAGAGCGAC-3′. HEK293T cells were transfected with the lentiCRISPRv2 containing the APC sgRNA and packing vectors pMD2G and psPAX2 at a 2:2:1 ratio for viral production. Then, RKO or WiDr cell lines were transduced with the APC-lentivirus and selected with puromycin (Sigma-Aldrich) to generate the stable cell lines. For generation of PKM2 knockdown (KD) cell lines, PKM2 shRNAs were designed by Bionics . APC KO-RKO and -WiDr cells were transfected with pGPU6/hygro (shCON) or two independent pGPU6/hygro-PKM2 (shPKM2) vectors, and then selected in a medium containing hygromycin B . Of those, PKM2 shRNA #1 was used for real-time PCR analysis, glucose consumption, lactate secretion assays, and mouse xenograft assay. SW480 cells were transfected with the control shRNA or PKM2 shRNA #1 and then selected in a medium containing hygromycin B .t-test or Spearman correlation analysis. Results shown are representative of at least three independent experiments. Differences reached statistical significance with *P < 0.05, **P < 0.01 and ***P < 0.001. Statistical computations were performed using Prism software (Graph Pad).All data are expressed as the mean ± standard deviation (s.d.), and the number of samples is indicated in each figure legend. The statistical significance of differences was assessed using the Student’s 19 To evaluate the global effects of KYA1797K on cellular proteins related to CRC, we performed proteomic analyses for DLD1 cell extracts by using two-dimensional polyacrylamide gel electrophoresis (2-DE)  and ATP synthase are associated with the regulation of oxidative phosphorylation.31 The reduction of PKM2, LDHA, and ATP synthase by KYA1797K was confirmed in DLD1 cells and several other CRC cell lines  Fig. . Twelve DE) Fig. . These pDE) Fig. . Among tDE) Fig. ; PKM2, Lnes Fig. . The pro 32 Fig. , indicat 32 Fig. . Consist 32 Fig. . In addi 32 Fig. .Fig. 1SuAPC compared with those in CRC cells harbouring WT APC , and AXIN2 in CRCs compared to those expressed in adjacent normal tissues . PKM2 and LDHA were elevated with β-catenin in 70.2% (73/104) of CRCs compared with normal colon tissues Fig. . Furtherues Fig. . Gene seGLUT1, PFK1-M, and PFKBP1 were similarly increased by Wnt3a treatment  by KYA1797K treatment in the tumour area of the small intestine from Apcmin/+ mice compared with those of vehicle-treated mice  on its promoter, we tested its involvement in the transcriptional regulation of PKM2. The transcriptional activity of the PKM2 reporter harbouring the genomic DNA upstream of the transcription start site of the PKM2 was increased by APC KO -Tcf4 , a clinically important growth factor in CRCs, on PKM2 expression in CRC cells. The induction of PKM2 was specific to Wnt/β-catenin signalling in RKO CRC cells . The results may be attributed to the fact that the expression of c-Myc was affected by various mitogenic signals such as Sonic hedgehog (Shh) and EGF42 as well as Wnt/β-catenin signalling and other unknown factors. Therefore, we explored TCF-4, shown the correlation with PKM2 and β-catenin, as a mediator between PKM2 and the Wnt/β-catenin signalling in the Warburg effect of CRCs.PKM2 also reported to be regulated by EGF or EGFR, as shown by its upregulation and translocation into the nucleus followed by interaction with β-catenin for cell proliferation in brain tumours.APC mutation-induced Wnt/β-catenin signalling in the Warburg effect and tumorigenesis in CRCs: APC mutation increases β-catenin and enhances the β-catenin/Tcf4 binding on PKM2 promoter regions, which subsequently augments its transcription. The increased PKM2 further enhances the expression of other glycolytic enzymes and induces the Warburg effect together with the reactivation of β-catenin signalling in colorectal tumorigenesis.Most of our study focused on the regulation of PKM2 via Wnt/β-catenin signalling; however, we found that the Wnt/β-catenin pathway was also subjected to regulation via PKM2, as indicated by the decrease of β-catenin by PKM2 knockdown. Therefore, a positive loop that accelerates the activation of Wnt/β-catenin signalling and PKM2 expression could be present in the tumorigenesis accompanying cancer metabolism and the Warburg effect in colorectal tumorigenesis. On the basis of the results described in this study, we suggest the following model Fig.  for the APC mutation directs cancer metabolism and the Warburg effect via PKM2 in CRC. Therefore, targeting cancer metabolism, especially the key factor PKM2, could be an ideal approach for the treatment of CRC.Overall, our study suggests that aberrant activation of Wnt/β-catenin signalling such as by pathologically important Supplementary information


High probability of metastasis limited the long-term survival of patients with hepatocellular carcinoma (HCC). Our previous study revealed that Galectin-3 was closely associated with poor prognosis in HCC patients.The effects of Galectin-3 on tumour metastasis were investigated in vitro and in vivo, and the underlying biological and molecular mechanisms involved in this process were evaluated.Galectin-3 showed a close correlation with vascular invasion and poor survival in a large-scale study in HCC patients from multiple sets. Galectin-3 was significantly involved in diverse metastasis-related processes in HCC cells, such as angiogenesis and epithelial-to-mesenchymal transition (EMT). Mechanistically, Galectin-3 activated the PI3K-Akt-GSK-3β-β-catenin signalling cascade; the β-catenin/TCF4 transcriptional complex directly targeted IGFBP3 and vimentin to regulate angiogenesis and EMT, respectively. In animal models, Galectin-3 enhanced the tumorigenesis and metastasis of HCC cells via β-catenin signalling. Moreover, molecular deletion of Galectin-3-β-catenin signalling synergistically improved the antitumour effect of sorafenib.The Galectin-3-β-catenin-IGFBP3/vimentin signalling cascade was determined as a central mechanism controlling HCC metastasis, providing possible biomarkers for predicating vascular metastasis and sorafenib resistance, as well as potential therapeutic targets for the treatment of HCC patients. In fact, both EMT and angiogenesis are complex processes with the potential involvement of multiple signalling pathways; therefore, a better understanding on molecular underpinnings of them will allow more progress in metastatic HCC-target therapy.Epithelial-to-mesenchymal transition (EMT) plays a key role in the early phase of metastasis, during which cells lose cell–cell interactions, due to the lack of E-cadherin expression, and acquire increased motility, invasiveness and extracellular matrix-degrading capability.LGALS3, belongs to the β-galactoside-binding protein family. As a cytosolic protein, Galectin-3 can easily cross the intracellular and plasma membranes into the nucleus and mitochondria or be externalised, regulating several biological processes.17 Numerous studies have indicated that Galectin-3 plays a significant role in tumour progression in distinct manners, such as angiogenesis, chemo-resistance, immunosuppression and metastasis.19 In addition, activation of β-catenin pathway by Galectin-3 has been reported in multiple cancer types including breast, lung, colon cancers, leukaemia and sarcomas.20 Our previous study showed that Galectin-3 was a prognostic factor in patients with HCC,21 indicating that Galectin-3 may be a crucial regulator of HCC progression, even the metastatic process. In the present study, we aimed to comprehensively investigate whether Galectin-3 is involved in the metastasis of HCC and evaluate the possible molecular mechanisms underlying this process.Galectin-3, a multifunctional protein encoded by n = 60) were obtained from patients with primary HCC at the Sun Yat-sen University Cancer Center from 2005 and 2008. After resection, fresh tissues were immediately immersed in RNAlater® , incubated overnight at 4 °C, and subsequently stored at −80 °C until RNA isolation. For immunohistochemistry (IHC) or immunofluorescence (IF) staining, the training set, a total of 287 HCC tissues were collected at the Cancer Center of Sun Yat-sen University from 2005 to 2008. Patients with tumour node metastasis (TNM) stage III received postoperative adjuvant sorafenib therapy. The criterion for sorafenib resistance is that CT scans show the progression of tumour lesions after treatment for more than 1 month. The criterion for sorafenib sensitivity is that CT scans show the tumour lesions regress or are stable after treatment for more than 1 month, and this phenomenon lasted for more than 4 weeks. The testing set was an HCC tissue microarray purchased from Outdo Biotechnology Company , which included 90 pairs of cancerous and adjacent non-cancerous tissues. These tissues in this set were collected between June 2007 and November 2008. None of the patients in these two sets received no prior anticancer treatments. All the tissue samples in the two sets were collected within 30 min after operation and snap-frozen in liquid nitrogen.For real-time quantitative reverse transcription-polymerase chain reaction (qPCR), HCC tissues and adjacent normal tissues (n = 5 each) and hypodermically injected with Hep3B-vector, Hep3B-Galectin-3, Hep3B-Galectin-3-shControl, Hep3B-Galectin-3-shβ-catenin, Huh7-shControl, Huh7-shGalectin3, Huh7-shGalectin-3-vector and Huh7-shGalectin-3-β-catenin (5 × 106 cells in 100 μL PBS). Tumour growth was monitored once per week using an in vivo imaging system  at day 7, 14 and 21. Mice were killed by cervical dislocation after being anaesthetised with 10% chloral hydrate at day 22. All the xenografts were harvested, and tumour weight was measured. IHC staining was performed to examine the expression of CD34, IGFBP3 and vimentin.All animal procedures complied with the ARRIVE guidelines and were carried out according to the National Institutes of Health guide for the care and use of Laboratory animals. All cells used in in vivo study were stably expressed luciferase. To explore the effect of Galectin-3 on tumorigenesis and the role of β-catenin in this process in mice, forty 6-week-old female BALB/c nude mice  were randomly divided into eight groups (n = 5 each) and injected with Hep3B-vector, Hep3B-Galectin-3, Hep3B-Galectin-3-shControl, Hep3B-Galectin-3-shβ-catenin, Huh7-shControl, Huh7-shGalectin-3, Huh7-shGalectin-3-vector and Huh7-shGalectin-3-β-catenin (2.5 × 106 cells in 100 μL PBS) via tail vein injection. Tumour metastasis was monitored once per week using an in vivo imaging system at day 14, 21 and 28. All mice were killed by cervical dislocation after being anaesthetised with 10% chloral hydrate at day 29, and lung weight was used to measure the lung metastasis. H&E staining was performed to examine the numbers of micro-metastatic pulmonary nodules.For metastasis model construction, forty 6-week-old female BALB/c nude mice were randomly divided into eight groups (n = 10 each) and hypodermically injected with Huh7-shControl, Huh7-shGalectin-3, Huh7-shGalectin-3-vector and Huh7-shGalectin-3-β-catenin (5 × 106 cells in 100 μL PBS). Each group was randomly dived into two groups  at day 7. DMSO or sorafenib (Sigma–Aldrich) was orally administrated  until day 28. Tumour growth was monitored once per week using an in vivo imaging system from day 12 to day 26. All mice were killed by cervical dislocation after being anaesthetised with 10% chloral hydrate at day 27. Xenografts were harvested and tumour weight was measured.To assess whether blockade of Galectin-3-β-catenin signalling has an effect on the sensitivity of HCC cells to sorafenib, forty 6-week-old female BALB/c nude mice were randomly divided into four groups  kinetic curves were firstly performed to determine peak signal time of for the tumour growth and metastasis mouse models by using in vivo imaging system, which were about at 10 min and 15 min after intraperitoneal injection of luciferin, respectively. Then, the luminescence of each mouse in tumour growth and metastasis models were recorded at 10 and 15 min after luciferin injection, respectively. Finally, the total photo flux of each mouse in all sets were quantified by Living Image software.For IHC staining, formalin-fixed paraffin-embedded sections were deparaffinised in xylene, hydrated with ethanol gradient, and washed briefly in tap water. Endogenous peroxidase was blocked with methanol containing 0.3% hydrogen peroxide for 30 min. To restore antigenicity, slices were boiled in 10 mM citrate buffer (pH 5.8) in a microwave oven for 30 min. Next, the sections were incubated with goat serum diluted in PBS at 22 °C for 30 min. Subsequently, the sections were incubated with BATF3 specific primary antibody diluted 1:200 at 4 °C overnight . The next day, the sections were washed with fresh PBS and incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 30 min. Finally, the sections were stained with 3,30 diaminobenzidine (DAB) substrate  and counterstained with Mayer haematoxylin. Five random fields per sample were assessed using a light microscope . The intensity of the IHC staining was evaluated using the following criteria: 0, negative; 1, low; 2, medium and 3, high. The extent of IHC staining was scored as 0, 0–10% stained; 1, 10–40% stained; 2, 40–70% stained and 3, 70–100% stained. The final scores were calculated by multiplying the scores of the intensity by those of the extent. All samples in each set were divided into four grades according to IHC score: 0, −; 1–3, +; 4–6, ++ and 7–9, +++. Furthermore, “−” and “+” were grouped into “low expression”, while “++ and + ++” were grouped into “high expression”.For cell IF staining, cells were fixed in 4% paraformaldehyde for 15 min, washed twice with cold PBS and then incubated with antibody at 4 °C overnight. After washing with PBS, the cells were incubated with an appropriate fluorescent dye-conjugated secondary antibody for 1 h, and then nuclear staining with DAPI was performed for 10 min. Observe the cells under a fluorescent microscope.Tissue IF staining was performed using a Pano-Panel Kit according to the manufacturer’s instructions . Tissues or cells were examined for the expression of Galectin-3, β-catenin, CD34, IGFBP3 or vimentin. Images of IF staining were taken and quantitatively analysed using tissue microarray scanner Vectra 2 software .The following rabbit anti-human primary antibodies were used for IHC and IF staining: Galectin-3 , β-catenin , CD34 , IGFBP3 (Abcam) and vimentin .22 Total cellular protein was extracted in lysis buffer by radioimmunoprecipitation assay. The protein concentration was determined by using the bicinchoninic acid kit . Load the same amount of protein onto 10% SDS-polyacrylamide gel electrophoresis gel. After electrophoresis, the protein was blotted onto a polyvinylidene fluoride membrane. The membrane was blocked in 5% skim milk and incubated with the primary antibody overnight at 4 °C, and then treated with secondary antibody at 37 °C for 2 h. The primary rabbit anti-human antibodies used were as follows: ECAD , NCAD , vimentin (Proteintech Group), MMP1 , MMP3 (Abcam), IGFBP3 (Abcam), β-catenin , Akt , phospho-Akt , GSK-3β , phospho-GSK-3β , TBP (Proteintech Group) and β-actin .The protocol used for western blotting is described elsewhere.IGFBP3 and VIM promoter region as follows: 5′-TGTTACTTGTCAAAGCCACTTT-3′ and 5′-ATAGTCTCCTTTTATCTCCCTTG-3′.The ChIP assay was performed according to the manufacturer’s instructions . Anti-β-catenin  and anti-TCF4  antibodies were used to immunoprecipitate the chromatin in HCC cells. qPCR was performed using primers identified for the TCF4-binding site in the https://genome-cancer.ucsc.edu). Gene expression levels based on the mRNA sequencing data of TCGA are shown as the mean ± standard error of the mean (SEM) of triplicate determinations. Survival, Gene Ontology (GO), Gene Set Enrichment (GSEA) and correlation analyses in HCC patients were performed according to the TCGA mRNA sequencing data and follow-up information. All analyses were performed using the R Package ‘Survival’.The TCGA dataset for HCC was obtained from the UCSC Cancer Browser . P < 0.05 was considered statistically significant. Other materials and methods used in the present study are listed in Supplementary Materials and Methods.Seventeen Data are shown as the mean ± S.D. and were analysed using the Student’s 21 In the present study, we constructed a large-scale model of HCC by increasing the number of cases to 278  and obtaining an HCC tissue microarray consisting of a further 90 cases  for validation. In both selected sets, mitotic index has been demonstrated as a vital prognostic biomarker of tumour progression and metastasis . We further analysed the correlation between Galectin-3 and CD34 expression based on their IHC staining scores in the HCC samples in the training, testing and vascular invasion sets. As expected, a strong correlation between the expression of these two proteins was exhibited in all three sets, especially in the vascular invasion set  organisation, regulation of cell adhesion and vasculature development Fig. , all of 9 we performed an IF assay for phalloidin, an F-actin stain, to assess the effect of Galectin-3 on cell–cell adhesion and mobility. As shown in Fig. Next, we examined the expression level of Galectin-3 in five HCC cell lines. According to the results shown in Fig. 17 Accumulating evidences suggested that β-catenin signalling was responsible for tumour angiogenesis and EMT induced by aberrant protein expression.26 Furthermore, the GSEA results indicated that Galectin-3 had a significantly positive association with the Wnt signalling pathway  proliferation in the colony formation and CCK8 assays, which was disrupted by β-catenin overexpression , we also found that this mutation pattern mainly existed in well-differentiated HCC in this study . Moreover, the nuclear accumulation of β-catenin in patients with CTNNB1 mutation was higher than those without mutations according to IHC staining (Supplementary Fig. It has been reported that β-catenin signalling is also constitutively activated through activating mutations of its gene, CTNNB1. To discriminate the effects of Galectin-3 cascade versus CTNNB1 mutations in the activation of β-catenin pathway, we also assessed the mutational status of β-catenin in in exon 3. Consistent with other literatureIn summary, we show that Galectin-3 modulated angiogenesis- and EMT-driven metastasis via activation of the β-catenin signalling cascade by targeting IGFBP3 and vimentin in the HCC tumour microenvironment. Clinically, high expression levels of the components of the Galectin-3-β-catenin-IGFBP3/vimentin axis were closely correlated with vascular metastasis-driven sorafenib resistance and poor survival in large-scale HCC samples. The findings are of great significance for the further understanding of the mechanism underlying HCC metastasis. The Galectin-3-β-catenin axis may serve as a biomarker for predicting vascular invasion and poor prognosis, in addition to providing a set of effective targets for anti-metastatic HCC therapies. However, the clinical application of this axis requires further investigation.Supplementary file


Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by epithelial cell damage, myofibroblast activation, and collagen deposition. Multiple studies have documented that the Wnt/β-catenin pathway is aberrantly activated in IPF and plays a vital role in myofibroblast differentiation and activation. Kinases such as Src initiate Wnt/β-catenin signaling by phosphorylating β-catenin at tyrosine residues, which facilitates β-catenin accumulation in the nucleus and promotion of fibrosis progression. Nintedanib has been approved for the treatment of IPF as a multitargeted tyrosine kinase inhibitor. Nintedanib has been demonstrated to directly block Src, and whether it attenuates pulmonary fibrosis through regulating the Wnt/β-catenin pathway remains unclear. In this study, we found that nintedanib attenuated myofibroblast activation through inhibiting the expression of genes downstream of Wnt signaling such as Cyclin D1, Wisp1, and S100a4. Further experiments showed that nintedanib inhibited Wnt3a-induced β-catenin nuclear translocation through suppressing Src kinase activation and β-catenin Y654 phosphorylation. Additionally, Src knockdown fibroblasts exhibited a phenotype similar to that of the nintedanib treatment group, and the inhibitory effects of nintedanib were consistent with those of the Src kinase inhibitor KX2-391. In summary, our study shows that nintedanib exhibits an anti-fibrosis effect, partly by inhibiting the Src/β-catenin pathway. Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrosing interstitial pneumonia with an unknown etiology that occurs primarily in older adults . The annSeveral signaling pathways are thought to play specific roles in fibrogenesis.Wnt/β-catenin signaling has been shown to be a major regulatory pathway in adult organ reparation . In the In vitro studies have also revealed that it has signiﬁcant effects on the suppression of myofibroblast proliferation, migration, and transformation through blocking tyrosine kinases  family, the fibroblast growth factor receptor (FGFR) family, and the platelet-derived growth factor receptor (PDGFR) family . It was  kinases . In addi kinases , TGF-β . FastKing gDNA Dispelling RT SuperMix was obtained from Tiangen Biotech Co., Ltd. . The inhibitor KX2-391, RIPA lysis buffer (middle) and the BCA kit were purchased from Beyotime Biotechnology . The primary antibodies described in the study included anti-fibronectin, anti-collagen I, and anti-β-tubulin antibodies ; anti-GAPDH, anti-p(Y416)-Src, and anti-Src antibodies ; anti-α-actin antibody ; anti-β-catenin and anti-lamin B1 antibodies ; and anti-p(Y654)-β-catenin antibody . The secondary antibodies HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were from Abcam .Nintedanib (>99%) was purchased from HWRK Chem Co., Ltd. . For each experiment, nintedanib was freshly prepared by dissolving it in DMSO . Bleomycin (BLM) was acquired from Nippon Kayaku . Wnt3a was purchased from Peprotech . TRIzol reagent and DEPC-treated water were obtained from Thermo Fisher Scientific corporation . RNase, DNase, and DNA Away H2 in a humidified atmosphere.The mouse lung myofibroblast cells line (Mlg) were grown in DMEM  supplemented with 10% FBS . Cells were maintained at 37°C with 5% CO2 in a humidified atmosphere as described previously (Primary pulmonary myofibroblasts (PPF) isolated from male C57BL/6 J mice were cultured in DMEM supplemented with 10% FBS at 37°C with 5% COeviously . Cells aSix- to eight-week-old male C57BL/6 J mice were obtained from the Laboratory Animal Centre, Academy of Military Medical Sciences of People's Liberation Army . A total of 18 mice weighing between 20–23 g were used in the experiments. The mice were housed under controlled temperature (22–26°C) and a 12-h light-dark cycle. All animal care and experimental procedures complied with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Nankai University (Permit No. SYXK 2014-0003). Animal studies are reported in compliance with the ARRIVE guidelines .−1). Nintedanib was intragastrically administered daily for 1 week beginning 7 days after BLM injury. The control and model groups received an equal volume of vehicle (0.5% CMC-Na) using the same schedule and route of administration. Mice were sacrificed at day 14 after BLM administration for the evaluation of pulmonary fibrosis.Intratracheal BLM administration was performed as described previously . In briein vitro experiments, nintedanib was used at two concentrations (1 and 2 μM) according to published studies and MTT assays . All luciferase results were obtained from three independent experiments in different times, and the ordinate of the scatter plot represents the ratio of firefly-luciferase to Renilla-luciferase.TCF/LEF promoters were cloned into the pGL4.49 luciferase reporter vector , and Mlg cells were transfected with luciferase reporter plasmids using PEI  to form the TCF/LEF-firefly luciferase (TOPFlash) assay system. Renilla-luciferase was used as an internal control. Cells were treated 18 h after transfection with a series of nintedanib for 8 h. The luciferase activity of cell lysates was determined using a luciferase assay system  as described by the manufacturer. Total light emission was measured using a GloMaxβ-actin) using the −ΔΔCT (cell samples) or −ΔCT (tissue samples) method in the experiments  for 10 min and blocked with 5% BSA for 60 min in a humidified chamber. Mlg cells were incubated with α-SMA antibody (1:100 dilutions) and β-catenin (1:200 dilutions) overnight at 4°C, respectively. After washing three times with PBS, the cells were incubated with fluorescein (FITC) AffiniPure goat anti-mouse IgG (H+L) or rhodamine (TRITC) AffiniPure goat anti-rabbit IgG (H+L) . Cell nuclei were labeled with DAPI , and the cells were photographed with a TCS SP8 confocal  microscope.The siRNA duplexes targeting mouse Src mRNA were obtained from Tsingke Biology Technology . The siRNA sequence was 5'-GAAGCUGAGGCAUGAGAAG-3'.® RNAiMAX Reagent  as described by the manufacturer. Cells were harvested and analyzed for mRNA and luciferase activity 48 h after transfection.The siRNAs  were transiently transfected into Mlg cells using LipofectamineThe data and statistical analysis complied with the recommendations for the experimental design and analysis in pharmacology   and treated Mlg with different doses of nintedanib. Next, we determined the RNA levels of the following activation makers: Col I, Fn, and α-SMA. The results showed that the expression of Col I, Fn, and α-SMA induced by Wnt3a was significantly decreased by nintedanib  and the initiation of EMT, and it has been correlated with increased β-catenin-dependent transcription in both A549 cells and primary AECs . As mentin vitro  of Nankai University (Permit No. SYXK 2014-0003).Conception and design: CY, HZ, and XiaohL. Collection and assembly of data: XiaowL, RD, and HY. Data analysis and interpretation: SG, QJ, KH, and RL. Manuscript writing: XiaopL and LZ. All authors read and approved the final manuscript.This study was supported by the National Science and Technology Major Projects for “Major New Drugs Innovation and Development” [Grant 2019ZX09201001], Fundamental Research Funds for the Central Universities, Nankai University , and Tianjin Science and Technology Project [Grant No. 17PTSYJC00070].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.


In normal physiology, endothelial cells (ECs) form a vital barrier between the blood and underlying tissue controlling leukocyte diapedesis and vascular inflammation. Emerging data suggest that neuronal guidance cues, typically expressed during development, have roles outside the nervous system in vascular biology and immune responses. In particular, Class III semaphorins have been reported to affect EC migration and angiogenesis. While ECs express high levels of semaphorin 3F (SEMA3F), little is known about its function in mature ECs. Here we show that SEMA3F expression is reduced by inflammatory stimuli and increased by laminar flow. Endothelial cells exposed to laminar flow secrete SEMA3F, which subsequently binds to heparan sulfates on the surface of ECs. However, under pro-inflammatory conditions, reduced levels of SEMA3F make ECs more prone to monocyte diapedesis and display impaired barrier function as measured with an electric cell–substrate impedance sensing system and a microfluidic system. In addition, we demonstrate that SEMA3F can directly inhibit the migration of activated monocytes. Taken together, our data suggest an important homeostatic function for EC-expressed SEMA3F, serving as a mediator of endothelial quiescence. Endothelial cells (ECs) form a vital barrier between the blood and underlying tissue, and are crucial for maintaining vessel homeostasis by controlling vascular tone, blood fluidity, endothelial permeability and the regulation of plasma constituents and leukocyte transendothelial migration . The obsIn particular, members of the Class III SEMA family have been reported to affect endothelial biology ,6,7. ClaIn this article, we confirmed the high expression of SEMA3F in ECs and demonstrated its modulation by shear stress and inflammatory factors. We further investigated the functional importance of SEMA3F and propose the involvement of SEMA3F in endothelial barrier function and monocyte migration. We set off by studying the expression of Class III SEMAs in ECs using available transcriptomics data on GPL570 platform, a commonly used human transcriptome microarray platform. Of the Class III SEMAs, SEMA3A, SEMA3C and SEMA3G are abundantly expressed by some of the ECs A. High aGiven the high expression of SEMA3F in ECs, we next assessed if inflammatory and hemodynamic conditions affected the expression of SEMA3F. For this we used human umbilical vein endothelial cells (HUVECs), in which we confirmed the expression of Class III SEMAs by qPCR A. Treatm2) for 1 day or 7 days and gene expression to static cultured ECs was compared. As expected, laminar shear stress induced the expression of shear-dependent transcription factor Kruppel Like Factor 2 (KLF2) after 1 day and further after 7 days, while oscillatory shear stress failed to do so  was validated using qPCR A. EndothThe morphological change strongly indicates the possibility of impaired endothelial barrier function after reduction of SEMA3F expression. To test this postulation we assessed the effect of SEMA3F KD on endothelial barrier function through the use of electric cell–substrate impedance sensing (ECIS)  and the Next, we assessed whether loss of barrier function upon SEMA3F knock down alters monocyte transendothelial migration with monocyte chemotaxis over Transwell filters containing endothelial monolayers with SEMA3F knock down or control cells. Consistent with the reduced barrier function, we found that loss of SEMA3F led to increased monocyte transendothelial migration F.Since SEMA3A can inhibit directional migration of monocytes , we inveIn this study, we confirmed the high expression of SEMA3F in ECs found with gene expression meta-analysis of omni datasets. We revealed a KLF2-dependent regulation of SEMA3F under laminar shear stress. Using an shRNA knock down model, we pinpointed an essential role for SEMA3F in maintaining the endothelial barrier function and inhibiting the mobility of monocytes. Collectively, these results support that SEMA3F expressed by ECs can serve as a mediator of quiescent ECs. The function of Class III SEMAs in EC biology has received a good amount of attention with SEMA3A being the most thoroughly investigated. SEMA3A binds to PLXNA2 (instead of PLXNA1 and PLXNA3 in the case of SEMA3F), which activates the Rho-family small GTPase Rnd1 and induces a collapse of the cytoskeleton ,16. The Instead of using exogenous SEMA3F, we studied the effect of reduced level of SEMA3F on EC barrier function, which should be more physiologically relevant. In line with SEMA3F being shown to inhibit stress fiber formation ,24, afteOne of the most common reasons for endothelial dysfunction is an adverse hemodynamic environment, evidenced by the fact that more atherosclerotic plaques can be found at bifurcations of vessels and regions with low vessel wall shear stress ,28. The One key aspect of endothelial function is the ability to synthesize and maintain a network of membrane-bound proteoglycans and glycoproteins at the endothelium lumen, called the glycocalyx . This glIn regard to leukocyte guidance, several members of the Class III SEMAs have been studied in the context of inflammation and leukocyte recruitment. Studies have revealed that these Class III SEMAs can dampen inflammation or mediate resolution of inflammation ,41,42,43In conclusion, we demonstrated a novel function of SEMA3F in endothelial quiescence. As a shear-inducible secretory factor, SEMA3F is essential for the endothelial barrier function and is able to regulate monocyte migration. Together these studies provide novel insight into the role of endothelial expressed SEMA3F as a mediator of endothelial homeostasis contributing to vascular health.Curated gene expression data of all human cell types that were profiled on Affymetrix Human Genome U133 Plus 2.0 Array platform (Accession: GPL570/HG-U133_Plus_2) were obtained using Genevestigator software . For eacv/v) and antibiotics. THP-1 cells  were cultured in RPMI 1640  supplemented with 10% FCS (v/v), antibiotics and 0.05 mM 2-mercaptoethanol. Primary HUVECs were isolated from in house human umbilical cords and were cultured in EGM2 medium  supplemented with antibiotics. Gelatin (0.5% w/v in water) coated plates or flasks were used for HUVECs. All experiments using HUVECs were repeated at least 3 times using cells from different donors. All cells were maintained in a 37 °C incubator with 5% CO2.HEK293T cells  were cultured in DMEM (Gibco) supplemented with 10% FCS  or RIPA  buffer were added for RNA analysis or protein analysis, respectively.Culturing cells under laminar or oscillatory flow was performed using an iBiDi flow system according to the manufacture’s instruction. Briefly, HUVECs were seeded into closed perfusion chambers  and allowed to adhere for 15 minutes. The 2 inlets of the slide were then connected to 2 syringes, which acts as the reservoir for medium. The tops of the 2 syringes were connected to an iBiDi pump, which introduces pressure on one of the syringes in alternate manner. A central valve was placed on the tube, which allowed alternation of the flow direction. For the laminar flow condition, the alternation of pressure introduction was synchronized with the valve-controlled alternation of flow direction, resulting in unidirectional flow over the cells with constant shear stress. For oscillatory flow, the pump still alternated similar to laminar flow, while the valve alternated every 1 second, creating oscillatory flow over the cells. The pump setup allowed perfusion of culture medium over the monolayer of ECs at a shear stress of 10 dyn/cmSecond generation lentiviral system was used for delivery of a plasmid containing the KLF2 gene or a plasmid containing short hairpin RNA (shRNA) construct targeting SEMA3F mRNA (SEMA3F KD) or KLF2 mRNA (KLF2 KD). Plasmids without the KLF2 gene (mock) or with a scrambled shRNA (scrambled) were used as controls. Briefly, packaging plasmids (pPAX), envelope plasmids (pVSVG) and plasmids containing shRNA constructs were transfected into HEK293T cells using the polyethylenimine transfection method. The culture medium containing lentivirus particles was harvested 2 days and 3 days after transfection and was filtered with 0.45 µM filters to exclude cell remnants. The lentiviral particle containing medium was then used to transduce HUVECs with dilution ratio of 1:10 in EGM2 medium. The inoculum was removed after 1 day. Puromycin  selection was done 2 days after the lentiviral transduction. Cells that were resistant to puromycin for 24 hours were subcultured and used in subsequent experiments.For quantification of mRNA expression, cells were lysed in Trizol  and total RNA was isolated using RNeasy miniKit  according to the manufacturer’s instructions. First strand complementary DNA synthesis was done using M-MLV reverse transcriptase kit  with oligodT as primer. Quantitative polymerase chain reactions (qPCR) were done using SYBR green/polymerase mix  on a Biorad CFX384 system. GAPDH served as the reference gene. List of primers used in qPCR is provided in w/v in TBST). The primary antibodies were prepared in 5% BSA solution: rabbit anti-human SEMA3F  and rabbit anti-human GAPDH . After overnight incubation of primary antibody at 4 °C, secondary antibody  was added and incubated for 1 hour. HPR signal was detected with a Pierce™ ECL Western Blotting Substrate  and captured by the Chemidoc system . The images were analyzed in ImageJ software. For Western blot analysis, cells were lysed with the ice cold RIPA buffer  supplemented with a Complete Protease Inhibitor Cocktail . The cell debris was precipitated by centrifuging at 14,000 rpm for 20 min and supernatant was harvested. Protein concentration in samples was measured using Pierce BCA Protein Assay Kit . For electrophoresis, 10 µg samples in 20 µL were prepared with loading buffer  with or without DTT, depending on whether the reductive environment was needed. After electrophoresis separation on a 4–15% polyacrylamide gel , proteins on gels were transferred onto PVDF membranes  using BioRad turbo transfer system . The membranes were blocked using 5% BSA solution , Gibco, Paisley, UK) for 10 minutes. Cells were then permeabilized with 0.1% triton-X100 for 2 minutes for F-actin and blocked by 5% BSA (w/w in HBSS) for 30 minutes. Except when staining for heparan sulfate and SEMA3F, then cells were not permeabilized and directly incubated with blocking solution. The primary antibody was prepared in 0.5% BSA (w/v in HBSS+): CDH5 mouse anti-human antibody , SEMA3F rabbit anti-human antibody  and heparan sulfate , 1:110). Overnight incubation of the primary antibody was done at 4 °C. The secondary antibody was also prepared in 0.5% BSA , together with Rhodamine Phalloidin  and Hoechst  to stain F-actin and cell nuclei. Fluorescence images were captured by the Leica sp5 confocal microscopic system and analyzed using ImageJ software.For immunofluorescence staining, HUVECs were cultured on µ-Slide 8 well  and fixated with 4% formaldehyde  were added to the wells by mixing the cell suspension with medium in the wells. The measuring was then resumed for at least 30 hours. For both baseline and experiment timepoints, impedance was measured at multiple frequencies every 5 minutes. Measurements at 4000 Hz were used for data analysis, because impendence is contributed mainly from cell junction resistance at this frequency.Electric cell–substrate impedance sensing (ECIS) assays were done using the ECIS Ztheta  device with the standard 8 well arrays  or 96 wells array , which measures the change in the EC monolayer barrier function in real time . The pla®  with 4 mg/mL type 1 collagen  in the extracellular matrix channel. After allowing the cells to adhere for 1 hour, culture medium was replace by a mix of Endothelial Cell Growth Medium MV2  and Pericyte Growth Medium  in a ratio of 1:1. The device was placed on a rocker platform with 7° angle of motion and eight minutes timed operation to allow continuous flow of medium in the microvessels. After 24 hours, the medium was refreshed and the HUVECs were cultured for 3-4 more days. To measure vessel permeability, the culture medium in the extracellular matrix (ECM) channels was replaced by HBSS+ buffers and the culture medium of the microvascular channels were replaced with HBSS+ buffer containing 125 µg/mL Albumin-Alexa 555 . Following this, the OrganoPlate® was placed in the environmental chamber  of a fluorescent microscope system (Nikon Eclipse Ti) and time-lapse images were captured. The permeability coefficient was calculated by determining the fluorescent intensities in the microvascular channel and in the ECM channel of the capture images. The fluorescent intensity of the ECM channel was normalized with the fluorescent intensities in the microvascular channel of each measured time point. This showed the change in intensity ratio inside the gel channel as a function of time. The scatter plot was fitted with a linear trend line to determine the slope. Finally, using Fick’s First Law the apparent permeability was determined as:http://rsbweb.nih.gov/ij/, NIH, United States) and Matlab (MathWorks).HUVECs with or without SEMA3F knockdown were seeded in gelatin coated microvascular channels of custom-made gradient design OrganoPlate4 cells/cm2 on the Transwell insert one day before the experiments and then kept in EGM2 (100 µL in the top chamber and 500 µL in the bottom chamber). Two hours before migration, EGM2 was replaced by migration medium (RPMI 1640 with 0.25% BSA) to let ECs adapt to the new medium. When indicated, 15 h before the experiment, THP-1 cells were treated with 2 ng/mL TNFα. Before the experiment, THP-1 cells were washed with migration medium and resuspended to a concentration of 1 × 107 per mL. Calcein AM  was added so that THP-1 cells would carry green fluorescence. After labeling, THP-1 cells were washed twice in migration medium and resuspended to a concentration of 1 × 106/mL. For SEMA3F pretreatment, 500 ng/mL SEMA3F  was added to the suspension and cells were incubated for 30 minutes. After 30 minutes, THP-1 cells were added to Transwell inserts and the inserts were placed on wells with 500 µL migration medium supplemented with 10 nM MCP1  with or without 500 ng/mL SEMA3F. The cells were allowed to migration for 2 hours. Thereafter, the cells in the bottom chambers (containing migrated THP-1 cells) were pelleted and lysed by 1% TritonX-100. Fluorescent signal of Calcein AM was measured by a colorimetric analyzer , representing the number of THP-1 cells migrated.Chemotaxis of THP-1 monocytes was measured using 24-well Transwell culture inserts with a 5 μm pore size filter , either coated with 10 µg/mL Fibronectin  or containing an endothelial monolayer. For transendothelial migration experiments, ECs were seeded at density of 4 × 10