Accumulating evidence implies that cell fusion is one of the driving forces of cancer invasion and metastasis. However, considerably less is still known about the triggering factors and underlying mechanisms associated with cancer-host cell fusion, particularly in inflammatory tumor microenvironment. In this study, we confirmed that inflammatory factor TNF-α could enhance fusion between squamous cell carcinoma cells 9 (SCC-9) and human umbilical vein endothelial cells (HUVEC). Further study revealed that TNF-α could promote up-regulation of syncytin-1 in SCC-9 and its receptor neutral amino acid transporter type 2 (ASCT-2) in HUVEC. Syncytin-1 acted as an important downstream effector in TNF-α-enhanced cancer-endothelial cell fusion. TNF-α treatment also led to the activation of Wnt/β-catenin signal pathway in SCC-9. The activation of Wnt/β-catenin signal pathway was closely associated with the up-regulation of syncytin-1 in SCC-9 and increased fusion between SCC-9 and HUVEC while blocking of Wnt/β-catenin signal pathway resulted in the corresponding down-regulation of syncytin-1 accompanied by sharp decrease of cancer-endothelial cell fusion. Taking together, our results suggest that Wnt/β-catenin signal pathway activation-dependent up-regulation of syncytin-1 contributes to the pro-inflammatory factor TNF-α-enhanced fusion between oral squamous cell carcinoma cells and endothelial cells. It is thought that cell fusion provides a profound and unifying explanation for aneuploidy, heterogeneity, metastasis, and drug resistance in cancer12567It is well recognized that cell fusion plays a crucial role in a variety of physiological events, such as fertilization, placentation, skeletal muscle formation, and tissue regenerationRecently, inflammation has also been suggested as a possible trigger for cell fusion11014Wnt/β-catenin is one of the most classic signal pathways16161718In the present study, we show that the pro-inflammatory factor TNF-α can activate Wnt/β-catenin signal pathway and thereby enhance the fusion between oral squamous cell carcinoma cells and endothelial cells via up-regulation of fusogenic protein syncytin-1. These results provide new insight into the interaction between oral cancer cells and endothelial cells, demonstrate a signal transduction pathway that links inflammation, Wnt/β-catenin signal pathway and cell fusion in tumor microenvironment, and also predict a novel potential role of chronic inflammation in tumor progression.We co-cultured equal numbers of RFP-labeled SCC-9 and GFP-tagged HUVEC cells for 24 h without any fusogenic reagents. Double fluorescent staining showed that hybrid cells were observed in co-culture system , demonstin vitro  folds under the 10 ng/ml TNF-α stimulation compared with untreated cells in vitro . FACS anExpression of syncytin-1 was detected in surgical specimens from 28 patients with Oral Squamous Cell Carcinoma (OSCC). Immunohistochemistry showed that syncytin-1 was expressed in 26 out of 28 human OSCC specimens (93%) and mainly localized in cellular membrane and/or cytoplasm, but the level of syncytin-1 expression showed different ; AccordiTo investigate whether TNF-α could regulate the expression of fusogenic protein syncytin-1 and its receptor ASCT-2, we examined the syncytin-1 and ASCT-2 expression in TNF-α stimulation group and control group, respectively. Western blot analysis showed that the constant stimulation of 10 ng/ml TNF-α actually induced a continuous and dramatic increase of syncytin-1 in SCC-9 and ASCT-2 in HUVECs during an observation period of 24 h. Quantitative analysis showed that the ratio of syncytin-1/GAPDH and ASCT-2/GAPDH are significantly higher than the control counterparts at each time-point ,f, demonSyncytin-1 has been regarded as an important fusogenic protein in placentogenesis. In this study, we inhibited the endogenous expression of syncytin-1 in SSC-9 cells by short hairpin RNA (sh-RNA) to investigate the biological function of syncytin-1 during cell fusion. Our results showed that knockdown of syncytin-1 significantly inhibited the mRNA and protein expression of syncytin-1 in SCC-9 cells by qRT-PCR assay  and westAfter knockdown of syncytin-1, RFP-tagged HUVECs were co-cultured with GFP-labeled sh-RNA of syncytin-1 in SCC-9 and GFP-labeled SCC-9, respectively. Cell counting analysis showed the transfection of syncytin-1 sh-RNA obviously inhibited the number of fused cells between SCC-9 and HUVEC in co-culture systems on day 1, 3, 7 . FACS asWnt/β-catenin pathway has been reported to play important roles in several molecular events, such as tumor metastasis, EMT, stem cell renewal and cell reprogramming. After activation or inhibition of Wnt/β-catenin signal pathway, fusion between SCC-9 and HUVECs were evaluated by using artificial counting and FACS on co-culture day 1, 3, and 7 in this study. As shown in When SCC-9 cells were pre-treated with β-catenin-specific sh-RNA (sh-β-catenin-SCC-9), the number of double-labeled fluorescent staining cells fell sharply to nearly 1/3~1/2 of the control co-culture group. In accordance with this, the bio-fluorescently-stained cells increased 2~3 folds when β-catenin was overexpressed/enhanced by transfection of en-RNA in SCC-9 (en-β-catenin-SCC-9) . The FACTo investigate whether syncytin-1 was responsive to Wnt/β-catenin pathway activation, the protein level of syncytin-1 was detected after treatment of the SCC-9 cells with different reagents. As shown in Cell-cell fusion, an essentially cellular event for normal development and tissue homeostasis, has been involved in cancer progression and attracted extensive attention in molecular oncology in recent years14It is well known that the presence of chronic inflammation is one of the most important characteristics in tumor microenvironment22212425261128Cell fusion is a strictly regulated, poorly understood, multistep process that involves cell-cell recognition, cell-cell adhesion and membrane-membrane fusion230Wnt/β-catenin is one of the most classic signaling pathways, and has been extensively linked with in tumor metastasis, EMT, cell reprogramming, and cancer stem cells (CSCs)16181224Collectively, the schema graph and the fusion rate of our experiments were showed in 2.SCC-9 cell line was kindly donated by professor Zhuan-Bian, which was purchased from American Type Culture Collection . The SCC-9 was cultured in DMEM/F12  added with 10% FBS . Human umbilical vein endothelial cells (HUVECs) were kindly provided by Professor Yi-Fang Zhao and Dr Hai-Xiao Zou; The HUVECs were cultured in EC basal medium 2  which was added with 4% fetal bovine serum(FBS) and EGM-2 growth factor mixture. The HUVECs was restricted in passage 3 to passage 7 when applied in our study. And all of the cells were cultured in an incubator which was kept in 37 °C and contained 5%COTM II kit  and on a 7500 ABI system . All the qRT-PCR data was normalized to GAPDH expression. The tests were repeated at least three times to confirm the consequences.The total RNA from SCC-9 and HUVECs were extracted via using TRIzol reagent . cDNA was developed from total RNA by making use of reverse transcription kit . The sequences were: syncytin-1: primer: 5′-GAAGGCCCTTCATAACCAATGA-3′; reverse: 5′-GATATTTGGCTAAGGAGGTGATGTC-3′; The qRT-PCR was performed by the instructions on the SYBR Premix Ex TaqTotal protein of SCC-9 and HUVECs was extracted by using M-PER  supplemented with Protease inhibitor and phosphatase inhibitor on ice. Then the BCA protein assay kit (Thermo Fisher Scientific Inc. USA) was employed to test the quantity of each sample. Afterwards, loading buffer (5×) was added to all of the protein solutions to form mixed liquor. And then solution were heated for 10 minutes at 95 °C. Next, Aliquots of 10 μg of protein were added to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 30 mins at 60 V and 1 h at 110 V. On the following the proteins were transferred to polyvinylidene difluoride (PVDF) membrane in transfer buffer for 2 h at 200 mA. The membranes were blocked with 5% non-fat milk in Tris-buffered saline which contained 0.05% Tween 20 (the PVDF membrane should be blocked with TBST with 5% BSA when phosphorylated protein was detected) at room temperature for 1 h. Straight after that the membranes were incubated with anti-GAPDH antibody(1:6000) , anti-syncytin-1 antibody , anti-ASCT-2 antibody , anti-P-β-catenin antibody , anti-β-catenin antibody overnight at 4 °C. Then the bound antibodies were tested by horseradish peroxidase-conjugated, anti-rabbit IgG or anti-mouse IgG . Western blot analysis was repeated at least three times to confirm the consequences.28 samples of surgical specimens from patients with oral squamous cell carcinoma were gathered and were fixed with 10% formalin with embedded in paraffin followed. Then antigen retrieval by microwaving was adopted to deparaffinized sections. In the following, the sections were blocked with 10% goat serum. Next, sections were incubated with antibody to human syncytin-1 and biotin-conjugated secondary antibody and avidin-peroxidase were followed. On the other hand, Nuclei were counterstained with haematoxylin.For the use of surgical specimens for our research purposes, prior patients’ consents and approval were obtained from the Ethics Committee of School & Hospital of Stomatology, Wuhan University. Written informed consent was obtained from all subjects for publication of this study. All experiments were performed in accordance with approved guidelines of School & Hospital of Stomatology, Wuhan University.Wnt/β-catenin signal pathway could be activated by medications such as recombinant human Wnt3α (100 ng/mL) , While to the contrary, DKK-1 (100 ng/mL)  was often used to Block this pathway. So these drugs were elegantly added in SCC-9 to achieve the goal of block or enhance the pathway for 6 h, 12 h and 24 h, respectively.Recombinant lentivirus was used to silence and enhance/overexpress the expression of β-catenin in Wnt/β-catenin signal pathway. The sequences of recombinant lentivirus which was used to silence the expression of β-catenin: 5′-GCAGGTGTTCTGCTGATATCA-3′. Then 10 ng/mL TNF-α was added to the silenced SCC-9 in 6 h, 12 h and 24 h; On the other side, we divided the normal and silenced cells into two group, and then TNF-α(10 ng/mL)  was sustained for 6 h, 12 h, 24 h. Then we detected the expression of syncytin-1 respectively. Analogously, SCC-9 with enhanced β-catenin was used to test the expression of syncytin-1.The lentiviral particles of GFP- and RFP- were purchased from GenePharma, Shanghai, China. The sequence of lentiviral particles of GFP-: 5′-TTCTCCGAACGTGTCACGT-3′; The sequence of lentiviral particles of RFP-: 5′-TTCTCCGAACGTGTCACGTTTC-3′. On the following, the cultured SCC-9 was added with lentiviral supernatants which contained RFP vector. Alike, the cultured HUVEC was added with lentiviral supernatants which contained GFP vector. After transduction for 72 h, the RFP-SCC-9 and GFP-HUVEC were selected with 10 μg/ml puromycin . The efficiency of transduction was validated by immunofluorescent microscopy .The four different sh-RNA sequences  targeting syncytin-1, β-catenin and negative sh-RNA sequence (sh-RNA-mock) were purchased from Genepharma . The sequence of recombinant lentivirus which was used to silence the expression of syncytin-1: 5′-GCAGCGTCCCCGGAAATATTGA-3′. For constructing human syncytin-1 and β-catenin sh-RNA plasmids, These sequences were inserted into the BamHI/EcoRI sites of pGPU6/GFP/Neo Puro lentivectors. Then 293 T cells were co-transduced. The lentiviral particles were collected and used to infect SCC cells. The infected cells were identifed by 10 μg/ml puromycin. The steps of transfection en-RNA of β-catenin were as same as sh-RNA of β-catenin.We co-cultured RFP-SCC-9 and GFP-HUVEC with the mix of 1:1 for 4 groups: group one was cultured with none of the stimulus, while the other three groups were added with TNF-α, Wnt3α and DKK-1. And the results were collected by co-cultured for 1, 3 and 7 days. We also co-cultured sh-syncytin-1 with RFP-HUVEC. The dual-fluorescent positive cells were observed in the fluorescence microscope ; After co-cultured for 14 days, the dual-fluorescent positive cells were confirmed by FACS . We then co-cultured sh-β-catenin and en-β-catenin SCC-9 with RFP-HUVEC for 1, 3, 7 and 14 days, and dual-fluorescent positive cells were counted and confirmed as above.p values < 0.05. All results were repeated at least three times to confirm the results.All statistical analyses were processed by using the Student′s t-test, one-way ANOVA, two-way ANOVA. GraphPad Prism 5.01 . FlowJo 7.6.1, FlowJo 10.0 . Statistical packages were used to perform statistical data analysis and data were expressed as mean ± standard deviation. The criterion of statistical significance was that How to cite this article: Yan, T.-L. et al. Up-regulation of syncytin-1 contributes to TNF-α-enhanced fusion between OSCC and HUVECs partly via Wnt/β-catenin-dependent pathway. Sci. Rep.7, 40983; doi: 10.1038/srep40983 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Biochemical analysis of nuclear extracts from cancer biopsies revealed the existence of low molecular weight (LMW) pβ-Cat552, increased to the exclusion of full size (FS) forms of β-catenin. LMW β-catenin lacks both termini, leaving residues in the armadillo repeat intact. Further experiments showed that TCF4 predominantly binds LMW pβ-Cat552 in the nucleus of inflamed and cancerous cells. Nuclear chromatin bound localization of LMW pβ-Cat552 was blocked in cells by inhibition of proteasomal chymotrypsin-like activity but not by other protease inhibitors. K48 polyubiquitinated FS and LMW β-catenin were increased by treatment with bortezomib. Overexpressed in vitro double truncated β-catenin increased transcriptional activity, cell proliferation and growth of tumor xenografts compared to FS β-catenin. Serine 552-> alanin substitution abrogated K48 polyubiquitination,  β-catenin nuclear translocation and tumor xenograft growth. These data suggest that a novel proteasome-dependent posttranslational modification of β-catenin enhances transcriptional activation. Discovery of this pathway may be helpful in the development of diagnostic and therapeutic tools in colitis and cancer.Nuclear activation of Wnt/β-catenin signaling is required for cell proliferation in inflammation and cancer. Studies from our group indicate that β-catenin activation in colitis and colorectal cancer (CRC) correlates with increased nuclear levels of β-catenin phosphorylated at serine 552 (pβ-Cat Wnt/β-catenin - induced gene transcription plays a central role in self-renewal, proliferation, differentiation, polarity, morphogenesis, and development4. Aberrant Wnt/β-catenin signaling is found in several tumors, including colorectal cancer (CRC)5. β-catenin signaling is increased in over 90% of CRC due to mutations in either β-catenin exon 3 or adenomatous polyposis coli (APC), believed to enhance β-catenin stability by reducing degradation7. Ultimately β-catenin translocates into the nucleus and binds transcription factor TCF4 (T cell factor 4) to drive transcription of Wnt regulated genes11.β-catenin is a cytoplasmic protein that participates in intercellular adhesion and Wnt-mediated transcriptional activation  stabilize β-catenin armadillo repeats making this domain essential for Wnt signaling. Together, these studies indicate that β-catenin regions essential for Wnt signaling include the Armadillo repeat core and proximal C terminal regions 20. Transgenic studies in mice where endogenous β-catenin was replaced by mutant forms with D164A substitution and deleted C-terminus show the ability of this mutant to suppress transcription despite preserved adhesive function21. Studies in Drosophila with mutated Armadillo protein (truncated to only 12 armadillo repeats) show increased transcriptional activation after stimulation22. In general, studies highlight the importance of Armadillo and C terminal transactivation domains in β-catenin signaling while suggesting that other regions are dispensible.The primary structure of β-catenin is composed of N and C terminal regions and a central core of 12 armadillo repeats spanning residues 134−678. Cadherins, APC and TCF family transcription factors bind to β-catenin within the core region, whereas GSK3β and α-catenin bind sites within N terminal amino acidset al. indicate that Akt phosphorylates β-catenin at serine 552 located in the open loop within the tenth Armadillo repeat. In PTEN mutant mice (Mx1Cre/PTENfl/fl), increased numbers of epithelial cells with nuclear phospho-β-cateninSer552 (pβ-Cat552) were detected in small bowel polyps. Staining for pβ-Cat552 co-localized with stem cell markers  and increased TCF4 transcriptional activity seen in Mx1Cre/PTENfl/flxTOP-GAL mice23. Using an antibody specific for pβ-Cat552, we detected enhanced nuclear staining in epithelial cells in human colitis and colorectal cancer24. Given that anti-pβ-Cat552 staining identifies an epitope with the core region, we further explored its role in activation of β-catenin signaling.In humans and rodents, increased PI3K (phosphatidylinositide 3-kinase) activation seen with diminished PTEN (phosphatase and tensin homolog) levels enhances β-catenin activation. Studies from He Studies here interrogate alterations in β-catenin that occur during Wnt/β-catenin signaling in cancer and mucosal inflammation. The data reveal that post-translational modifications of β-catenin in the ubiquitin-proteasome pathway yield a truncated β-catenin molecule containing a serine 552-phosphorylated core region without N and C termini. This proteolytic processing of β-catenin is required for binding with TCF4 and subsequent transcriptional activation.5. To examine the molecular events occurring during enhanced β-catenin signaling in the colon, intestinal epithelial cell (IEC) nuclear isolates from patients with CRC and colitis were assayed by WB (western blot) using antibodies specific for β-catenin epitopes at N and C termini as well as within the region of armadillo repeats (core and pβ-Cat552). Data in Fig. 552 epitopes in cytosolic and membrane fractions. Examination of nuclear fractions indicates that levels of FS β-catenin detected by C-terminal and core region-specific antibodies were similar in normal and CRC tissue. N-terminal antibody detected enhanced levels of β-catenin in this sample, while other WB failed to detect increased N-terminal β-catenin  are associated with increased nuclear accumulation of β-catenin and enhanced Wnt/β-catenin signaling in tumor cells552antibodies was increased in CRC tissue compared to normal (arrows). Given that both core and pβ-Cat552antibodies identify epitopes within the armadillo repeats, we postulated that β-catenin may be cleaved into a low molecular weight (LMW) form in transformed cells.Interestingly, a smaller, 52–56 kD fragment of β-catenin detected by core region and pβ-Cat52 antibody detected FS β-catenin in normal colon compared to predominantly LMW β- catenin present in relatively high levels in CRC. In most cases, the LMW form of nuclear β-catenin detected by anti-pβ-Cat552 antibody was found to the exclusion of higher molecular weight forms  interfering RNA) treatment. Together, these results indicate that antibodies to core and pβ-Cat552 epitopes identify a LMW form of β-catenin present in normal cells and increased in states of enhanced Wnt signaling.To confirm the specificity of antibodies detecting LMW β-catenin, siRNA knockdown of β-catenin in normal human colonic epithelial cells (NCM460) was utilized. As shown in Suppl. Fig. 26, we examined its role in β-catenin processing. Cytosolic and nuclear fractions of colon cancer cell line HT29 were used for immunoprecipitation (IP) with an antibody specific for the K48 poly-Ub and then probed for β-catenin epitopes within N and C termini as well as pβ-Cat552. Data in Fig. 27 increased levels of K48 ubiquitinated β-catenin in the cytosol. As expected, N and C terminus antibodies detected molecular forms of poly-Ub-β-catenin at MW 120 kD. These findings were consistent with the predicted molecular weight (MW) of β-catenin (88 kD) and attached side chain composed of four ubiquitins (8.5 kD×4 = 34 kD). By comparison, the antibody specific for pβ-Cat552 recognized poly-Ub-β-catenin of approximately 86–90 kD MW. These data were consistent with the combined MW of LMW β-catenin (52–56 kD) and four ubiquitins (34 kD). The detection of K48 polyubiquitinated FS and LMW pβ-Cat552 in bortezomib-treated samples suggests that both FS and LMW pβ-Cat552 are degraded in the proteasome following K48 polyubiquitination. In one of the repeated experiments with HT29 cells  polyubiquitin chain (poly-Ub) has been shown to target proteins to the proteasome for degradationies Fig. . In all ies Fig. . In Supphttp://web.expasy.org/peptide_cutter)28. Next, a number of inhibitors specific for relevant proteases were used to treat HT29 colon cancer cells. Data show that inhibition of chymotrypsin-like activity with epoxomicin29, bortezomib, VR2330 (200 nM), and MG31231 reduced TCF/LEF (T cell factor 4/lymphoid enhancer-binding factor) luciferase activity 32. In CRC biopsy samples treated with proteasome inhibitor MG132 ex vivo, as well as in HT29 cells treated with bortezomib, we found that proteasome inhibition greatly reduced nuclear levels of pβ-Cat552 bound to TCF4  conditions promotes expression of stem cell genes whereas low Wnt (Methods) conditions inhibit stem cell expansion/gene transcription. In data presented in Suppl. Fig. 552 show greater levels of pβ-Cat552 localized to chromatin-bound fractions in cells grown under high Wnt compared to low Wnt conditions 28. From the total of 28 possible sites flanking N and C termini of the armadillo repeats, we choose high specificity sites  tyrosin142 and  phenylalanin 683. To test if treatment with chymotrypsin  would generate peptides with molecular weight close to 52–56 kDa we used recombinant β-catenin. As seen on Suppl. Fig. 2+) sepharose pull down (His tag specific) experiment presented on Suppl. Fig. Given findings that nuclear LMW β-catenin levels were increased in colon, pancreas, lung and liver tumors, we suspected that protein cleavage was associated with β-catenin transcriptional activity. To test this notion, NCM460 cells were transfected with constructs encoding FS β-catenin, and β-catenin truncated at N and C termini. The “double truncated” β-catenin, referred to as ∆∆ β-catenin was generated based on the predicted chymotrypsin cutting sites outside of armadillo repeats  or where the C-terminal sequence has been cleaved (identified by His tag). Both of the residual peptides weighed approximately 75kD. To confirm these interpretations, we performed pull down experiments in which total cell lysates were immunoprecipitated with anti-Flag antibody or precipitated with His tag specific Coies Fig.  revealed35. TCF4 IP studies showed that levels of pβ-Cat552 bound to TCF4 were highest in ∆∆β-catenin cells compared to ∆N89 or FS β-catenin . Cells were transfected with ∆N89 β-catenin to allow comparisons to mutated, “constitutively active” β-cateninnin Fig. . Similarnin Fig. . Total cnin Fig.  show equ36. Alterations in cyclin D1 turnover in cancer can lead to nuclear accumulation of cyclin D1 independent of changes in cyclin D1 mRNA expression. In normal, cyclin D1 protein is translocated in cytosol where it is utilized by proteasome37.To investigate the ability of ∆∆ β-catenin to increase expression of β-catenin target genes, mRNA expression from NCM460 transfected with FS and ∆∆ β-catenin was compared to vehicle control. As presented in Suppl. Fig. Together, these data indicate that LMW β-catenin efficiently localizes to chromatin-bound fractions where it binds TCF4 and drives TCF/LEF transcriptional activity.To further investigate whether β-catenin truncation conveys enhanced cell invasiveness, NCM460 cells transfected with control, FS β-catenin, ∆N89 β-catenin and ∆∆β-catenin vectors were examined in methylcellulose colony formation and proliferation assays. Findings in Suppl. Fig. 38. To examine the impact of β-catenin  serine 552 phosphorylation on localization to chromatin bound fractions, RKO tumor cells were transfected with distinct ∆∆ β-catenin constructs containing wild type (FS and ΔΔ) or Ser552->Ala substituted (FS 552A and ∆∆ 552A) sequences. RKO colon cancer cells were used to avoid mutations in the β-catenin signaling pathway. All constructs were Flag tagged at the C terminus. As seen in Fig. 552 correlated with transcriptional activity.Phosphorylation of β-catenin at serine 552 has been reported to affect nuclear translocationTo examine the impact of serine 552 phosphorylation on β-catenin ubiquitination in the cytosol, RKO cells . In NFκB signaling, the inhibitor IκBα is degraded in the proteasome which frees NF-κB to translocate into the nucleus and regulate gene transcription40). In contrast, ubiquitinated ∆∆ β-catenin might translocate in the nucleus, where we propose de-ubiquitinating enzymes generate LMW pβ-Cat552 that localizes to the chromatin-bound fraction , we found increased levels of a serine 552 phophorylated LMW form of β-catenin in the nucleus suggesting that β-catenin processing had occurred. Biochemical studies revealed that FS β-catenin was ubiquitinated in the cytosol by K48 poly-chain (Ub-β-catenin) Figs  and 7C peus Figs  and 6A seus Figs . Detectimib Fig.  suggestsion Figs . Once acons Figs –S8. It iome Fig. . We diag552 in colon cancer cells suggests that this pathway does not preclude the well-characterized degradation pathway altered by mutations in APC and β-catenin. In these cells, a high level of “stabilized” full length β-catenin is available for phosphorylation and cleavage in the pathway presented here  in the cytosolic proteasome  and then phosphorylation of serine 33/37 by GSK3βome Fig. . FS pβ-Csol Figs  as was Lons Figs  and S8B sol Fig. .The findings reported here support the conclusion that β-catenin undergoes an important cleavage step essential for signaling in the nucleus. This event appears to be most prevalent in transformed tissue where β-catenin activation is increased related to mutations in the canonical Wnt/β-catenin pathway . The present findings unveil an important nuance of this pathway, that β-catenin undergoes a significant modification on its way to nuclear translocation and TCF4 binding. Careful studies were conducted to support the conclusion that generation of LMW β-catenin resulted from regulated chymotrypsin-like activity in the proteasome Fig. . Given tThe antibodies used are listed in Suppl. Table Reagents used: calyculin A ; bortezomib and VR23 ; TNF ; MG132, epoxomicin, bafilomycin, chloroquine, cathepsin K inhibitor I, calpeptin, KYP2047 and polybrene ; zeocin and puromycin ; siPORT NeoFX transfection reagent ; jetPRIME transfection reagent ; luciferase reagent ; recombinamt β-catenin .ex vivo stimulation, biopsy specimens were obtained from healthy patients undergoing routine colon cancer surveillance. Colorectal cancer (CRC) specimens were obtained from patients undergoing surgery. Collection of all patient materials for this study was approved by Institutional Review Board protocol (IRB #13–0559-F3R). Pancreas and lung biopsy samples were obtained from Northwestern Memorial Hospital (Chicago). Liver biopsy samples were homogenized in the laboratory of Professor Josep Llovet and semi-purified protein frozen at −80 °C delivered overnight in dry ice.For all human studies, informed consent was obtained from every patient and samples were coded. Collection of all material was approved by the Northwestern University or University of Kentucky Institutional Review Boards, in accordance with their guidelines and regulations. Human colonic biopsy specimens were obtained from patients undergoing diagnostic or surveillance colonoscopy for known or suspected ulcerative colitis (UC) or cancer. Specimens were collected from Northwestern Memorial Hospital (Chicago) and the University of Kentucky and Good Samaritan Hospitals . For comparison and Human colon epithelia samples were delivered from the operating room in ice cold PBS. Samples were washed once with ice cold PBS and incubated at 4°C with rotation in PBS with 10mM DDT and 50nM calyculin A for 30min, 4°C. The samples were centrifuged at 300 rpm for 5 min. Cells were frozen in liquid nitrogen and stored at −80°C until use.Human colon biopsies from CRC patients were treated as above. The tissue was divided into two parts and treated with MG132 (in HBSS) as indicated Fig. . Samples6/mouse) in a 1:1 mixture of media and Matrigel   (n = 7 per group). Tumor dimensions were measured using a caliper and tumor volumes were calculated as mm3 = π/6×(larger diameter)×43.All animal experiments were approved by the University of Kentucky Institutional Animal Care and Use Committee and were conducted in accordance with their regulations and guidelines. Male athymic nude mice (5–6 weeks old) were purchased from Taconic . To establishe CRC xenografts, mice were subcutaneously injected with tumor cells  were received by a cell licensing agreement with INCELL Corporation , and were routinely propagated under standard conditions in M3:10A medium supplemented by 10% of fetal bovine serum (FBS) with addition of the conditioned medium (33%) from previously cultured NCM460 cellslog phase were treated with a series of specific inhibitors of proteasomal chymotrypsin-like activity , proline endopeptidase , lysosomal endopeptidases , inhibitors of cathepsin K, 100nM, and proteinase K . The cells were incubated under standard conditions for 8 hrs with proteasome inhibitors and overnight with other inhibitors. Cells were harvested and fractionated.SW480 cells  were cultured in Leibovitz’s L-15 medium with 10% FBS under standard conditions. HT29 , RKO , Caco2  and HCT116  cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS under standard conditions. HT29 cells in siRNA to human β-catenin was obtained from Ambion . siRNA was introduced in NCM460 cells with siPORT NeoFX transfection reagent. Forty eight hours after transfection cells were harvested and used for experiments. siRNA nonsense mix  was used as a control.31 with primers listed in Suppl. Table 34. The insert was also re-cloned into a pLV vector. Primers are listed in Suppl. Table The pLV lentiviral plasmids encoding FS, ∆89 β-catenin, ∆N142 and ∆∆ β-catenin with His tag on N terminus and Flag tag on C-terminus were made based on human β-catenin pcDNA3.1 neo (gift from Dr. Eric Fearon: Addgene plasmid # 16828)The reporter construct containing TCF/LEF luciferase (TCF/luc) was generated in the DNA/RNA Delivery Core, SDRC at Northwestern University  by inserting six copies of the TCF/LEF response element in the lentiviral pGF1 vector . The cells were infected with TCF/luc viral particles as described above. Luciferase activity was detected with luciferase reagent.34 and pcDNA3.1 expressing C terminal FLAG tagged FS β-catenin with serine 552 substituted to alanine (FS 552A) (gift from Dr. Dexing Fang38). Double truncated (∆∆ and ∆∆ 552A) and ∆N142 β-catenin constructs were created with primers listed in Suppl. Table Wild type FS β-catenin insertion was re-cloned in pcDNA3.1 with zeocin resistance from pcDNA3.1 expressing C terminal FLAG tagged FS β-catenin (gift from Dr. Eric Fearon: Addgene plasmid # 16828)Proliferation assays were performed by using the CyQUANT® Cell Proliferation Assay Kit  according to the manufacturer’s instructions.4 per well (6 wells for each cell line). Cells were stained with trypan blue and viable cells were counted on a hemocytometer 5 times every 24hrs after initial 48hrs of incubation.Cells were placed in 96 well plates at 1045. Colonoids were established in WENR media (100ng/ml Wnt3a and 1μg/ml R-spondin)33. After 48hrs, media were changed to reduced Wnt3a and R-spondin (25ng/ml Wnt3a and 250ng/ml R-spondin) for 5 days.Colonic crypts were isolated from C57BL\6 mice via collagenase digestion and embedded in Matrigel as previously described46. All buffers used contained Protease Arrest™ protease inhibitor cocktail , and phosphatase inhibitor cocktail I and II  at 1:100. Human epithelial cells were homogenized in glass homogenizer in buffer I , lysates were passed through a 250μm tissue strainers , centrifuged at 4°C for 10 min at maximum speed in table top centrifuge. The supernatants were collected and used as the cytosolic fraction. Pellets were re-suspended in buffer II  and incubated on ice for 30min, then centrifuged as above. The supernatants were used as the membrane/organelle fraction. Pellets were dissolved in buffer III , 100mM NaCl), and with 2U of Benzonase  per 100µl of lysate and incubated for 30 min at room temperature (RT). Following centrifugation the supernatants were used as nuclear fractions.The subcellular protein fractionation  protocol was modified from described procedures2 and 3μl of micrococcal nuclease (300 units) per 100μl, supplemented with protease and phosphatase inhibitors, was added to the pellet. After vortex for 15sec at highest setting mixture was incubated at room temperature (RT) for 15min and centrifuged at 16,000×g (highest setting of microcentrifuge) for 5min. Supernatant was collected and used as chromatin bound fraction of nuclear proteins.Cultured cells and colonoids were fractionated according to Pierce manufacturer’s protocol . In particular, chromatin-bound protein extraction was performed after nuclear soluble proteins isolation. Nuclear Extraction Buffer (NEB-company formulation) with 5μl of 100mM CaClProtein concentration was measured by BCA assay .The purity of the fractions was confirmed by WB with anti-α-tubulin, anti-e-cadherin, anti-laminB1, anti-histoneH3, and anti-fibrillarin antibodies  were added in each IP reaction and the mixture was incubated overnight at 4 In experiments presented in Fig. For WB, proteins were transferred on Immobilon FL  by semi-dry transfer  and membranes blocked in Pierce Protein-Free T20 blocking buffer  for 1hr, and incubated overnight at 4°C in 1:1000 primary antibody solution. Membranes were extensively washed, incubated in 0.02μg/ml secondary antibody for 1hr., washed again and developed using West Pico, Dura or Femto reagent .Cobalt sepharose pulldown of total lysates and chromatin-bound fractions from FS, ∆142 and ∆∆ β-catenin overexpressing NCM460 cell lines was performed according to manufacturer’s protocol . For each reaction 500μg of lysate and 20μl of sepharose were used. Precipitates were run on SDS-PAGE and WB probed with anti-Flag antibody.1mg of cytosolic or nuclear protein lysate was used to precipitate with 1:4(v:v) of 100% trichloroacetic acid. After 10min incubation at 4°C samples were centrifuged and pellets were washed with ice-cold acetone. The pellets were dried, reconstituted in RIPA buffer and protein concentration measured by BCA assay. IP was performed as above.All WB and IP experiments were repeated at least three times.Total RNA was isolated from colonoids or cultured cells using the RNeasy Mini Kit  and reverse transcribed using High Capacity cDNA Reverse Transcription Kit . Real time PCR used the ABI Step OnePlus real-time PCR system and Power SYBR green PCR master mix (Applied Biosystems). Primers were designed by Primer Express software 3.0 (Applied Biosystems) based on nucleotide sequences from the National Center for Biotechnology Information data bank (Suppl. Table 2 (pH 8.0) and 2ng/µl chymotrypsin, sequencing grade  added to sample. After overnight incubation at 25°C protein was precipitated with acetone  and resolved at SDS-PAGE along with intact 5µg of β-catenin. Gel was stained with silver according manufacturer protocol .DDT was added  to 5µg of recombinant β-catenin dissolved in 25% glycerol, 50mM Tris-HCl, 150mM NaCl, 0.25mM DDT pH7.5. Protein was incubated 20min at 50–60 °C for 20 min. Reduced protein mixture was cooled to RT and iodoacetamide was added to a final concentration of 15mM. Mixture was Incubated in dark for 15min at RT. Volume was adjusted to 50µl with 100mM Tris-HCl, 10mM CaClAll experiments were performed at least three times. Results are expressed as mean ± S.E.M. where applicable. A two-tailed Student’s t-test was used to compare the intergroup. Differences between groups were considered statistically significant at p<0.05.All data generated or analyzed during this study are included in this published article (and its Suppl. Information files).Supplemental materials


DF) is a rare mesenchymal lesion with high risk of local recurrence. Specific β‐catenin mutations (S45F) appeared to be related to this higher risk compared to T41A‐mutated or wild‐type (WT). We explored the influence of both mutations and WT on structure stability and affinity of β‐catenin for α‐catenin and the pattern of gene expression that may influence DF behavior. Using 33 surgically resected primary DFs harboring T41A (n = 14), S45F (n = 10), or WT (n = 9), we performed a comparative molecular analysis by protein/protein interaction modeling, gene expression by DASL microarrays, human inflammation gene panel, and assessment of immune system‐based biomarkers by immunohistochemistry. Mutated proteins were more stable than WT and formed a weaker complex with α‐catenin. Consensus unsupervised gene clustering revealed the presence of two DF group‐mutated (T41A + S45F) and WT (P = 0.0047). The gene sets ‘Inflammatory‐Defense‐Humoral Immune Response’ and ‘Antigen Binding’ were significantly enriched in T41A. The deregulation of 16 inflammation‐related genes was confirmed. Low numbers of T cells and tumor‐associated macrophages (TAM) infiltrating the tumors and low/absent PD‐1/PD‐L1 expression were also identified. We demonstrated that mutated DFs (T41A or S45F) and WT are two distinct molecular subgroups with regard to β‐catenin stability, α‐catenin affinity, and gene expression profiling. A different inflammation signature characterized the two mutated groups, suggesting mediation either by T41A or by S45F. Finally, all mutated cases showed a low number of TIL and TAM cells and a low or absent expression of PD‐1 and PD‐L1 consistent with β‐catenin activation insensitive to checkpoint blockade.Desmoid‐type fibromatosis ( ADLaverage dynamic lengthDFdesmoid‐type fibromatosisFDRfalse discovery rateFFPEformalin‐fixed paraffin‐embeddedGSEAgene set enrichment analysisMDmolecular dynamicsNESnormalized enrichment scorennumberPARprogression arrest rateRFSrecurrence‐free survivalTAMtumor‐associated macrophagesTILtumor‐infiltrating lymphocytesWTwild‐type1et al., et al., et al., CTNNB1 gene encoding for β‐catenin are typically carried by most sporadic DFs. Three different β‐catenin point mutations, T41A, S45F, and S45P, have been described in the vast majority of DFs and have been associated with the prevention of protein phosphorylation and subsequent protein stabilization with translocation to the nucleus, where β‐catenin regulates transcription through its interaction with TCF/LEF  is a rare mesenchymal infiltrative lesion that exhibits high risk of local recurrence despite surgery with negative resection margins. The high propensity to relapse, the need for multiple surgeries with consequent functional and cosmetic impairment, the observation of prolonged stability in the absence of specific treatments, and the description of spontaneous regression have let the sarcoma community to change the management of this disease toward a more conservative tendency  surgical resection except one. All samples were obtained after informed consent from patients. The study was approved by the Independent Ethics Committee of Fondazione IRCCS Istituto Nazionale dei Tumori .For this study, we selected 33 primary DFs surgically treated at our institution. All samples had the following characteristics: (a) DF diagnosis by an expert pathologist (SP); (b) known 2.2et al., et al., et al., et al., et al., et al., et al., et al., et al., 4IGG pdb) was used to build all WT and mutant β‐catenin/α‐catenin protein–protein complex and to determine the dynamics and energetics of their interactions via MM/PBSA MD simulations  models of WT and mutant isoforms of β‐catenin were obtained by a combination of homology modeling techniques with molecular dynamics (MD) refinements , normalized by quantile algorithm, and exported without correcting for background or scale. All microarray data are MIAME compliant and the raw data were deposited into the NCBI's Gene Expression Omnibus (GEO) database (http://www.ncbi.nmlm.nih.gov/projects/geo/) with accession number GSE65021The sample areas containing more than 90% tumor cells without necrosis or surrounding normal tissue were chosen for the analysis and macrodissected. Total RNA was isolated using Qiagen RNeasy FFPE Kit  following the manufacturer's instructions and quantified by Nanodrop‐1000 instrument . RNA quality was assessed by RT‐qPCR amplifying amplicons of different size for ACTB housekeeping gene (Mittempergher 2.4www.bioconductor.org) and the BRB‐ArrayTool developed by Richard Simon and the BRB‐ArrayTools Development Team . Unsupervised tumor subtype identification was performed by ConsensusClusterPlus  was determined by the global test through 1000 permutations as computed by BRB tool using default parameters. Pathway analysis on 905 GO terms was performed using gene set enrichment analysis (GSEA). The degree of gene sets’ enrichment in the WT and mutated DF was represented by a normalized enrichment score (NES) and NES with FDR < 0.05 and > |1.5| was considered statistically significant. Graphical representation of GSEA findings was provided by GOBubble plot function of GOplot R package  and the z‐score of each gene set. For comparison purposes, the gene list derived from 14 sporadic DF (not characterized for CNTBB1 mutation) and five paired normal samples plus six solitary fibrous tumor specimens was analyzed .The Nanostring nCounter System analysis was performed by Pharma Diagen  using the Human Inflammation panel (249 genes). Statistical analysis was conducted by comparing WT versus both mutated cases and then the mutation T41A versus S45F. Raw data from Nanostring platform were normalized by resorting to a two‐step strategy using the R NanoStringNorm package  according to the manufacturer's instructions. The following antibodies were used: β‐catenin 14 , CD3 polyclonal , CD4 4B12 , CD8 C8/144B , CD20 L26 , CD21 1F8 , CD56 123C3 , CD68 KP1 , CD163 10D6 , FOXP3 259D/C7 , PD‐L1 SP142 , PD1 NAT105 , and EZH2 5246S .33.1et al., The N‐terminal domain of β‐catenin, which contains the residues T41 and S45, mediates protein degradation and interacts with α‐catenin in the cell–cell adhesions mechanically coupling the actin cytoskeletons of adjacent cells ; rather, both mutant proteins showed a slightly increased stability compared to the WT counterpart, the T41A and S45F being more stable than the WT β‐catenin by 1.33 kcal·mol−1 and 1.26 kcal·mol−1, respectively.Computer‐based simulations revealed that the thermodynamic stabilities  of the two mutant T41A and S45F β‐catenin isoforms are comparable (ΔGbind) between α‐ and WT β‐catenin is shown in the first row of Table −1, respectively), ultimately resulting in the formation of a weaker complex  = 2.15 ± 0.04 Å for the T41A‐mutant and ADL(S45β‐E205α) = 2.14 ± 0.06 Å for the S45F‐mutant complexes).The presence of either missense substitutions  results in a plummet of the stabilizing interactions of the corresponding α‐/β‐catenin complexes. Indeed, the substitution of two polar residues, both featuring an –OH group available for hydrogen binding, with two residues devoid of such feature led to global conformational changes, which involve protein regions at a distal position from the mutant loci  and WT (Cluster 2: 6/9 samples are WT)  and normal samples/solitary fibrous tumor specimens (n = 11) were applied to our dataset as custom gene sets in GSEA; this analysis identified in WT β‐catenin cases a significant enrichment of genes present in normal tissues . This nucleosome‐binding protein HMGN1 is a potent alarmin that promotes T cell‐mediated antitumor immunity  versus WT DFs, among the 139 biomarkers resulted to be expressed in all the 33 samples, only HMGN1 was found statistically significantly overexpressed in mutated DFs , CD163+, and/or CD68+, distributed around the vessels .Our profiling analysis is quite unique. Indeed, only a recent gene expression study compared mutated versus WT DFs, indicating that mutated DFs did not cluster separately and did not have a different clinical behavior from WT tumors , further studies are needed to confirm our data.CC, FP, Sabrina Pricl, and Silvana Pilotti involved in conception and design; CC, AB, NP, LD, EL, MF, and SB acquired data; FP, Sabrina Pricl, SC, and PV analyzed and interpreted the data; AG, Silvana Pilotti, SS, MF, and EP participated in revising the article. All the authors gave final approval of the version to be submitted.Table S1. Binding free energies (ΔGbind) and binding free energy differences (ΔΔGbind) for the WT, T41A and S45F β‐catenin in complex with α‐catenin.Click here for additional data file.Table S2. List of the 382 genes differentially expressed between mutated‐ and wt‐DT patients.Click here for additional data file.Data S1. Computational details.Click here for additional data file.


In this study we show that re-arrangements at Catenin Delta 2 (CTNND2) locus, including gene duplications, are very common in clinically significant PCa and may underlie δ-catenin overexpression. We find that δ-catenin in PCa cells exists in a complex with E-cadherin, p120, and α- and β-catenin. Increased expression of δ-catenin leads to its further stabilization as well as upregulation and stabilization of its binding partners. Resistant to degradation and overexpressed δ-catenin isoform activates Wnt signaling pathway by increasing the level of nuclear β-catenin and subsequent stimulation of Tcf/Lef transcription targets. Evaluation of responses to treatments, with androgen receptor (AR) antagonist and β-catenin inhibitors revealed that cells with high levels of δ-catenin are more resistant to killing with single agent treatment than matched control cells. We show that combination treatment targeting both AR and β-catenin networks is more effective in suppressing tumor growth than targeting a single network. In conclusion, targeting clinically significant PCa with high levels of δ–catenin with anti-androgen and anti β-catenin combination therapy may prevent progression of the disease to a castration-resistant state and, thus, represents a promising therapeutic strategy.The mechanism of prostate cancer (PCa) progression towards the hormone refractory state remains poorly understood. Treatment options for such patients are limited and present a major clinical challenge. Previously, δ-catenin was reported to promote PCa cell growth  A member of the p120-catenin subfamily, δ-catenin was originally discovered as a neuron-specific protein and later was shown to also play an important role in cancer. Studies report that δ-catenin is up-regulated in various human carcinomas including breast, lung, prostate, esophagus and colorectal cancer . In a nuElevated expression of δ-catenin may be due to gene amplification, transcriptional activation and mutations in non-coding regulatory region , 8. MutaIt is well established that δ-catenin can promote signaling through a canonical Wnt/β-catenin pathway activating LEF1-mediated transcription. One report noted that in PCa cells, δ-catenin interacts with E-cadherin in a competitive manner with p120 . Each prA number of post-translational modifications of δ-catenin that affect its stability, function and subsequent downstream effects have been described , 13, 18.The status of δ-catenin and its function in PCa have been studied extensively. Its overexpression is well documented , 24, 25,In this study, by analyzing the landscape of structural DNA alterations in a large series of PCa treated by radical prostatectomy we discovered that δ-catenin is frequently re-arranged in cases with clinically significant disease. The re-arrangements mostly represented duplications that are likely to account for higher expression levels of the protein in those cases. In a cell model overexpressing δ-catenin we found that it stimulates levels of its binding partners E-cadherin, p120, α and β-catenin and downstream targets of Tcf/Lef transcription factor. In LNCaP cells ectopically expressing δ-catenin, only the higher molecular weight form of δ-catenin protein was detected. The form was very stable and was not a product of phosphorylation, sumoylation or ubiquitination. When treated with inhibitors of β-catenin, overexpressing δ-catenin clones showed improved survival relative to parental cells. A combination treatment with anti-androgen and β-catenin inhibitor resulted in more efficient killing of cells with high levels of δ -catenin than either agent alone. Collectively our data suggest that PCa cells with overstimulated δ–catenin/β-catenin network are resistant to androgen deprivation and β-catenin inhibition. Combination therapy targeting both pathways simultaneously may be considered for PCa with high levels of δ-catenin.Overexpression of δ-catenin is found in PCa of Gleason score (GS) 7 and higher , 24, 25.To gain an insight into potential cause of overexpression of δ-catenin we next compared CTNND2 gene status in surgically treated clinically significant and clinically insignificant PCa using mate pair whole genome sequencing , 30. CliIn order to elucidate perturbations at Wnt- β catenin signaling pathway due to overexpression of δ-catenin and examine their contribution to treatment sensitivity we generated a number of cell models with altered levels of δ-catenin. We first compared protein complexes involving key catenin proteins in a panel of PCa cell lines using non-denaturing gel electrophoresis Figure . The banIn contrast, another androgen-dependent gene, NKX3.1, was upregulated in δ-catenin knocked down clones Figure . This elLNCaP cells, compared to other PCa cell lines, showed relatively high expression of every protein involved in catenin network , consistβ-Catenin, when activated is known to translocate from cytoplasm to the nucleus to facilitate transcription of target genes –39. To eWe next examined whether higher levels of δ-catenin in over-expressing clones affect the composition of the catenin binding complex. By a pull down assay using antibody to each protein in the putative complex as a bait, we found that E-cadherin, α-catenin, p120, δ-catenin and β-catenin exist in one complex Figure . There wWestern blot analysis of cells expressing exogenous δ-catenin not only revealed its elevated levels but also pointed to a major product of a higher molecular weight than that in parental LNCaP cells Figure . SurprisUbiquitination is an important step in the process of targeting proteins for proteosomal degradation  and was Among other protein modifications, sumoylation is known to affect localization, transcriptional regulation and enzymatic activity . It was The significance of Wnt/β-catenin pathway in PCa is well documented –57, and Collectively, these results indicate that combination treatment targeting AR and β-catenin pathways in cells with high levels of β-catenin is more promising approach than androgen ablation alone.in vivo [Progression of PCa to androgen-independent state, is a main cause of treatment resistance, tumor progression and PCa-related death. Castrate resistant PCa is associated with a poor prognosis and mean survival time of 16–18 months . There ain vivo , 70. AR in vivo . Furtherin vivo , 72. Thein vivo , 73–75. in vivo . Inversein vivo , 25, canin vivo . Other rin vivo .in vivo to stimulate its expression [The level of Slug, known to control epithelial to mesenchymal transition, was diminished in LNCaP clones overexpressing δ-catenin Figure . This ispression , and levpression  which ispression –82. Sincpression , 84. Thepression . Earlierpression . Contrarpression . Our resAn increase in β-catenin level was concomitant with overexpression of δ-catenin in LNCaP cells Figures  and 4. TCooperation of AR network and β-catenin in regulating the growth of prostate cells is well established , 69, andIn summary, our study reports on complexity of interaction between catenin proteins within Wnt signaling pathway and its cross-talk with AR network. We show that δ-catenin in PCa induces β-catenin which, in turn, impacts responses to existing androgen deprivation therapy and currently under development β-catenin inhibitors. Further studies are needed to refine therapeutic interventions in hormone refractory PCa driven by Wnt/β-catenin.The following antibodies were used for western blotting and co-immunoprecipitation: δ-catenin , β-catenin , p120 , α-catenin , cyclin D1 , NKX3.1  and Slug . Inhibitors pyrvinium (cat. P0027), CCT031374 (cat. SML1399), ALLN (cat. A6185), 2-D08 (cat.. SML1052) and casodex (cat. B9061) were purchased from Sigma. λ-PP (cat. P0753S) was from New England Biolabs.Prostate cell lines LNCap, BPH1, 22RV1, VCaP and DU145 were purchased from ATCC. LNCaP, BPH1 and 22RV1 cells were maintained in RPMI1640 with 2 mM L-glutamine and 10% fetal bovine serum in a CO2 (5%) incubator at 37°C. VCaP and DU145 cells were grown in DMEM medium supplemented with 10% fetal bovine serum. Stable LNCaP clones overexpressing δ-catenin were generated by transfection of CTNND2 Lentifect™ Lentiviral Particles , single clones were selected by addition of puromycin at final concentration of 2 μg/ml. Expression level was confirmed by Western blot analysis.VCaP cells with knock down levels of δ-catenin were generated using CTNND2 gene specific knock out kit via CRISPR  according manufacturer's instructions. TurboFectin  was used to transfect gRNA vectors, scramble negative control and donor vector. The integration of functional cassette was confirmed by PCR using genomic DNA from selected puromycin-resistant clones and specific primers: CGAAGAGG TTCACTAGGCGCGCCTTTCT (forward) and CGAAGAGGTTCACTAGGCGCGCCTTTCT (reverse).PCR was performed on DNA isolated from PCa samples  using reCells for protein analysis were lysed in NTEN buffer  supplemented with complete protease inhibitor cocktail  and phosphatase inhibitor cocktail II . Protein concentration was determined using Pierce BCA Protein Assay Kit . Protein was separated by SDS-PAGE, transferred to nitrocellulose membrane and blotted with corresponding antibodies. For pull down experiments cell lysates were incubated with precipitating antibodies immobilized on protein A/G PLUS-Agrose beads  for 4-16 hours. Samples were washed with NTEN buffer containing protease inhibitor cocktail. Immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose membranes for Western blotting.Native gel electrophoresis was performed to detect protein complexes. Sample Prep Kit was used to extract protein and gel electrophoresis was done using NuPAGE Bis-Tris Gels  according to manufacturer's instructions.5 cells in the serum free medium were added to the inside of each membrane, FBS-containing medium was added to each matched well on 24-well plate. After 48 hour incubation, noninvasive cells were removed from the upper surface of the transwell membrane, and cells which migrated through the membrane were incubated with Cell Stain Solution, washed with Extraction Solution, absorbance at 560 nm was measured using GloMax-Multi Detection System (Promega).The cell migration assay was performed using polycarbonate membrane with 8 μm pore size . 2 × 103 cells were seeded on 6-well soft agar plate, maintained at 37°C for 10–20 days, washed with PBS and stained with Giemsa solution. The number of colonies (containing >50 cells) was counted using the following formula: colony formation efficiency = number of colonies/number of cells seeded.For colony formation assay 5 × 10MTS cell proliferation assay was performed following manufacturer instructions . Cells were plated in 96-well plates and absorbance values were obtained using a GloMax multi detection system (Promega).4 cells were seeded in 24-well plate in growth medium and maintained for 48 hours. For treatments growth medium was replaced with RPMI1640 containing 10% charcoal stripped FBS  and cells were maintained at 37°C for 24 hours more. Pyrvinium and/or casodex were added at indicated concentrations and cells were incubated with drugs for designated time. Cell viability was measured using CellTiter 96 AQuious assay kit  according manufacturer's instruction.5 × 10t-test was used to analyze the results. Differences were considered to be statistically significant at p < 0.05.All data were presented as mean ± SD. The mean was the average of at least triplicate samples in each experiment. Each experiment was repeated at least three times. Student's


This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that β2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3--2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both β2-GPI and deglycosylated β2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of β2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that β2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, β2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that β2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for β2-GPI in anti-angiogenic therapy.Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The β The fgulation ,5. Our peceptors ,8, altho2-GPI has the ability to inhibit the VEGF-induced cell growth and migration in human aortic endothelial cells (HAECs) [Vascular endothelial growth factor (VEGF) signaling has an important role as a pro-angiogenic factor, permitting physiological revascularization. Therefore, drug or biological components targeting the VEGF signaling pathway have been extensively used as potential anti-angiogenic agents ,10. Rece (HAECs) . Alterat (HAECs) ,13.2-GPI regulates the VEGF-induced angiogenesis within vascular endothelial cells still remain unclear. Therefore, we aimed to determine the effect of β2-GPI on the VEGF-induced angiogenesis in HAECs; also, we investigated whether the phosphorylation of ERK1/2, Akt, and eNOS was regulated by β2-GPI. This study could provide new ideas for therapeutic strategies that ameliorate the vascular pathology observed in neovascularization and endothelial remodeling.Angiogenesis plays a major role in the pathogenesis of several diseases such as rheumatoid arthritis , cerebra2-GPI antibody was prepared as described previously [VEGF-A was purchased from Sigma-Aldrich . Rabbit anti-βeviously . The groHuman aortic endothelial cells (HAECs) were purchased from Cascade Biologics  and were cultured at 37°C in Medium 200 (Cascade Biologics) supplemented with low serum growth supplement  containing 2% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and 1% antibiotic mixture according to the manufacturer’s instructions.2-GPI was purified from human plasma by methods that have been previously used [2-GPI was isolated and purified by a 3% perchloric acid precipitation and a Heparin-Sepharose affinity chromatography . The purity of the β2-GPI was determined through 10% SDS-PAGE and Western blot analysis. The purified β2-GPI showed a single band in the SDS-PAGE, at approximately 50 kDa. For the deglycosylation assay, β2-GPI was denatured in 0.5% SDS and 40 mM DTT at 37°C for 10 min. After boiling the sample, peptide-N-glycosidase F  was added and incubated at 37°C for 72 h. The deglycosylated β2-GPI was detected by SDS–PAGE.βsly used . Briefly4 cells/well) and incubated in media with a LSGS containing 0.5% FBS for 24 h at 37°C. After treatment of β2-GPI or anti-β2-GPI antibody in the presence or absence of VEGF for 72 h, cells were incubated with 3--2,5-diphenyl tetrazolium bromide  for another 4 h. Then, medium was removed and the formazan crystals were dissolved in isopropanol. The amount of solubilized blue formazan was quantified by previously described methods [2-GPI or anti-β2-GPI antibody in the presence of VEGF for 72 h. Cells were then labeled with BrdU and quantification was performed using a cell proliferation ELISA colorimetric kit  according to the manufacturer’s instructions.HAECs were seeded in 96-well plates (2 × 103 cells/well) were plated onto the μ-slide and treated with β2-GPI and VEGF for 14 h. After incubation, the morphology of cells was visualized, the degree of tube formation in each group was estimated by the presence of total length, and images were analyzed by the Metamorph tube formation module .The μ-slide  coated with growth factor-reduced basement membrane extract  was allowed to polymerize at 37°C for 30 min. Cells  of National Yang-Ming University. The care of animals was conducted in accordance with the guidelines established by the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals.Forty-eight male C57BL/6 mice  were randomly allocated to one of the four groups (n = 6). Mice were housed with sterilized stainless steel cover and bedding, under 12 hour circadian cycle of artificial light, 22±2°C temperature, and 40–60% relative humidity. Food and drinking water were supplied in vivo Matrigel plug assay, male C57BL/6 mice were anesthetized by intraperitoneal (i.p.) injection of 15 μl of 2.5% avertin before the experiment. Following euthanasia, mice were injected subcutaneously with 500 μl Matrigel containing VEGF (20 ng/ml), heparin (50 U/ml), and either β2-GPI (200 μg/ml) or phosphate buffered saline (PBS) as a control. After 14 days, mice were euthanized by CO2 inhalation and Matrigel plugs were dissected out to quantify hemoglobin using the Drabkin’s reagent kit  according to the manufacturer’s instructions. As a separate experiment of the in vivo angioreactor angiogenesis assay, we performed a procedure identical to the one described above, except for the Matrigel with or without VEGF and β2-GPI, which was put into sterilized angioreactors. Physical appearance, behavior and local clinical signs of the animals were daily observed throughout experiment. Mice were sacrificed by CO2 inhalation if they became clinically ill (weight loss more than 20% or hunching behavior). All mice were treated humanely throughout the experimental period.For the 2-GPI on the VEGF-induced expression of cellular signaling proteins were determined by Western blot analysis. HAECs were lysed in a buffer containing 1% Triton X-100, 50 mM HEPES, 6 mM EDTA, a phosphatase inhibitor , and a complete protease inhibitor cocktail . Whole cell extracts were collected by centrifugation at 12,000 × g for 20 min at 4°C. Protein concentration was determined using the Bradford assay , with BSA as a standard. Equal amount of proteins were subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes  after gel electrophoresis. Immunoblots were blocked with 5% non-fat milk for 1 h and then incubated with primary antibodies for 16 h at 4°C. After washing, the transferred blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies  for 1 h at 4°C. The bound IgG protein bands were visualized using an enhanced chemiluminescence detection kit system . The relative intensity of the protein bands was quantified by densitometry using the Image Quant software .The effects of βt-test was used to evaluate statistically significant differences between two groups. Statistical analyses between three or more groups were performed using one-way ANOVA with Tukey's method as a post hoc test. A p < 0.05 was considered statistically significant.The results are expressed as mean ± SEM of at least three independent experiments. A Student's 2-GPI dose-dependently decreased the VEGF-induced proliferation of HAECs  and deglycosylated β2-GPI (200 μg/ml) significantly inhibited the VEGF-induced tube formation . These results show that β2-GPI inhibits the VEGF-induced eNOS phosphorylation in HAECs.We also examined whether βt 15 min . In cont2-GPI is able to inhibit endothelial migration and VEGF-induced cell growth [2-GPI on angiogenesis of HAECs is still unknown. β2-GPI is a glycoprotein with a circulating concentration of approximately 200 μg/ml in human plasma [2-GPI could alter endothelial cell function, which could prevent or ameliorate the vascular pathology observed in patients with angiogenesis or neovascularization. The results of this study support the idea that β2-GPI counteracts the adverse effects of the VEGF-induced angiogenesis in HAECs.Neovascularization is associated with diverse pathological processes such as atherosclerotic plaque rupture, ischemic retinopathies, and carcinogenesis –26. Angil growth , the effn plasma . We post2-GPI are involved in its anti-angiogenic activity, we determined the effect of a deglycosylated β2-GPI on angiogenic tube formation. We showed that the deglycosylated β2-GPI has the same inhibitory effect as β2-GPI in VEGF-treated HAECs. This suggests that the carbohydrate residues of β2-GPI are not involved in the VEGF-induced angiogenic activity, consistent with findings reported by Yu P et al., 2008 [2-GPI structure contains a distinct kringle domain at the C-terminal, which carries a lysine-rich sequence motif that binds negatively charged lipids or anionic lipid-containing target membranes [2-GP1 binds to the surface of endothelial cells through TLR2 or annexin 2 [2-GPI in pathophysiological events [It has been shown that glycosylation affects the angiogenic activity of several proteins –30, althl., 2008 . The 2-Gembranes ,36. Prevnnexin 2 ,37. Accul events .2-GPI behaves as a cell viability maintaining factor for endothelial cells [2-GPI has a protective effect against oxidative stress-induced endothelial cell death [2-GPI-deficient mice [2-GPI may play a role in vascular endothelial integrity. During fibrinolysis, fibrin-catalyzed cleavage of plasminogen produces clot-digesting plasmin and the antiangiogenic molecule, angiostatin [2-GPI, a protein form that has been proteolytically cleaved at Lys317/Thr318 residues, have been found in the plasma of leukemia patients [2-GPI is able to bind plasminogen and inhibits endothelial cell growth in vitro, and suppressed neovascularization and tumor growth in vivo [2-GPI. In the present study, we show that native β2-GPI suppresses the VEGF-induced endothelial cell proliferation and angiogenesis in HAECs. The antiangiogenic activity of endothelial cells provides a potential linkage to the inhibition of neovascularization in vivo. In this study, β2-GPI also shows a potent antiangiogenic activity in vivo, as demonstrated by matrigel plug and angioreactor assays.βal cells . Furtherll death . Recentlent mice . Therefoiostatin . Varyingpatients . Moreove in vivo –45. Thes2-GPI in angiogenesis, we highlighted changes in its signaling pathway and attempted to predict its functional implications. VEGF is known to be one of the most potent angiogenic factors that promote cell migration, cell proliferation, tumor angiogenesis, and tumor cell growth [2-GPI is able to inhibit endothelial cell growth through cyclin proteins and the MAPK signaling pathway [2-GPI inhibits the VEGF-induced angiogenesis by suppressing the phosphorylation of ERK1/2, Akt, and eNOS in HAECs.Given the molecular basis underlying the inhibitory effects of βl growth , 46–49. l growth , 50–52.  pathway . Activat pathway ,55. On t pathway . Moreove pathway –59. Accu pathway –62. In t2-GPI suppresses the VEGF-induced angiogenesis in vitro and in vivo. Furthermore, we shed light to the mechanisms by which β2-GPI affects its underlying signaling pathways; specifically, by suppressing the phosphorylation of ERK1/2, Akt, and eNOS. These results suggest a potential role for β2-GPI in neovascularization and its therapeutic application for the prevention of angiogenesis-related diseases.In summary, our study provides evidence demonstrating that β


Thus, we hypothesized that hypoxia can regulate the differentiation and pluripotency of MMSCs through the Wnt/β-catenin pathway. To test this hypothesis, MMSCs from rats at embryonic day 18.5 were cultured in normoxic (21% O2) and hypoxic (1% O2) conditions. The effects of hypoxia on differentiation, stemness, proliferation, and apoptosis of cultured MMSCs and on the activity of the Wnt/β-catenin pathway were tested. Our results revealed that the hypoxic condition increased the number of epithelial cells (E-cadherin+ or CK18+) as well the expression of markers of renal tubule epithelia cells , decreased the number and proliferation of stem cells (SIX-2+ or CITED1+), and induced apoptosis. Additionally, hypoxia reduced the expression of Wnt4 as well as its downstream molecules β-catenin, LEF-1, and Axin2. Activation of the Wnt/β-catenin pathway by LiCl or BIO modified the effects of hypoxia on the differentiation and self-renewal of MMSCs. Thus, we concluded that hypoxia induces the differentiation and inhibits the self-renewal of MMSCs by inhibiting the Wnt/β-catenin pathway. The observations further our understanding of the effects of hypoxia on kidney.Hypoxia is an important and influential factor in development. The embryonic kidney is exposed to a hypoxic environment throughout its development. The Wnt/ Because the effect of hypoxia on development of the kidney is controversial, understanding the kidney's responses to oxygen deprivation is critical.Prenatal hypoxia is a common complication during pregnancy, which leads to negative foetal outcomes such as birth defects . It has 2 or even lower oxygen tension, owing to atypical blood vessel networks [Coordinated interactions between the embryo and its environment are critical for development. Early-stage mammalian embryos develop in hypoxic environments and live at low oxygen concentrations (ranging from 2% to 9%) throughout foetal development , 5. A hynetworks . Oxygen The development of the mammalian kidney is guided by reciprocal inductive interactions between the ureteric bud  and the metanephric mesenchyme  . In ratsβ-catenin pathway is considered to play a critical role in development of the kidney. Upregulation of Wnt4, which mediates epithelialization of the metanephric mesenchyme, has been detected after exposure of mesenchymal stem cells to hypoxia [β-catenin (downstream of Wnt pathway) induces tubulogenesis [β-catenin blocks MET in cultured MMSCs [β-catenin pathway. Activation of Wnt/β-catenin with lithium chloride (LiCl) or 6-bromoindirubin-3-oxime (BIO) attenuated the effect of hypoxia. Thus, we conclude that hypoxia promotes the differentiation and depresses the self-renewal of MMSCs by inhibiting the Wnt4/β-catenin pathway.Among many other signalling pathways, the Wnt/ hypoxia . Disrupt hypoxia , 18. In ogenesis  and consed MMSCs , 21. Thued MMSCs , 22. E-ced MMSCs –26. Our Twenty timed-pregnant female Sprague-Dawley rats were provided by the Animal Center of Shanghai Medical College, Fudan University . At embryonic days 18-19, the age of the embryos was counted from the day of the vaginal plug. This study was performed in strict accordance with the Guidelines on the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research and the Guidelines of Animal Care. The protocol was approved by the Committee on the Ethics of Animal Experiments of Fudan University.5 cells/ml on 30 mm culture dishes or 96-well plates or 3-well chamber slide system in Dulbecco's modified Eagle's medium (DMEM)/F12 (1 : 1) including 10% foetal calf serum and grown at 37°C, 5% CO2, and 95% air (approximately 21% O2). After attachment, cells were treated with 1% O2 in a variable oxygen control incubators (Thermo Scientific) which could generate hypoxic conditions and monitor real-time O2 sensitively or maintained at 21% O2 for 3 days. To activate the Wnt/β-catenin pathway, LiCl (20 mM) or BIO (5 nM) was added to the culture medium.The primary MMSC cultures were prepared as previously described . Brieflyβ-Catenin signalling pathway . Briefly, after culture under hypoxic or normoxic conditions for 3 days, MMSCs were collected, and protein was extracted. The proteins were separated by 12.5% SDS–PAGE, electrotransferred to a PVDF membrane , and subsequently blocked with 5% (w/v) fat-free milk in TBST for 1.5 h at room temperature. The membranes were blotted sequentially with primary antibodies, including anti-GAPDH antibody , anti-SIX-2 antibody , anti-CITED1 antibody , anti-E-cadherin antibody , anti-Wnt4 antibody , anti-β-catenin antibody , anti-LEF1 antibody , and anti-HIF1α antibody , and then with secondary antibodies  conjugated with horseradish peroxidase. The last step of western blotting antibody detection was performed with western blot chemiluminescent HRP substrate reagent (Thermo Fisher). The expression levels of different proteins were normalized to those of the internal control (GAPDH) [Western blotting was performed to examine CITED1, SIX-2, E-cadherin, and the related proteins in the Wnt/ (GAPDH) .−ΔΔCt method [The total RNA was isolated from MMSCs using TRIzol . The RNA levels of SIX-2, Wnt4, LEF1, Axin2, PKG1, Glut1, CDH6, Aqp1, and OPN were detected with a SYBR-Green RT-qPCR kit  according to the manufacturer's instructions . The levels of mRNA expression were normalized to those of the housekeeping gene (GAPDH). Simple relative quantification of target gene expression normalized to GAPDH was performed using the 2t method . The priμl for 106 cells, CK18: Abcam, Catalog number ab82254) and subsequently washed with PBS supplemented with 1% FBS. After removing the primary antibody by washing twice, samples were reincubated with either a FITC-, Cy3, or Alexa flour 594-conjugated secondary antibody at a dilution of 1 : 500 using an exposure time of 20 min. Apoptosis was detected using Annexin V-FITC/PI staining kit  according to the manufacturer's instructions. Before starting FCM, cells were again rinsed twice with wash buffer. FCM was carried out using a FACSCalibur .Cells were harvested from the culture dishes using trypsin EDTA and fixed with 4% paraformaldehyde, pH 7.4, dissolved in PBS. Single cells for flow cytometry (FCM) were incubated for 30 min with their respective primary antibodies (1 5 cells/ml in 500 μl complete medium) were seeded into each well of chamber slide system. After 24 hours of normoxic incubation, the chamber was removed, and cells were fixed with methanol for 10 minutes. The cells were preincubated with 5% normal goat serum to block nonspecific binding and then incubated with primary antibodies  at 4°C overnight. Extensive washing was followed by incubation with FITC- or Cy3-conjugated secondary antibody (1 : 200) for 1 h at room temperature. Slides were mounted with antifade mounting medium  with DAPI . Images were acquired with a confocal laser scanning microscope (Zeiss 510).The MMSCs (2 × 105 cells/ml in 500 μl complete medium) were seeded into each well of chamber slide system and incubated under hypoxic or normoxic condition for 3 days. Apoptosis was detected using a TUNEL apoptosis assay kit . Cells growing on slides were fixed in 1% formaldehyde for 30 min, permeabilized (using 1% Triton-X100 in PBS) for 10 min, and rewashed before application of TUNEL reagents diluted to 50% with TUNEL dilution buffer. After washing, positive staining was detected with label solution. Proliferation of cells was detected using keyFluor488 Click-iT EdU kit . Cells were incubated with 10 μM EdU for 1 hour prior to being harvested and then fixed and permeabilized as described above. For EdU staining, the slides were incubated with Click-iT reaction cocktail containing Alexa Fluor 488 for 30 min at room temperature according to the manufacturer's instructions.The MMSCs (2 × 104 cells/well in 100 μl complete medium) were seeded into 96-well cell plates. After 72 hours of hypoxic or normoxic incubation, 10 μl AlamarBlue ready-to-use solution was added to each well and the plates were incubated at 37°C for a further 2 hours. The absorbencies with wavelengths set at 570/600 nm were measured by SpectraMax fluorescence multiwell plate reader. Breeding ratio (%) = (117216 × A570 sample − 80586 × A600 sample)/(117216 × A570 control − 80586 × A600 control) × 100%.The effect of hypoxia on the viability of MMSCs was determined using AlamarBlue cell viability assay kit  as previously described. Briefly, the MMSCs (1 × 10t-test (comparison of two groups) and one-way ANOVA followed by Tukey's post hoc test (comparison of more than two groups) were regarded as significant when p < 0.05.Data are presented as means ± SEM and were analysed using SPSS software. Statistical analyses including the independent α-SMA) was detected in most of isolated cells. In contrast, no significant expression of the epithelial cell marker (E-cadherin) was observed  with a mesenchymal cell marker  and its targets genes PKG1 and Glut1 under 1% O2 condition  or BIO (5 nM), the observed upregulation of LEF1 and Axin2 suggested activation of the Wnt signalling pathway  in comparison to conventional cell culture conditions (21% O2) for 3 consecutive days. Many more E-cadherin- or CK18-positive cells were observed among cells exposed to hypoxia, compared with those subjected to normoxic conditions    . The RNA < 0.01) .n = 3, all p < 0.001)      .n = 3, both p < 0.01)  . These rβ-catenin signalling pathway.Collectively, these findings support our hypothesis that hypoxia facilitates the differentiation and suppresses the renewal of MMSCs by inhibiting the Wnt/2 levels ranging from 2% to 9% [2 tension  on differentiation and self-renewal of the MMSCs. We found that unlike 1% O2, 5% and 7% O2 had no effects on either differentiation or self-renewal of the MMSCs. Additionally, the metabolic/molecular biology studies performed on cell lines show that mitochondrial respiration can provide sufficient amount of energy under 1% O2 to permit cell proliferation [2 to research the effect of hypoxic condition on the MMSCs. We demonstrated that hypoxia promotes the differentiation and attenuates the stemness of MMSCs through blocking of the Wnt/β-catenin pathway.Hypoxia is a critical microenvironmental factor for normal embryonic development. Mammalian embryonic development occurs at low intrauterine O2% to 9% . Particu2% to 9% . Even in2% to 9% . Howeverferation . Thus, w2 tension has been shown to inhibit the differentiation of human embryonic stem cells [2) induce ureteric bud branching during kidney development in vitro organ culture [2) induced the differentiation of mesenchymal cell progenitors into epithelial cells, as evidenced by increased expression of the epithelial cell marker E-cadherin and an increased number of E-cadherin- and CK18-positive cells. It seems that hypoxia may induce ureteric bud branching through stimulating the differentiation of MMSCs. However, we found that hypoxic conditions (1% O2) inhibited ureteric bud branching in vitro organ culture [2 that we applied exceeding the tolerance of organ to hypoxia. Thus, further investigation will be needed to explore the effect of hypoxia on development of kidney.The effect of hypoxia on the differentiation of stem cells varies among individual tissues. Hypoxia is known to promote cell differentiation and development in many tissues. It has been reported that hypoxia increases formation of terminal branches during tracheal development  and enhaem cells . In the  culture , 5. Howe culture . The rea2) maintains a higher level of undifferentiated human embryonic stem cells [2 environment has also been demonstrated to have positive effects on the maintenance of stemness of murine stem cells [In addition to inducing differentiation, maintaining stemness and proliferation of stem cells to preserve an available pool of undifferentiated progenitors is also important for development. Some reports have demonstrated that hypoxic culture 1–4% O% O2 mainem cells . A reducem cells . Moreoveem cells . Anotherem cells . Our preβ-catenin, as evidenced by the attenuated level of Wnt4 and its downstream molecules β-catenin, LEF1, and Axin2. Previous investigations have revealed that stimulation of the Wnt/β-catenin signalling pathway in stem cells represses differentiation and maintains a state of self-renewal [β-catenin induces nephron differentiation [β-catenin activity is essential for the fully epithelialized state of the renal vesicle [β-catenin on the differentiation of MMSCs may be distinct at different stages of development. Our data were collected from MMSCs harvested from embryonic kidney at embryonic days 18-19, which is near birth. A simple explanation is that hypoxia inhibited the Wnt/β-catenin pathway, whose activation blocks the differentiation of MMSCs at the late embryo stage in rats. Further studies may be required to explore the effects of hypoxia on the differentiation of MMSCs as well as on the Wnt pathway at early embryo stages.The development of the kidney is a developmental process in which mesenchymal to epithelial transition (MET) occurs. A mesenchymal stem cell pool responds to paracrine and autocrine factors that regulate whether cells remain within the niche or epithelialize. Among these factors, Wnt4 is considered to be a critical regulator in this process. Our investigation found that hypoxic culture inhibited Wnt/-renewal , 42. To ntiation . Howeverntiation . Further vesicle . In thisDifferentiation is a major hurdle for the successful translation of stem cell research to clinical applications. The ability of hypoxic preconditioning to induce the differentiation of stem cells toward mature cell lineages provides support for the application of hypoxic pretreatment before transplantation of stem cells. Indeed, accumulating evidence is suggesting that transplantation of hypoxia-preconditioned stem cells is more effective in tissue repair. It has been shown that the transplantation of hypoxia-preconditioned mesenchymal stem cells (MSCs) enhances vessel formation and skeletal muscle fibre regeneration after hindlimb ischemia and stimulates angiogenesis and neurogenesis after cerebral ischemia , 45. It In summary, the observations herein further our understanding of the effects of hypoxia on kidney development by demonstrating that hypoxic conditions stimulate the differentiation of MMSCs by inhibiting canonical Wnt signalling. Additionally, this study aids in understanding the therapeutic potential and possible clinical impact of hypoxia-preconditioned MMSCs.


Sgo1 expression pattern in developing and adult mouse”  Sgo1 gene expression pattern utilizing Sgo1_LacZ_Knock in mouse line and immunohistochemistry in wild type mice. The data presents Sgo1 expression pattern during development, and in post-developmental proliferative and quiescent tissue. The article describes following tissues: developing heart, neural tube, adult colon, cerebellum, cerebral cortex, liver, and testis.The data shown in this article are related to the research article entitled “Characterization of  In both stages, Sgo1 was expressed robustly in the heart and the neural tube . The Sgo1+/LacZ mice have a flippase recognition target (FRT) flanked lacZ cassette inserted between exons 4 and 5 of the Sgo1 gene in a C57Bl6/N strain. A splicing acceptor site precedes the FRT for inclusion of lacZ cassette into the transcript and following polyadenylation sequence, resulting in a truncated Sgo1 mRNA transcript, and protein. The crossings were made with Sgo1+/+ and Sgo1+/LacZ and the appearance of vaginal plug was designated as Embryonic day (E) 0.5. Embryos at E9.5, E10.5, E12.5, E15.5, E18.5, and 4-month old adult mice, were collected for the experiment.2.2Sgo1+/LacZ were permeated via rinse buffer  overnight at 4 °C with gentle rocking and the wild type samples for DAB staining were dehydrated to 70% ethanol gradually at this time point.Mice were sacrificed according to Canadian Council on Animal Care (CCAC) guidelines. The uterus was extracted and placed in cold phosphate buffer solution (PBS) during embryo extraction. The yolk sac of each embryo was used to genotype the mice using PCR. The samples were then left in PBS on ice for 5–10 min to reduce blood in the sample. The samples were incubated in 4% paraformaldehyde (PFA) at 4 °C with gentle rocking for; E9–E10.5: 5 min, E12–E15.5: 10 min, individual organs: 5 min. After rinsing in PBS to remove residual PFA, 2.3The X-Gal buffer and heterozygous embryos were warmed to 37 °C. X-Gal  dissolved in dimethyl-formamide (100 mg/ml) was then added to the X-Gal buffer  immediately before use to a final X-Gal concentration of 1 mg/ml. The samples were incubated in this X-Gal staining solution for 24 h at 37 °C in total darkness with abundant movement for equal exposure to the staining solution. The samples were thoroughly washed in the rinse buffer on ice until the solution was completely free of yellow coloration from the X-gal buffer. They were then fixed in 4% PFA for minimum 48 h. The samples were gradually dehydrated to 70% ethanol for storage and imaging. For paraffin embedding, the samples were dehydrated gradually to 100% ethanol and incubated respectively in 50/50 ethanol/xylene, 2× 100% xylene, 50/50 xylene/paraffin for maximum 5 minutes each step and were finished with 2× paraffin at 60–62 °C for one hour. The samples were sectioned at 10 μm and counterstained with Nuclear Fast Red (NFR). For cryosectioning, the samples were submerged in a 1:1 ratio of 50% sucrose:Optimal Cutting Temperature (OCT) overnight and frozen in OCT with ethanol slurry on dry ice (OCT: Leica). The samples were equilibrated to the cryotome׳s working temperature (−20 °C), then sectioned at 12 μm to be counterstained with NFR.2.4DAB-immunostaining was performed according to the manufacturer׳s instruction . The wild type samples were sectioned at 10 μm. Antigen retrieval was performed with citrate buffer  at 68 °C for 20 min and 0.3% Triton at room temperature for 30 min. The endogenous peroxidase activity was minimized with hydrogen peroxide incubation for 15 minutes for DAB staining. Two mouse monoclonal anti-SGO1 antibodies  were used separately and compared in representative slides to support the specificity of the SGO1 localisation. TUBB3 and RBPMS antibodies were acquired as the courtesy of Alyson Fournier׳s lab at Montreal Neurological Institute. The primary antibodies were targeted with rabbit anti-mouse IgG secondary antibody conjugated to horseradish peroxidase for DAB staining and goat anti-mouse Alexa 488 for fluorescent staining. DAB slides were counterstained with methyl green and mounted with Permount medium. Fluorescent slides were mounted with DAPI incorporated anti-fade medium.2.5Protein lysates were extracted from the corresponding tissues with RIPA buffer and concentration was quantified using BCA assay. The protein extracts were reduced with 5% end concentration of beta-merceptoethanol and denatured at 95 °C for 5 min before the SDS-PAGE. After the transfer, the nitrocellulose blot was blocked with 5% bovine serum albumin and incubated with anti-SGO1 ab58023 antibody followed by goat anti-mouse HRP conjugated antibody. The blot was developed with ECL reagent.


In order to enhance the compatibilization and interfacial adhesion between epoxy and liquid carboxyl-terminated butadiene acrylonitrile (CTBN) rubber, an initiator was introduced into the mixture and heated to initiate the cross-linking reaction of CTBN. After the addition of curing agents, the CTBN/epoxy blends with a localized interpenetrating network structure were prepared. The mechanical properties and morphologies of pre-crosslinked and non-crosslinked CTBN/epoxy blends were investigated. The results show that the tensile strength, elongation at break and impact strength of pre-crosslinked CTBN/epoxy blends are significantly higher than those of non-crosslinked CTBN/epoxy blends, which is primarily due to the enhanced interfacial strength caused by the chemical bond between the two phases and the localized interpenetrating network structure. Both pre-crosslinked and non-crosslinked CTBN/epoxy blends show a bimodal distribution of micron- and nano-sized rubber particles. However, pre-crosslinked CTBN/epoxy blends have smaller micron-sized rubber particles and larger nano-sized rubber particles than non-crosslinked CTBN/epoxy blends. The dynamic mechanical analysis shows that the storage modulus of pre-crosslinked CTBN/epoxy blends is higher than that of non-crosslinked CTBN/epoxy blends. The glass transition temperature of the CTBN phase in pre-crosslinked CTBN/epoxy blends increases slightly compared with the CTBN/epoxy system. The pre-crosslinking of rubber is a promising method for compatibilization and controlling the morphology of rubber-modified epoxy materials. Tg) of the epoxy matrix decreased with the addition of CTBN. Akbari et al. [Epoxy resin is one of the most popular thermosetting materials used as a matrix for adhesives, coatings, sealants and composites because of its excellent mechanical and chemical properties ,2. The mi et al.  preparedSome morphological parameters of the rubber, such as the content, particle size and volume, can strongly influence the mechanical properties of rubber-modified epoxy resins. The toughness of rubber/epoxy blends increased with the increasing rubber content and reached a maximum at 15–20 phr CTBN ,21. LargAs the curing reaction proceeds, the molecular weight of the epoxy increases and the rubber phase precipitates, leading to the formation of a two-phase morphology . The morIn our previous study, we prepared an in situ pre-crosslinked CTBN/epoxy blend with much better mechanical properties compared to its traditional CTBN-modified counterpart. A local interpenetrating structure was formed in the pre-crosslinked CTBN/epoxy blend, which greatly improved the compatibilization and interfacial adhesion between the two phases, and thus resulted in good mechanical properties of the blend . HoweverDiglycidyl ether of bisphenol-A  was purchased from Shanghai Resins Co., Ltd. . The curing agent isophorone diamine (IPDA) was purchased from Aladin Co., Ltd. . Benzoyl peroxide (BPO) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. . The liquid rubber used in this study was a CTBN copolymer containing 9% acrylonitrile (CTBN-9) and 0.615 mmol/g of carboxyl, and was purchased from Zibo Qilong Chemical Industry Co., Ltd. . All materials were used as received, except that BPO was recrystallized from ethanol solution prior to use.Pre-crosslinked or non-crosslinked CTBN/epoxy blends containing 0, 5, 10 or 15 phr CTBN (relative to 100 g epoxy) were prepared in appropriate quantities as described by Zhou et al.  and Xu e−1 at a resolution of 4 cm−1.The FTIR spectra of pre-crosslinked and non-crosslinked CTBN/epoxy mixtures without the curing agent were recorded by a Nicolet 6700 spectrometer  operating from 4000 to 500 cmThe tensile tests were performed using a universal testing machine  at a crosshead speed of 2 mm/min according to ASTM D-638. The Izod impact strength was determined using a cantilever impact tester  according to ASTM D-256. The average of at least five samples was reported.The tensile fractured and cryofractured surfaces of samples were observed using an S-4800 scanning electron microscope  with an accelerating voltage of 15 kV. The cryofractured surfaces were prepared in liquid nitrogen, and then etched in toluene for 10 h. The surfaces were sputter-coated with gold prior to observation.3) were determined using a DMA Q800  at a frequency of 1 Hz and a heating rate of 5 °C/min from −100 to 200 °C.The dynamic mechanical properties of samples  and weight-average diameter (Dw) of the dispersed rubber particles in different rubber-modified epoxies were calculated using the following equations: ni is the number of particles in the diameter range Di, and PDI is the polydispersity index.The number-average diameter . However, pre-crosslinked CTBN/epoxy blends have smaller micron-sized rubber particles and larger nano-sized rubber particles than non-crosslinked CTBN/epoxy blends. There is a slight increase in the storage modulus and glass transition temperature of the rubber phase after the pre-crosslinking. The use of pre-crosslinked rubber is a promising method for controlling the morphology of rubber-modified epoxy materials.