Discorea nipponica Makino, and its molecular mechanism of action. In this study, all the ten human osteosarcoma cell lines were sensitive to dioscin treatment in a dose- and time-dependent manner. Dioscin inhibits proliferation and induces cell cycle arrest as well as apoptotic cell death in osteosarcoma cells. More importantly, oral administration of dioscin (60 mg/kg) showed significant therapeutic effect on osteosarcoma growth without obvious side effects in vivo. In addition, dioscin possesses the ability to suppress stem-cell-like phenotype of osteosarcoma cells. Mechanistically, dioscin inhibits osteosarcoma stem-cell-like properties and tumor growth through repression of Akt/GSK3/β-catenin pathway. Moreover, β-catenin expression in osteosarcoma patients was associated with clinical prognosis. Conclusively, the present study provides comprehensive evidence for the inhibition of dioscin on osteosarcoma stem-cell-like properties and tumor growth through repression of Akt/GSK3/β-catenin pathway, which suggests dioscin as a promising therapeutic regimen. And β-catenin may be a potential therapeutic target as well as a significant prognostic marker for osteosarcoma patients in clinic.Osteosarcoma is the most common primary bone tumor in children and adolescents. Many patients with osteosarcoma always develop drug resistance to current chemotherapy regimens, which induces a poor prognosis. And cancer stem cells (CSCs) have been reported to possess the properties to self-renew and maintain the phenotype of tumor, which may lead to clinical treatment failure. Thus, it is an urgent task to develop several potentially useful therapeutic agents, which could target CSCs in osteosarcoma. This study aims to clarify the in vitro and in vivo anti-osteosarcoma effects of dioscin, the primary component derived from  The prognosis of patients with osteosarcoma can be improved with the combination of surgery and chemotherapy, and the 5-year survival rate has reached 60−70% in the recent years2. However, a considerable number of patients either are not sensitive to chemotherapy or develop drug resistance with current chemotherapy regimens3. Therefore, one strategy to avoid chemo-resistance and improve clinical outcomes is to identify effective therapeutic agents that can increase drug-response rates.Osteosarcoma is the most common primary bone tumor, which mainly affects children and adolescents7. One of the most successful examples for natural agents is artemisinin (qinghaosu), which is proved to possess significant antimalarial effect by Youyou Tu who was awarded the 2015 Nobel Prize in Physiology or Medicine for her discoveries concerning a novel therapy against malaria.It has been a long history for agents derived from natural sources to be used for clinical treatment, which shows multiple biological activities, such as anti-inflammation and anti-tumor propertiesDiscorea nipponica Makino is a traditional Chinese medicinal herb and has been used as an antitussive and antiasthmatic agent for a long period of time. Dioscin, a kind of steroidal saponin extracted from Discorea nipponica Makino, has been reported to have many biologic effects, including attenuating renal ischemia/reperfusion injury, anti-inflammatory, and anti-allergic properties9. Recently, dioscin has shown significant anti-tumor activity in several types of cancer11. However, very little is known about the effects of dioscin on osteosarcoma, and the molecular mechanisms of its anti-tumor activity remain unclear. Therefore, the aim of our study was to investigate the in vitro and in vivo anti-osteosarcoma effects of dioscin and to clarify its molecular mechanism of action, which can help confirm its potential as a therapeutic agent in the treatment of osteosarcoma patients. Moreover, identifying the targets of dioscin may also help provide valuable biomarkers as prognostic predictive factors for osteosarcoma patients.Discorea nipponica Makino. And 3--2,5-diphenyl tetrazolium bromide (MTT), adriamycin were purchased from Sigma-Aldrich. ICG-001 and XAV-939 were purchased from Selleck Chemicals.Dioscin  was extracted from 12. U2OS/MTX300 cells, a methotrexate-resistant derivative of the U2OS human osteosarcoma cell line, were provided by Dr. M. Serra , which was continuously cultured in the presence of 300 μg/l MTX13. All the other cells were cultured in DMEM  and supplemented with 10% fetal bovine serum , penicillin , and streptomycin (100 mg/l) at 37 °C in a 5% CO2 humidified incubator.The human osteosarcoma cell lines, U2OS, HOS, MNNGHOS, 143B, MG63, SJSA1, and G292, were obtained from the American Type Culture Collection (ATCC). The ZOS and ZOS-M cell lines have been described previouslyA total of 2000 cells were plated in 96-well flat-bottom plates and exposed to dioscin at different concentrations in a final volume of 180 μl. At the indicated times, 20 μl of 5 mg MTT/ml in PBS were added to each well, and the plates were incubated at 37 °C for 4 h. After removal of the medium, 150 μl DMSO was added to each well to dissolve the formazan crystals. Absorbance at 490 nm was determined using a microplate reader.m dioscin at 37 °C in a 5% CO2 incubator for 24 h and were subsequently collected and analyzed using a Cytomics FC 500 instrument (Beckman Coulter) after propidium iodide staining. The percentage of cells in the different phases of the cell cycle were determined with cell-cycle analysis software.Cells were treated with 2.5 μm dioscin for 48 h and were subsequently collected, washed twice with PBS, and incubated with Annexin V-FITC and propidium iodide for 30 min in the dark. Cell apoptosis was analyzed using FACScan , and CellQuest software was used to analyze the original data.The cells were treated with 2.5 μ2 incubator. Cell colonies were fixed in methanol and stained with crystal violet. The number of colonies that contained >50 cells was counted under microscope.Cells were seeded in triplicate at a density of 800 cells/well in six-well flat-bottom plates with 2 ml DMEM containing 10% fetal bovine serum. Cells were incubated with or without dioscin treatment for 48 h and then cultured for 10 days at 37 °C in a 5% COCells were seeded at 50% density in six-well flat-bottom plates, and after overnight incubation the cells were treated with vehicle or dioscin for 24 h. Then, the cells were fixed and then stained with the Hoechst 33258 dye. The morphologic changes and characters of apoptosis were observed using laser scanning confocal microscope (Olympus FV1000).14. And the secondary sarcosphere-formation assay was performed without further treatment after compounds treatment in primary sarcosphere-formation assay.Sarcosphere-formation assay was performed as previously describedFor immunohistochemistry, sections were incubated with antibody against Bcl-2 , Ki-67 , proliferating cell nuclear antigen (PCNA) , β-catenin , Gli1 , NICD1 , phospho-Akt (Ser473) , phospho-GSK3β (Ser9)  at 4 °C overnight. Primary antibodies were detected by the Dako EnVision Kit  according to the manufacturer’s protocol. The staining intensity was evaluated and scored by two independent pathologists. The extent of staining was scored as 0, 0% of cells stained; 1, 1–25% of cells stained; 2, 26–50% of cells stained; 3, 51–75% of cells stained; or 4, >75% of cells stained. Staining intensity was scored as 0, negative; 1, weak; 2, intermediate or 3, strong. The final staining score was defined as the product of the extent and intensity scores.After they were treated with vehicle or dioscin, cell samples were fixed using 4% paraformaldehyde solution for 15 min at room temperature and then extracted with buffer containing 0.5% Triton X-100 for 5 min. The cells were then incubated with primary antibodies against SOX2 , β-catenin , phospho-Akt (Ser473) , phospho-GSK3β (Ser9)  at 4 °C overnight. Next, samples were incubated with secondary antibody at room temperature for 1 h. Finally, nuclei were counterstained with DAPI. Immunofluorescence was detected using laser scanning confocal microscope (Olympus FV1000).TM RT reagent Kit (Takara), according to the manufacturer’s protocol. Real-time PCR amplification was performed using SYBR®Premix (Takara) on Real-Time PCR System (ABI ViiATM7Dx). Primers are the following:After cells were treated with vehicle or dioscin, total cellular RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The cDNA was synthesized by PrimeScript15. Antibodies against p21 , Cleaved PARP , Bcl-2 , Bcl-xL , Gli1 , NICD1 , β-catenin , Lamin B1 , phospho-Akt (Ser473) , Akt , phospho-GSK3α/β (Ser21/9) , GSK3α/β , GAPDH , β-actin  were used.The analysis was performed according to standard procedures described previously6 cells in 200 µl PBS) were injected subcutaneously near the scapula of the nude mice. Eight days after injection, the mice were randomly separated into appropriate groups . Mice were treated with the vehicle or dioscin (60 mg/kg)10 by oral administration every day. ADM was injected intraperitoneally at a dose of 6 mg/kg once a week. Body weights of the mice as well as the length and width of the tumors were measured every 4 days, and the tumor volume was calculated using the formula V = 1/2 (width2 × length). All mice were killed at the end of the experiment, and the tumor weights were measured after careful resection. H&E staining of the mice organs was performed according to standard procedures.All studies were approved by the medical ethical committee of the First Affiliated Hospital of Sun Yat-sen University. And we completed the animal experiments according to the guidelines in Center of Experiment Animal of the First Affiliated Hospital of Sun Yat-sen University. Female nude mice, 4 weeks old, were purchased from Nanjing Biomedical Research Institute of Nanjing University. 143B cells  curve analysis was employed to define an optimal cutoff value for high β-catenin expression by using the 0, 1-criterion. The IHC score with the shortest distance from the curve to the point , which represents both maximum sensitivity and specificity, was selected as the cutoff valuettest or log-rank test (SPSS software 20.0), and a p-value of <0.05 was considered significant.All data were obtained from three independent experiments and expressed as the mean ± SD. The differences were analyzed using two-tailed Student’s Discorea nipponica Makino, and its chemical structure  is shown in Fig. m  followed by flow cytometry. And we observed a G2/M phase arrest . The Dioscin group received 60 mg/kg of dioscin every day by oral administration. The ADM 6 mg/kg group was set as a positive control. A significant tumor size reduction in mice treated with dioscin and ADM was observed at the termination of the study, which was reflected by the tumor growth curve. The mean volumes of the tumors were 1981.10 mm3 for the Vehicle group, 533.55 mm3 for the Dioscin group (p < 0.001), and 787.33 mm3 for the ADM group (p < 0.01) , and 0.64 g for the ADM group (p < 0.01)  and tumor weights (p = 0.069) was observed in the Dioscin group than the ADM group, which suggested that dioscin may have a greater inhibitory effect on osteosarcoma than ADM, one of the currently used chemotherapy drugs.The above experimental evidence has shown the inhibitory effect of dioscin on osteosarcoma in vitro. We sought to further explore whether dioscin could inhibit the growth of osteosarcoma in vivo. Firstly, 143B cells were subcutaneously injected into nude mice until a tumor volume of approximately 200 mm01) Fig. . The ave01) Fig. . AlthougMoreover, IHC assay for mice tumor samples was performed to examine the level of in vivo apoptosis induced by dioscin treatment and its inhibitory effect on osteosarcoma cell proliferation. And the results showed that the tumor treated with dioscin had a significant decrease in the expression of Bcl2, Ki67, and PCNA Fig. . In addiConclusively, these results demonstrate that dioscin possesses anti-tumor properties in human osteosarcoma cells, without severe toxicity and side effects in vivo.3. And cancer stem cells (CSCs) have been reported to possess the properties to self-renew and maintain the phenotype of tumor, which may lead to clinical treatment failure19. The above results have shown the inhibitory effect of dioscin on osteosarcoma both in vitro and in vivo; we sought to investigate whether dioscin could suppress stem-cell-like properties of osteosarcoma cells. Sarcosphere-formation assay showed that dioscin treatment significantly induced smaller and fewer sarcospheres formation in U2OS (p < 0.001) and 143B (p < 0.001)   Fig. . Further05) Fig. .Fig. 3DiNext, we detected the expression of several stem cell markers after dioscin treatment in U2OS and 143B cells. As shown in Fig. In summary, dioscin decreases the stem-cell-like population and suppresses stemness properties of osteosarcoma cells. And dioscin negatively regulates self-renewal ability of osteosarcoma stem cells.22. Firstly, we examined the expression of three critical proteins  involved in CSC pathways27. And we found that only β-catenin decreased in a dose-dependent manner after dioscin treatment, while no significant changes could be observed in NICD1 and GLI1 LI1 Fig. . We furtLI1 Fig. .Fig. 4DiTo confirm the above result in vivo, IHC assay for β-catenin, NICD1, and GLI1 in mice tumor samples was performed to analyze the changes in CSC pathways in vivo induced by dioscin treatment. And the results indicated that the tumor treated with dioscin had a significant decrease in the expression of β-catenin, but not NICD1 or GLI1, when compared to the Vehicle group Fig. .24. To further verify whether dioscin inhibits osteosarcoma through repression of Wnt/β-catenin pathway, western blot and immunofluorescence were used to detect the level of cytosolic and nuclear β-catenin. As shown in Fig. It has been reported that β-catenin nuclear import is a critical step of Wnt/β-catenin signaling pathwayp > 0.05). Besides, the inhibition of dioscin on proliferation, colony formation and sarcospheres formation could be partly rescued by overexpressing β-catenin in osteosarcoma cells    Fig. . And we 05) Fig. .Taken together, these results indicate that β-catenin positively regulates stem-cell-like properties and tumor growth of osteosarcoma.31. Since GSK3 is a kinase within the β-catenin destruction complex, which could phosphorylate Axin-bound β-catenin and the phosphorylated β-catenin is then ubiquitinated and targeted for rapid destruction by the proteasome, preventing activation of β-catenin target genes33, GSK3 can act as a negative regulator of β-catenin. Moreover, GSK3 was the first Akt substrate reported34. Akt is reported to inactivate GSK3 by phosphorylation on an amino-terminal motif conserved in both GSK-3α (S21) and GSK-3β (S9)37. Therefore, we hypothesized that dioscin blocks the phosphorylation of Akt and subsequently activates GSK3 by decreasing the phosphorylation of GSK3, which promotes β-catenin degradation and reduces β-catenin nuclear import.To gain insights on the molecular mechanism by which dioscin suppresses Wnt/β-catenin pathway, we focused on glycogen synthase kinase 3 (GSK3) because GSK3 participates in different signaling pathways, most notably the Wnt/β-catenin pathway in cellsTo verify this hypothesis, we investigated whether the phosphorylation of Akt and GSK3 changed after dioscin treatment both in vitro and in vivo. And we found that phosphorylated-Akt (Ser473) and phosphorylated-GSK3α/β (Ser21/9) gradually decreased in a dose-dependent manner after dioscin treatment Fig. . More imIn summary, dioscin suppresses Wnt/β-catenin pathway mainly by blocking Akt activity, subsequently activating GSK3 and promoting β-catenin degradation.m) in osteosarcoma cell lines .Based on the above demonstration that β-catenin has an important role in osteosarcoma stem-cell-like properties and tumor growth, representative expression pattern of β-catenin protein in osteosarcoma cell lines and human osteosarcoma tissues was detected. Compared to the normal control human osteoblast hFOB1.19, the expression of β-catenin protein was significantly upregulated in osteosarcoma cell lines Fig. . MoreoveTo further investigate the value of β-catenin as a prognostic marker in osteosarcoma, the clinical significance of β-catenin in primary human osteosarcoma specimens was evaluated. Totally, surgical specimens from 107 cases of osteosarcoma were examined for β-catenin expression Fig. .p values were 0.086 and 0.044, respectively). Thus, the cutoff values were selected to achieve the maximum Youden index. After analysis, the cutoff scores for high β-catenin expression were 5.00 for survival status and 3.50 for lung metastases status, respectively. And tumors with scores higher than the obtained cutoff values were considered to have high β-catenin expression.To better assess the expression of β-catenin in primary human osteosarcoma, ROC curve was employed to define an optimal cutoff value for high β-catenin expression, based on the results of IHC assay and evaluation. As shown in Fig. p = 0.0178, Fig. p = 0.0082, Fig. Based on the definition of cutoff scores, Kaplan−Meier survival analysis revealed that patients with high β-catenin expression (β-catenin IHC score higher than 5.00) had worse overall survival than those with low β-catenin expression Here, we further demonstrated that β-catenin is significantly overexpressed in osteosarcoma cells, compared to the osteoblast hFOB1.19. And a higher expression of β-catenin was detected in osteosarcoma tissues, when compared to the normal tissues. Besides, the IHC assay showed that β-catenin protein is strongly expressed in 41.12% (44/107) clinical samples, and correlates with prognosis of osteosarcoma patients. Collectively, these results suggest that β-catenin could be a promising therapeutic target as well as a significant prognostic marker in osteosarcoma. More importantly, dioscin shows its significant therapeutic effect on osteosarcoma by targeting Wnt/β-catenin pathway, which indicates the clinical application value of dioscin for osteosarcoma patients.33. Moreover, Akt is reported to inactivate GSK3 by phosphorylation37. In this study, we demonstrated that dioscin inhibits stem-cell-like properties and tumor growth of osteosarcoma cells through Akt/GSK3/β-catenin signaling. However, how dioscin inhibits the activity of Akt and whether the inhibition is direct needs further study.GSK3 could phosphorylate Axin-bound β-catenin and then promote the rapid destruction of phosphorylated β-catenin, preventing the transcription of β-catenin target genes in the nucleusIn conclusion, the present study provides comprehensive evidence for the inhibition of dioscin on osteosarcoma stem-cell-like properties and tumor growth through repression of Akt/GSK3/β-catenin pathway, which suggests dioscin as a promising therapeutic regimen without obvious side effects for osteosarcoma patients. And this study not only identifies the Akt/GSK3/β-catenin axis as a critical regulator of osteosarcoma CSCs, but also clarifies that β-catenin may be a novel therapeutic target as well as a significant prognostic marker for osteosarcoma clinical treatment. The safety and efficacy of dioscin as a novel drug for osteosarcoma patients will be further evaluated in clinical studies.Supplementary Figure S1Supplementary Figure LegendSupplementary Table S1


Phyllanthus emblica L. (emblic leafflower fruits) have been thought of as the beneficial constituents for ameliorating endocrinal and metabolic diseases including diabetes. However, the effect of emblic leafflower fruits on diabetic vascular complications involved in ellagitannin-derived urolithin metabolites is still rare. In this study, acetylcholine-induced endothelium-independent relaxation in aortas was facilitated upon emblic leafflower fruit consumption in the single dose streptozotocin-induced hyperglycemic rats. Emblic leafflower fruit consumption also suppressed the phosphorylation of Akt (Thr308) in the hyperglycemic aortas. More importantly, urolithin A (UroA) and its derived phase II metabolites were identified as the metabolites upon emblic leafflower fruit consumption by HPLC-ESI-Q-TOF-MS. Moreover, UroA reduced the protein expressions of phosphor-Akt (Thr308) and β-catenin in a high glucose-induced A7r5 vascular smooth muscle cell proliferation model. Furthermore, accumulation of β-catenin protein and activation of Wnt signaling in LiCl-triggered A7r5 cells were also ameliorated by UroA treatment. In conclusion, our data demonstrate that emblic leafflower fruit consumption facilitates the vascular function in hyperglycemic rats by regulating Akt/β-catenin signaling, and the effects are potentially mediated by the ellagitannin metabolite urolithin A. Ellagitannins in Though the commercial insulin injection is a primary strategy for blood glucose control, the protection for organ functions is still indispensable in the therapeutic process of diabetes. The continuous hyperglycemia plays a crucial role in both the diabetic macroangiopathy and microangiopathy −) in extracted ion chromatogram  induced A7r5 VSMC model. In #P < 0.05), which was weakened by UroA treatment (5–40 μM). Because 40 μM UroA treatment led to obvious cell cytotoxicity  in A7r5 cells, the UroA concentrations from 5–20 μM were used in the following investigation. To further evaluate whether HG-induced cell proliferation was corresponding to the in vivo molecular evidence, the expressions of mRNAs and proteins were assessed by qPCR and western blot, respectively. As shown in Figures β-catenin were elevated in the HG-induced group (#P < 0.05), which was significantly suppressed by UroA (∗P < 0.05). Moreover, the mRNA expressions of the Wnt downstream proliferative targets, c-Myc and cyclin D1, increased in the HG group (#P < 0.05), which were also reduced by UroA treatment  or NaCl (20 mM) (MOCK group) before UroA administration. LiCl treatment elevated the expression of β-catenin, compared to the MOCK group (#P < 0.01). The results showed that LiCl-induced β-catenin accumulation was suppressed to 0.4- and 0.3-fold by UroA treatment with 10 and 20 μM, respectively, compared to the LiCl-treated group  suppressed the firefly luciferase expression to 0.6- and 0.5-fold, compared to the LiCl-treated group , which produced in tropical and subtropical areas including India, Malaysia, and southern China, are the subsidiary agricultural products and commonly consumed as a kind of seasonal fruits in these regions [ in vitro and in vivo suggest that ELF has antioxidant, antidiabetic, antimicrobial, lipid-regulatory, antiproliferative properties, and especially ameliorative effects on cardiovascular disorders [ in vitro cell model with ellagitannins is still limited. regions . Moreoveisorders . NoteworRecently, phenolic components were identified from ELF by column chromatography combined with nuclear magnetic resonance spectrum. It is acknowledged that gallotannins and ETs are the major phenolic constituents of ELF . Moreoveα-glucosidase and α-amylase in the intestine [β cells, while the insulin secretory function is partly retained in Yang's work.The antidiabetic function of ELF has been evaluated in animal models and humans , and thentestine . Howeverntestine . It coulThe inhibition of endogenous synthesis of NO and the elevation of vascular remodeling accelerators such as angiotensin II and endothelin-1 were considered as adverse factors in hyperglycemia. A published work demonstrated that the plasma NO contents were elevated for 1-2 weeks after STZ-treatment and afterwards reduced at four weeks , which w in vivo, rather than ETs. Poor bioavailability obstructs the overestimated benefits such as free-radical-scavenging capacity in vivo, which is shifting attention towards to the modulatory effects of urolithins on signaling pathways involved in the vascular dysfunction. The similar standpoint is illuminated in a review illuminating the relationship between ETs and vascular health [β-catenin cascade in the arterial tissues of hyperglycemia rats was blocked upon ELF consumption, accompanied by the suppression of c-Myc and cyclin D1 transcription. Importantly, UroA was detectable as a metabolite in urine after ELF consumption, indicating the ETs in ELF were converted to urolithins in vivo and leading us to keep a watchful eye on urolithins. The findings initially put forwards a new standpoint that the benefits of ELF for diabetes complications are presumably involved in the ellagitannin metabolite urolithins. To verify this hypothesis, the potential effects of UroA on Akt/β-catenin signaling were assessed using two in vitro cell models (Figures β/β-catenin signaling was commonly considered to promote cell proliferation and carcinogenesis [β-catenin and Tcf-4 play a key role in vascular remodeling by activating the c-Myc and cyclin D1 expressions, inhibiting VSMC apoptosis and promoting cell proliferation [β-catenin signaling were assessed using an in vitro high glucose VSMC model. In this cell context, Akt/β-catenin signaling was suppressed by UroA, similar to the results in the arterial tissues above. The evidence involved in the effects of UroA on Wnt/β-catenin signaling was also evaluated in a LiCl-induced A7r5 cell model, which is accordant to that in our previous study on hepatoma carcinoma cells [ in vitro data further enhance the viewpoint that urolithins converted from ETs upon ELF consumption could be considered as beneficial substances for vascular function in hyperglycemia.Bioconversion by the gut microbiota and Phase I/II metabolism is pivotal to characterize the potential chemicals for cardiovascular health. In these studies, ETs were frequently considered as the potential components. However, the internal metabolite of ETs is prevailingly identified as urolithin compounds, indicating that urolithins could be beneficialr health . Though  Figures  and 5. Iogenesis . Moreoveferation . The effma cells . Therefoβ-catenin, and transcription of c-Myc and cyclin D1 were suppressed, in both the artery of hyperglycemia rats and the HG-induced VSMC proliferation model upon ELF consumption or by UroA treatment, respectively. Furthermore, combining evidence from the component analysis in fruits and the metabolite identification in rats, urolithins derived from ETs in ELF were considered to be crucial for preventing VSMC dysfunction in STZ-induced hyperglycemia. In brief, we cautiously suggest that Akt/β-catenin signaling in hyperglycemia aortas is alleviated upon ELF consumption, which possibly attributes to, at least partly, urolithins derived from ETs in ELF.In the present study, the protective effects of ELF on STZ-induced VSMC dysfunction in rats were discovered for the first time. The correlation between ellagitannin metabolite UroA and HG-induced VSMC proliferation was also discussed in A7r5 cells. The primary finding of this study is that vasomotoricity damaged by STZ was improved upon ELF consumption. Moreover, the phosphorylation of Akt, accumulation of


Coronary artery disease (CAD) is the major cause of mortality and morbidity worldwide. The aim of this study was to explore the effect of resveratrol (RES) on Canonical β-catenin/Wnt and forkhead box O (FOXO) pathways in CAD patients.We performed this study on 10 metabolic syndrome patients with three-vessel CAD and 10 sex-aged matched healthy subjects. The effects of RES on β-Catenin, manganese superoxide dismutase (MnSOD), and peroxisome proliferator-activated receptor delta (PPAR-δ) expression were evaluated in peripheral blood mononuclear cells (PBMCs) of participants. RES could increase the MnSOD expression in CAD patients . After RES treatment, the MnSOD expression of patients is still non-significantly lower than controls. In both blank and RES treatments, a significant positive correlation between β-catenin and MnSOD mRNA expressions was found in controls, whereas no correlation between these gene expressions was found in untreated PBMCs of CAD patients. However, RES could modestly improve this pathway in CAD. RES could increase the MnSOD activity in healthy and CAD subjects . Furthermore, in both blank and RES treatments, the significant correlation was found between total β-catenin protein and the MnSOD activity in PBMCs of the controls but not in patients. The cross-talk between β-catenin/Wnt and FOXO pathways was impaired in PBMCs of CAD patients. RES treatment could lead to a modest increase in the MnSOD activity independent of β-catenin/FOXO pathway. Despite a modest improvement in the β-catenin/FOXO pathway after RES treatment, this pathway was not completely repaired in CAD patients. Metabolic syndrome is a constellation of cardiovascular and metabolic risk factors including obesity, insulin resistance, hypertension and dyslipidemia. Coronary artery disease (CAD) is considerably linked with these risk factors , 2. OxidIn order to reduce the deleterious effects of oxidative stress, several antioxidant protective networks and signaling pathways are operative in cells. The up-regulated gene-expression of free radical scavenging enzymes such as manganese superoxide dismutase (MnSOD) by members of the forkhead box O (FOXO) transcription factors is considered to be one of the paramount cell defensive mechanisms against oxidative damage , 6. It ie.g. red wine), peanuts, pomegranates and mulberries (Resveratrol (RES) , a natural polyphenol with antioxidant effects can be found in red grapes and its processed drinks  and age (40-55). The National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria was used to diagnose the metabolic syndrome  as preseAll patients underwent coronary angiography to assess the severity of CAD at Tehran Heart Center . The severity of CAD was defined in terms of number of involved vessels with significant stenosis (> 50 % narrowing in coronary arteries). The exclusion criteria were established as having malignancy, myocardial infarction, unstable angina, previous coronary intervention, inflammatory diseases and other known chronic diseases. The subjects receiving antioxidant therapy or vitamin supplements in the previous 12 months, and also smokers were not included in the study. Five patients were under treatment with cholesterol lowering and anti-hypertensive medications. The acetylsalicylic acid usage was stopped one week prior to coronary artery bypass graft (CABG) surgery. Healthy subjects were selected from the healthy population with normal electrocardiographic findings. This group had normal lipid profile with no history of diabetes, hypertension, endocrine disorders, and cancer. The study was approved by Tehran University of Medical Sciences (TUMS) Ethics Committee, and written informed consent was obtained from all subjects.Participants’ information was obtained by questionnaires on personal data and clinical measurements such as age, gender, drug consumption during the past months, and medical or family history of diabetes. Systolic and diastolic blood pressure was measured twice in the right arm of the subjects who had been resting for at least 10 min in a comfortable position . Body mass index (BMI) was calculated as weight in kilograms divided by the height in meters squared. The waist circumference was taken at the midpoint between the iliac crest and the lower rib margin.Blood samplingVenous Blood samples (25 cc) were obtained from participants after overnight fasting between 7:00 and 9:00 am. Then the samples were divided into two aliquots, in clot activator tube (5cc) and heparin-treated vacutainer (20 cc) in order to biochemical analyses and PBMC isolation, respectively. In addition, 1 cc blood was used for plasma ferric reducing antioxidant power (FRAP) assay. Serum and plasma samples were obtained after centrifuge at 300 g for 15 min at 4 ˚C, and were stored at -80 ˚C for further experiments. Laboratory measurementsSerum fasting levels of glucose (FBS), HDL cholesterol, low density lipoprotein (LDL) cholesterol, total cholesterol (TC) and triglyceride (TG) were measured by Pars Azmoon kits (Iran) using auto-analyzing system . PBMC separation6 cells/well in 12-well plates, in order to further experiments. RES  was dissolved in dimethyl sulfoxide (DMSO) , in which DMSO final concentration in culture medium was less than 0.025%. Blank group (untreated cells) received the same amount of DMSO.Human PBMCs were separated by the Ficoll-Hypaque (lympholyte-H)  gradient centrifugation method from heparinized blood samples. Freshly isolated PBMC were re-suspended in RPMI-1640 medium  containing 10% heat-inactivated fetal bovine serum , and 1% penicillin/streptomycin solution . The numbers of viable PBMCs were determined with trypan blue exclusion method. PBMCs were plated at density of 2 × 10Evaluation of cell viability under RES treatment 4/well) were seeded in 96-well plates and underwent overnight incubation in humidified atmosphere at 37 ˚C temperature with 5% CO2, then the medium was removed by centrifugation at 300 g for 15 min and replaced with a fresh medium containing two concentrations of RES (50 and 100 µM) prepared in FBS-free RPMI-1640. After 12 h and 24 h incubation, the cells were rinsed three times with Phosphate-buffered saline tablets , and then 5 mg/mL 3--2, 5 diphenyltetrazolium bromide (MTT)  was added into the wells. After 4 h the medium was removed and the blue formazan crystals were solubilized with 100 μL of DMSO. Optical density was recorded at 540 nm. Cell viability assessment was performed based on the reduction of MTT by mitochondrial dehydrogenase of viable cells. The percentage of viable cells was calculated relative to untreated cells. PBMCs  were seeded in 12-well plates. RNA extraction was performed using Total RNA Extraction Miniprep kit  according to the manufacturer’s protocol. The RNA concentration was determined by measuring the 260/280 nm absorbance ratio and its quality was evaluated using agarose gel electrophoresis. First-strand cDNA synthesis was carried out using 1 µg RNA with cDNA Revert Aid First Strand cDNA Synthesis Kit . The resulting cDNA were amplified using SYBR Green PCR Master Mix  by Rotor Gene real-time thermocycler  according to the manufacturer›s protocol. MnSOD, PPAR-δ, β-catenin and β-actin primers were purchased from QIAGEN . All gene-expression data were normalized to β-actin (ΔCT). The standard curves were generated from the pooled cDNA of the assayed samples.For RNA extraction, PBMCs , through colorimetric-based assay at 450 nm. Briefly, MnSOD prevents the reduction of WST-1  to WST-1 formazan by neutralizing superoxide anions radicals which are generated from the xanthine oxidase function (in the presence of xanthine and O2). MnSOD was detected from total SOD by administering potassium cyanide (KCN) 1 mM which inhibits both Cu/Zn-SOD and extracellular SOD. Total β-catenin protein (non-phosphorylated active form plus phosphorylated inactive form) was measured by enzyme immunoassay (EIA) according to the protocol described in the kit . Determination of PBMC numbers for enzyme activity and protein level measurement were performed according to kit manufacture protocol. The cells were harvested through trypsinization (for MnSOD enzyme activity) and scraping . Protein estimation of each sample was performed using the method of Bradford .FRAP assayTotal antioxidant capacity was determined by Benzie and Strain FRAP method . The antStatistical analysis-ΔCT (relative expression) from the measured ΔCT by Real Time PCR. Pearson correlation coefficients (r) were used to explain the associations between investigated genes. P-value of less than 0.05 was considered statistically significant. Data were analyzed using SPSS 20 . Descriptive analysis was operated for demographic and clinical data. All quantitative variables were tested for the normality by means of the Shapiro-Wilk test before analysis. Between groups comparisons were analyzed by Student›s t-test  and Mann-Whitney U test . The data of variables with normal distribution are expressed as means ± standard error of the means (SEM), and data of variables without normal distribution are expressed as median (interquartile ranges) (IQR). Comparative CT method  on PBMCs viability are shown in Influence of RES treatment on mRNA expression of β-catenin, MnSOD, and PPAR-δThe effects of RES on mRNA expression of the genes are shown in Measurement of MnSOD enzyme activity and total β-catenin protein The effect of RES on total β-catenin protein level and MnSOD enzyme activity in PBMCs of both healthy subjects and CAD patients are shown in FRAP values Plasma antioxidant levels of CAD patients and healthy subjects are shown in Oxidative stress is demonstrated as one of the paramount leading causes of atherosclerosis and consequent CAD . RES is This study was performed on PBMCs of three-vessel CAD patients with metabolic syndrome to assess the alterations of canonical β-catenin/Wnt and FOXO pathways in basal condition and in response to RES treatment. The main findings of our study are explained here: For the first time we demonstrated the impaired cross-talk between β-catenin/Wnt and FOXO pathways in PBMCs of CAD patients. Moreover, while RES could mildly improve the β-catenin/FOXO pathway in CAD patients, it could not repair this disrupted pathway. The β-catenin/Wnt pathway was intact in CAD patients but was not augmented by RES. Interestingly the MnSOD activity was significantly increased after treatment with RES independent from β-catenin/FOXO in CAD patients. The schematic representation of this impaired β-catenin /FOXO pathway and RES effects in CAD patients are displayed in Figure 7.et al.  levels. Total β-catenin protein and MnSOD enzyme activity of CAD patients appear to be lower than the healthy subjects. Evidence supports the fact that healthy subjects endure mild levels of oxidative stress , 27 whicet al.  agrees wMoreover, the fact that the number of involved coronary vessels of CAD patients is inversely associated with total antioxidant capacity ; which mThe slightly increased correlation between total β-catenin protein and MnSOD enzyme activity after treatment with RES reveals a modest augmentation of β-catenin/FOXO pathway in healthy subjects. The modest augmentation of β-catenin/FOXO pathway in our study might be justified by the findings of the other studies , 33 whice.g. MnSOD) in CAD patients might be regulated through protective signaling pathways other than β-catenin/FOXO pathway; e.g. NAD-dependent deacetylase sirtuin-1(SIRT1) /FOXO3a or Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor activation -2, 5 diphenyl tetrazolium bromide; KCN: Potassium cyanide; EIA: Enzyme immunoassay; FRAP: Plasma ferric reducing antioxidant power; JNK: Jun N-terminal kinases; GSK-3β: Glycogen synthase kinase 3β; SIRT1: NAD-dependent deacetylase sirtuin-1; Nrf2: Nuclear factor (erythroid-derived 2)-like 2; LRP5/6: Low density lipoprotein receptor-related protein 5/6; APC:Adenomatosis polyposis coli; CK1: Casein kinase 1.Authors᾽ contributionsMSB, AH, TG, RM, MB, MS and PP conceived of the study and participated in its design. All authors contributed writing and editing the manuscript. AH, MSB, and MB performed data interpretation and statistical analysis. MS helped to collect the samples. MSB, AH, TG, RM, MB and PP performed the experiments. All authors read and approved the final manuscript.


Mechanical force and Wnt signaling activate β-catenin-mediated transcription to promote proliferation and tissue expansion. However, it is unknown whether mechanical force and Wnt signaling act independently or synergize to activate β-catenin signaling and cell division. We show that mechanical strain induced Src-dependent phosphorylation of Y654 β-catenin and increased β-catenin-mediated transcription in mammalian MDCK epithelial cells. Under these conditions, cells accumulated in S/G2 (independent of DNA damage) but did not divide. Activating β-catenin through Casein Kinase I inhibition or Wnt3A addition increased β-catenin-mediated transcription and strain-induced accumulation of cells in S/G2. Significantly, only the combination of mechanical strain and Wnt/β-catenin activation triggered cells in S/G2 to divide. These results indicate that strain-induced Src phosphorylation of β-catenin and Wnt-dependent β-catenin stabilization synergize to increase β-catenin-mediated transcription to levels required for mitosis. Thus, local Wnt signaling may fine-tune the effects of global mechanical strain to restrict cell divisions during tissue development and homeostasis.DOI:http://dx.doi.org/10.7554/eLife.19799.001 Tissues and organs can both produce and respond to physical forces. For example, the lungs expand and contract; the heart pumps blood; and bones and muscles grow or shrink depending on how much they are used. These responses are possible because cells contain proteins that can respond to physical forces. One of the best studied of these is a protein called β-catenin, which increases the activity of genes that trigger cells to divide to promote the expansion of tissues. β-catenin is over-active in many types of cancer cells where it contributes to tumor growth. In addition to being switched on by mechanical force, β-catenin is also activated when cells detect a signal molecule called Wnt.Cells cycle through a series of stages known as the cell cycle to ensure that they only divide when they are fully prepared to do so. Benham-Pyle et al. investigated if physical force and Wnt activate β-catenin in the same way or if they have different effects on cell division. The experiments were conducted on dog kidney cells that had left the cell cycle and had therefore temporarily stopped dividing. Physical forces, such as stretching, resulted in β-catenin being modified by an enzyme called SRC kinase, which allowed the cells to re-enter the cell cycle. On the other hand, Wnt stabilized β-catenin and temporarily increased the number of cell divisions.When mechanical stretch and Wnt signaling were combined, the cells were more likely to re-enter the cell cycle and divide compared to either stimulus alone. These data suggest that physical force and Wnt signaling affect β-catenin differently and that they can therefore have a greater effect on cell or tissue growth when they act together than on their own.The findings of Benham-Pyle et al. show that β-catenin is not simply switched on or off, but can have different levels of activity depending on the input the cells are receiving. Future experiments will test whether these mechanisms also exist in three-dimensional tissues, which will help us understand how organs develop.DOI:http://dx.doi.org/10.7554/eLife.19799.002 Mechanical cues are critical for regulating cellular growth, morphology, and behavior in developing and adult tissues. Recently, cadherin-mediated cell-cell adhesions have been identified as core components that regulate the response of multicellular tissues to externally applied strain . Signifiβ-Catenin transcriptional activity drives cellular proliferation and is a downstream effector of the Wnt signaling pathway . β-CatenDrosophila Wingless. When Wnt ligand binds to Frizzled/LRP receptors, Axin is recruited to the plasma membrane and CKI/GSK3β are inactivated, resulting in inhibition of β-catenin ubiquitination and degradation  and then β-catenin transcriptional activities (G1 to S): inhibition of Yap1 and β-catenin transcriptional activities was sufficient to block cell cycle entry and G1 to S transition, respectively . However34, see also Materials and methods). The design and fabrication of the biaxial cell stretching device compatible with live imaging allowed for direct visualization of strained monolayers with an inverted fluorescence microscope. Briefly, quiescent monolayers were formed on compliant silicone substrates in a PDMS well, surrounded by a pneumatic chamber separated by a thin silicone wall. Vacuum pressure applied to the pneumatic chamber deflected the silicone wall outwards, resulting in biaxial stretch accompanied by equi-biaxial in-plane strain  was use2, In the absence of mechanical strain, quiescent epithelial monolayers maintained a steady turnover rate over 24 hr that was characterized by a low, but constant number of cells in S/G2  of the ISA does not provide sufficient cell numbers for biochemical characterization . Therefore, to test whether mechanical strain induced an accumulation of cells in S/G2 due to strain-induced DNA damage, monolayers were stained for the DNA damage associated histone variant phospho-γH2A.X  of the ISA does not provide sufficient cell numbers for biochemical characterization . Eight hours after mechanical strain, levels of pY654 β-catenin in the cytoplasm and nucleus had increased significantly in mechanically strained cells ; note that levels of pY654 β-catenin fluorescence at cell-cell contacts appeared to be low, as expected since pY654 β-catenin has a weaker affinity for binding E-cadherin  signaling . To testTo test whether inhibition of CKI phosphorylation affected β-catenin transcriptional activity in response to mechanical strain, monolayers were treated with the CKI inhibitor D4476 . D4476 rMechanical strain induced cell cycle re-entry and accumulation of cells in S/G2 but was insufficient to drive cells into mitosis, suggesting an additional level of regulation for cell transition from S/G2 to mitosis in the presence of mechanical strain. Since β-catenin transcriptional activity is required for progression through S phase, we considered whether further increasing the level of β-catenin activity, through inhibition of β-catenin degradation, might be sufficient to drive cells into, and through mitosis in the presence of mechanical strain.D4476-treated Fucci-MDCK monolayers were imaged on the biaxial live imaging cell stretcher for 24 hr following application of mechanical strain. In the absence of mechanical strain, the number of Geminin-positive cells in S/G2 in both control (DMSO) and D4476 treated monolayers was very low and did not change over 24 hr , Video 3To assess cell entry into mitosis, Fucci MDCK cells were tracked and the number of mitotic events per hour was measured as a function of the degradation of Geminin and cells switching from green to red fluorescence –6. In unCKI contributes in multiple ways to cell cycle progression and directly to mitotic entry . TherefoIn vivo, Wnt signaling blocks β-catenin degradation, resulting in β-catenin accumulation in the cytoplasm and nucleus and induction of β-catenin transcriptional activity . TherefoConditioned media was collected from control L cells or L cells expressing Wnt3A, as previously described . Both unSignificantly, monolayers under mechanical strain and treated with Wnt3A-conditioned media accumulated more Geminin-positive S/G2 cells than mechanically strained monolayers in the presence of control conditioned media . MoreoveIn unstrained monolayers, Wnt3A stimulation increased the number of mitotic events over 5 hr, which then gradually decreased over 24 hr , Video 7Mechanical cues are critical for the normal development, morphology, and function of multicellular tissues. Numerous pathways and molecular scaffolds have been identified as mechano-responsive, including cadherin cell-cell adhesion complexes , integriDrosophila development (Drosophila zone of non-proliferating cells (ZNC)  , and durmination . Our restrans adhesion interactions  β-catenin in the cytoplasm and nucleus. pY654 β-catenin has a weaker affinity for E-cadherin , and thu4 β-catengression  indicateIn contrast to the effects of inhibiting Src activity, inhibition of CKI activity increased β-catenin transcriptional activity  independLive imaging of Fucci-MDCK monolayers revealed that increased β-catenin transcriptional activity upon CKI inhibition produced a distinct cell cycle response to mechanical strain compared to un-treated monolayers  and 6. FCKI inhibition is a simple method to stabilize cytoplasmic β-catenin by short-circuiting the β-catenin degradation machinery, but it has the potential for off-target effects given the broad functions of CKI in cells. Wnt ligands are secreted signaling proteins that induce β-catenin transcriptional activity by inhibiting β-catenin phosphorylation by CKI/GSK3β . In the Dose-dependent effects and signaling thresholds have been previously described in early development , and sevDrosophila wing imaginal disc  cells , an MDCKVery dense, quiescent monolayers of MDCK cells were formed using a calcium switch method, as described previously . Briefly2  but the surface area strained is less since the surface area of the pillar used to stretch the substrates is smaller, depending on the amount of strain applied  placed on the PDMS surface, or imaging cell nuclei in a Fucci MDCK monolayer . A pressThe DNA damaging agent methyl methanesulfonate  was used as a positive control in DNA damage studies. Cells were treated with 0.1% (v/v) MMS for 2 hr prior to fixation and processing for imaging. The small molecule inhibitor iCRT3   paraformaldehyde for 15 min, permeabilized in 0.5% (v/v) Triton X-100 for 7 min, and blocked in PBS containing 0.2% (w/v) BSA and 1% goat or donkey serum at room temperature for 60 min. Primary antibodies used for immunofluorescence staining were: β-catenin , phospho-γH2A.X , p53 , p53BP1 , and pY654 β-catenin/mouse . EdU incorporation was performed using a Click-iT Plus EdU Alexa Fluor 647 Imaging Kit  as directed by the manufacturer.Immunofluorescence images were acquired with a Zeiss  Axiovert 200 inverted microscope equipped with a Mercury lamp and a 63X objective , and acquired with MicroManager Microscopy Software. Fluorescence live-cell images were acquired on a custom-built Zeiss Axiovert 200 M inverted wide field epifluorescence microscope (Intelligent Imaging Innovations (3i), Denver, CO, USA) equipped with a Hamamatsu C11440 Digital camera (Orca-flash4.0OLT), an x, y motorized stage with harmonic drive z-focusing, and a quad filter set for DAPI, FITC, Cy3 and Cy5 laser system . Live cells were imaged in DMEM without Phenol Red at 37°C with a 20X air objective for tracking cellular movement; images were acquired using Slidebook (3i) software.The chosen cell cycle markers, the transcriptional reporter, and  β-catenin localized in the cell nucleus. Therefore, the strategy for image quantification was nuclear segmentation (using Hoechst staining) followed by pixel intensity calculation for each nucleus; this method excluded cytoplasmic and plasma membrane staining. The nuclear pixel intensity of all biomarkers  was analyzed on a single cell basis using a previously described image-processing routine in MATLAB . The res2 (movie frame) over 24 hr between conditions using an unpaired t-test. In cases where the reported metric was an absolute distance to a nearest neighbor or a pixel intensity value relative to a no strain control, data were not assumed to be normally distributed and a Kolmogorov-Smirnoff (KS) test was used to compare distributions. All experiments on the ISA were repeated at least 3 times, and the data from all experiments used in the statistical analysis and figure presentations. For Fucci MDCK monolayers, at least 6 replicate movies were taken from at least 2 independent experiments and the data from all experiments used in the statistical analysis and figure presentations.Image quantification for signaling responses was represented as percent cells positive for the relevant biomarker and the threshold intensity separating positive and negative cells was kept constant between compared conditions. Statistics compared the mean percent of positive cells between multiple independent experiments using an unpaired t-test and the Holm-Sidak method to correct for multiple comparisons. When reporting # of occurrences of cell cycle progression, statistics compared the number of occurrences per 0.43 mm In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom, Reinhard Fässler (Reviewer #1), is a member of our Board of Reviewing Editors and the evaluation has been overseen by Vivek Malhotra as the Senior Editor.Thank you for submitting your article "Increasing β-catenin/Wnt3A Activity Levels Drive Mechanical Strain-Induced Cell Cycle Progression through Mitosis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Benham-Pyle and co-workers reported in an earlier paper that forces applied to a monolayer of MDCK epithelial cells induced rapid cell cycle entry and progression to S through the transcriptional activity of first Yap1 and then β-catenin. In the present paper, the authors extend their observations and show that force-induced Src activity and an additional elevation of β-catenin, achieved either by treating the monolayer with Wnt3a or a Casein Kinase I inhibitor (both of which stabilises β-catenin and increases nuclear β-catenin levels), are required to enable mitotic entry and successful cell division.Essential revisions:1) Confirm b-catenin phosphorylation and degradation with quantitative Western blots,2) Confirm that DNA damage plays no role in G2/S arrest with quantitative Western blots for active p53 and ATM/ATR,3) Confirm that indeed Src and not EGFR (PP2 inhibits both functions) is activated upon strain,4) Investigate whether increased strain can bypass the need for high, local Wnt signalling. If it can, test whether G2/S arrest is induced by inhibiting Src or enhanced by inhibiting CKI,5) Better discuss the relevance of this in vitro crosstalk of Wnt and force for cell division in vivo.eLife. Your revised article has been favorably evaluated by Vivek Malhotra (Senior editor) and three reviewers, one of whom, Reinhard Fässler, is a member of our Board of Reviewing Editors.Thank you for resubmitting your work entitled "Increasing β-catenin/Wnt3A Activity Levels Drive Mechanical Strain-Induced Cell Cycle Progression through Mitosis" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Specifically, the reviewers were disappointed that you did not test whether higher strain allows cells to proceed into mitosis in the absence of Wnt signalling, and whether the G2/S arrest is induced by inhibiting Src or enhanced by inhibiting CKI under higher strains. We appreciate that it is technically not trivial to apply the higher strain requested. Therefore, we ask to revise the text of the manuscript and discuss in extenso that increasing the strain above the level used in the current study may well occur in vivo  and that in such situations the role of Wnt may be less important or even fully dispensable for cells to pass through S/G2 and mitosis. In this context, the Streichan et al. citation serves as an excellent reference for the potential impact of higher strain the requirement of Wnt. […]Essential revisions:1) Confirm b-catenin phosphorylation and degradation with quantitative Western blots,We were unable to perform biochemical studies with the integrated strain array (ISA) used in the current studies, and our previous study .2  but the surface area strained is less since the surface area of the pillar used to stretch the substrates is smaller, depending on the amount of strain applied . Thus the cell population includes a uniform area under strain, surrounded by a variable area, depending on the level of strain, outside the pillar that is not strained. The small surface area, and hence small number of cells, coupled with 2 populations of strained and non-strained cells precludes rigorous biochemical analysis of the entire cell population. To circumvent these limitations, the quantitative methods used to measure protein levels from fluorescence images only obtained data from areas of cells over the pillar and hence under strain, and the custom MATLAB scripts provided an unbiased analysis of ALL cells in those areas comprising ~500 – 15000 cells. Exact cell numbers for each experiment have been included in the Source Data files for Figures and uploaded to the eLife web-site.We used the ISA in this study so that we could directly compare results to our previous publication . The ISA is designed for high-throughput analysis of up to 5 different strain levels, each applied simultaneously to 5 wells; the surface area of silicone substrates in each PDMS well is 0.81 cmIn addition, the highest level of strain that our devices can apply (~10%) resulted in 20-30% of cells activating β-catenin signaling and re-entering the cell cycle. Therefore, even if the strained populations could be isolated and pooled, data from a western blot of the whole cell population would be compromised by the heterogeneity in cell responses to strain, whereas the immunofluorescence methods and quantitation with MATLAB scripts provided the single cell resolution and sensitivity required to quantify the phenotype.To address these concerns, we have included statements in Results and Materials and methods sections to clarify the technical limitations of the ISA and its incompatibility with biochemical analyses. For added text, see Results section, “The small surface area (0.81cm2) of the ISA does not provide sufficient cell numbers for biochemical characterization…”. We also added the text above in the Methods and Materials section “We used the ISA in this study so…..”.2) Confirm that DNA damage plays no role in G2/S arrest with quantitative Western blots for active p53 and ATM/ATR,For the reasons stated in response to point #1, we could not add quantitative western blots to our revision.However, as suggested by the reviewers, we added additional markers for DNA damage – p53 and p53 Binding Protein 1 (p53BP1). In MDCK cells, activation of the DNA Damage Response results in increased accumulation of both p53 and p53BP1. While both p53 and p53BP1 levels increased significantly in cells treated with the DNA damaging agent MMS, no significant increases in levels of either p53 or p53BP1 were observed in strained monolayers . These rFor added text, see Results section, “The monolayers were stained for the DNA damage associated histone variant phospho-γH2A.X  and the DNA repair proteins p53 and p53 binding protein 1  … “, and in the Discussion section, “…..level of DNA damage in those cells, measured by levels of p53, p53BP1 and phospho-γH2A.X, was insignificant compared….”.3) Confirm that indeed Src and not EGFR (PP2 inhibits both functions) is activated upon strain,The experiments presented in the text with PP2 inhibition were repeated with the more specific Src Inhibitor SU6656 , and theResults using the more specific Src inhibitor SU6656 were similar to those observed with PP2 treatment. SU6656 treatment blocked strain-induced increases in: 1) pY654 β-catenin; 2) β-catenin transcriptional activity; and 3) cell cycle re-entry (EdU incorporation). We replaced the PP2 data previously presented in EGFR inhibition with PD153035 treatment reduced by 30-40%, but did not completely block (unlike SU6656), the strain-induced increase in pY654 β-catenin. However, PD153035 treatment had no effect on strain-induced β-catenin transcriptional activity or cell cycle re-entry. See Results section, “To test whether EGFR activation was involved in strain-induced accumulation of pY654 β-catenin and β-catenin signaling, monolayers were treated with the EGFR inhibitor PD153035…”4) Investigate whether increased strain can bypass the need for high, local Wnt signalling. If it can, test whether G2/S arrest is induced by inhibiting Src or enhanced by inhibiting CKI,The ISA and live strain device used here are capable of applying a maximum of ~15% and ~10% strain, respectively, and these maximum strain levels were used in our studies. These levels of strain are recognized as within the physiological level in vivo. We note that ~ 100% uni-axial strain has been applied previously to MDCK monolayers, and cells strained at that level did proceed through G2 into mitosis .5) Better discuss the relevance of this in vitro crosstalk of Wnt and force for cell division in vivo.In the original submission, we were reluctant to speculate too much about the in vivo relevance of our work. Nevertheless, in response to the reviewer’s urging, we expanded the discussion to include more in vivo relevance, and the potential importance of our observations. “It has been a long-standing hypothesis that growth-induced local mechanical cues could influence morphogenesis by modulating growth rates, the direction of growth, or differentiation…”[…]Specifically, the reviewers were disappointed that you did not test whether higher strain allows cells to proceed into mitosis in the absence of Wnt signalling, and whether the G2/S arrest is induced by inhibiting Src or enhanced by inhibiting CKI under higher strains. We appreciate that it is technically not trivial to apply the higher strain requested. Therefore, we ask to revise the text of the manuscript and discuss in extenso that increasing the strain above the level used in the current study may well occur in vivo  and that in such situations the role of Wnt may be less important or even fully dispensable for cells to pass through S/G2 and mitosis. In this context, the Streichan et al. citation serves as an excellent reference for the potential impact of higher strain the requirement of Wnt.We are happy to address this in the revised text in 2 ways:1). The cell stretchers that we use can apply a maximum of 8% (live imaging) and 15% (ISA) strain. Therefore we cannot apply strains of >30% as requested by the reviewers. We have added the following text to state this technical limitation:Results section: “The live cell stretcher and ISA were able to apply maximum strains of 8.5% and 15%, respectively. The maximum level of static biaxial stretch was applied and held for up to 24 hours,….”.Materials and methods section): “The ISA applies a maximum of 15% strain to cell monolayers, and maximum strain was used in all experiments.”Materials and methods section): “The live cell stretcher applies a maximum of 8.5% strain to cell monolayers, and maximum strain was used in all experiments.”2). The reviewers asked us “to revise the text of the manuscript and discuss in extenso that increasing the strain above the level used in the current study may well occur in vivo  and that in such situations the role of Wnt may be less important or even fully dispensable for cells to pass through S/G2 and mitosis.” We have added the following text, with the suggested references, to discuss the possibility that higher levels of strain could induce mitosis in the absence of Wnt signaling:Discussion section: “Our results indicate that local Wnt signaling in a tissue experiencing moderate levels of global mechanical strain could result in local hotspots of mitosis through increased levels of Wnt/β-catenin signaling.Drosophila and zebrafish . Thus, in tissues experiencing high levels of strain (30-50% strain), cell division and tissue expansion may not require Wnt signaling. Indeed, in quiescent MDCK monolayers, 50% uni-axial stretch was sufficient to increase the fraction of cycling cells and number of cell divisions . Therefore, while high levels of Wnt signaling and mechanical force could independently drive proliferation and tissue expansion, growth-generated mechanical forces during development could also combine with local (Wnt) signaling to refine areas of growth during tissue morphogenesis and during adult tissue repair.”Levels of strain higher than those applied here have been observed during dorsal closure, germband extension, and neurulation in


CARF (Collaborator of ARF)/CDKN2AIP was discovered as a novel ARF-binding protein. It has been established as an essential cell survival, p53-, and cell proliferation-regulatory protein. Although a moderate upregulation of CARF caused growth arrest and senescence, its excessively enriched levels were shown to facilitate aggressive proliferation and malignant transformation of cancer cells. Here, we examined the relevance of CARF levels in clinical tumors and found its amplification (both at gene and transcript levels) in a variety of invasive and metastatic malignancies. Consistent with the clinical readouts, enrichment of CARF in cancer cells promoted epithelial–mesenchymal transition (EMT). Cancer database and molecular analyses revealed that it activates Wnt/β-catenin signaling axis, as evident by enhanced nuclear localization and function of β-catenin marked by increased level of SNAIL1, SNAIL2, ZEB1, and TWIST1 and its downstream gene targets. Of note, targeted knockdown of CARF led to decrease in nuclear β-catenin and its key downstream effectors, involved in EMT progression. Consistent with this, CARF targeting in vivo either by naked siRNA or CARF shRNA harboring adeno-oncolytic virus caused suppression of tumor progression and lung metastasis. Taken together, we report clinical and therapeutic relevance of CARF in EMT and cancer invasiveness/metastasis, and propose it as a potent therapeutic target of aggressive cancers. However, risk of metastases and recurrence of disease remained largely inevitable and unpredictable, and hence warrant continued efforts to characterize the associated underlying mechanisms. Transformed cancer cells progressively loose cell–cell adhesion/epithelial traits and acquire invasive mesenchymal characteristics via a systemic cellular reprograming, viz. epithelial-to-mesenchymal transition (EMT). EMT facilitates spread of cancer from the site of origin to other organs via acquiring motility, activation of extra-cellular matrix (ECM)-degrading proteases enabling invasion and eventual dissemination through vascularization3. Conferring reorganization of cytoskeleton and cellular polarity, it plays indispensable role in biological processes including embryonic development and wound healing5, and has been tightly associated with cancer metastases, drug resistance, and recurrence8.Systematic diagnosis, screening, and targeted therapeutic approaches have considerably improved clinical response and survival of cancer patients in last decade. Furthermore, molecular profiling of aggressive tumors has not only led to improved prognosis, but also helped to minimize the therapeutic side effects in several common malignancies9. Transcription factors, viz. Slug, Snail, and Twist have been demonstrated to regulate this process and are in turn influenced by diverse signaling pathways including TGF-β1, RTK (Receptor Tyrosine Kinase) receptors, i.e., EGFR, ERBB2/HER2-AKT, ITGB1/FAK, NOTCH, and Wnt/β-catenin proteins10. In particular, deregulated function of TGF-β1, a regulatory cytokine has been associated with tumor initiation and metastases11. It is frequently amplified in metastatic breast and pancreatic cancers12 and found to promote malignant and metastatic characteristics via activation of Snail and Zeb113. Similarly, the RTK receptors, EGFR and ERBB2  have been shown to promote EMT12. Focal adhesion kinase (FAK) and AKT, serine/threonine kinases are upregulated and shown to promote EMT in various malignancies12. Upregulation of Wnt in cancer cells has been shown to stabilize β-catenin and promote its translocation to the nucleus, where it serves as a coactivator of TCF-LEF transcription factors and regulate several EMT regulating proteins including repression of E-cadherin and activation of Snail, Zeb1, and Vimentin13.During the process of EMT, cells progressively loose set of proteins required for cell-adhesion/tight-junction  and gains others  that offer mesenchymal propertiesCollaborator of ARF), as a novel interactor of tumor suppressor protein p14ARF (ARF)14. It has been shown that CARF activates p53, a key tumor suppressor in a ARF-dependent and independent manners16 leading to execution of growth arrest and pre-mature senescence, as marked by p21WAF1 activation in human cancer and normal cells, respectively18. Excessively enriched or super-high expression level of CARF was shown to promote pro-proliferation and malignant properties of cancer cells in a feedback regulatory manner involving p53-HDM2 and DNA damage regulating proteins 18. Most recently, we found that CARF led to transcriptional repression of p21WAF1 and contributes to malignant transformation remarkably detected in p53-deficient cells19. In light of above information, in the present report, we investigated clinical relevance of CARF in tumorigenesis and its progression. An initial survey marked amplified CARF levels in invasive and metastatic malignancies in public cancer patient databases and in clinical tumor samples. We generated CARF-enriched cells and performed in vitro and in vivo assays, and found that CARF enrichment leads to EMT progression via Wnt/β-catenin signaling axis as evidently found by β-catenin nuclear translocation and activation of TCF4/β-catenin transcriptional targets. CARF knockdown, on the other hand, abolished these activities both in in vitro and in vivo models demonstrating functional relevance of CARF in EMT progression and its importance as a therapeutic target for metastatic malignancies.We have previously cloned CARF (the n = 10) and without (n = 97) CARF amplification. As shown in Fig. 22, showed the least change in CARF-amplified patients. These data strongly suggested that CARF amplification is associated with metastatic properties of the cancer cells.In order to examine the status of CARF across malignancies, we surveyed its genomic alterations  in clinical tumor patient database (TCGA). As shown in Fig. We next examined the expression level of CARF in clinical tumor sections using tissue microarray. Of note, a significant and gradual increase in CARF was detected from progression of infiltrating to metastatic breast carcinoma as compared to the basal level in normal matched tissues Fig. . CompariIn order to get molecular insights to the role of CARF in EMT and cancer metastasis, we recruited a panel of in vitro cultured cancer cells and examined their level of CARF expression. We found that invasive ovarian (SKOV-3), lung (H-1299), and fibrosarcoma (HT-1080) cell lines derived from secondary metastatic tumor site possessed high level of CARF expression Fig. . AlthougWe next generated CARF-enriched U2OS and SKOV-3 cells, representing low and high endogenous CARF levels, respectively. To distinguish it from endogenous CARF, the ectopic CARF was tagged with GFP Fig. . Cell ph24. We next examined their correlation with CARF in TCGA cancer database. In a co-occurrence-based analyses, taking amplification of these key modulators in their top 10 datasets, we found an association of β-catenin amplification with CARF , Wnt/β-catenin, and NOTCH1 have been established as the key regulators of EMTF Figure . As no cF Figure . To testF Figure . Of noteF Figure . FurtherF Figure . Gene amF Figure . BesidesF Figure . These dTo investigate the above correlation in vitro, we examined β-catenin expression in U2OS/CARF-GFP cells and found its remarkable enrichment in the nucleus Fig. , suggest25 and Pyrvinium pamoate (PP), an allosteric activator of Casein Kinase 1α (CK1α)26 that stimulates ubiquitination and degradation of β-catenin by its phosphorylation27. As shown in Fig. Consistent to the increase in β-catenin transcript, and its nuclear translocation Fig. , we foun28. We opted to use sub-toxic concentrations (20–50 pM) of CARF siRNA for 48 h in in vitro assay. CARF-compromised SKOV-3 cells showed decrease in migration and invasion properties   mediated suppression of CARF in SKOV-3 cells (higher level of endogenous CARF expression) resulted in reduced survival, a housekeeping CARF function Fig.  as also es Figs. . Further Figures  and S8b. Figures , while E Figures . Additio Figures , N-cadhe Figures  expressiWe next determined the role of Wnt/β-catenin in CARF-induced EMT. As shown in Fig. To examine the physiological relevance of CARF-promoted EMT and its abrogation upon its suppression, we performed in vivo xenograft assay. Intravenously and subcutaneously injected nude mice with SKOV-3 cells (control groups), were treated with control and CARF siRNAs, as shown in the treatment regime Fig. . On day 7 p/s) was significantly attenuated in comparison to other groups demonstrating 186.2-, or 82.2-fold greater therapeutic efficacy than PBS (4.5 × 109 p/s), or RdB (2.0 × 109 p/s), respectively  levels in cancer patient datasets/tumor samples suggested clinical relevance of CARF upregulation with cancer metastasis  and elevated epithelial (E-cadherin) properties in these cells supported that CARF could be a potential therapeutic target in reversing the EMT process. Of note, whereas CARF enrichment caused nuclear translocation of β-catenin, its abolition was marked in CARF-compromised cells demonstrating that CARF has essential role in activation of β-catenin signaling involved in EMT. The data was endorsed by downregulation of several β-catenin-regulated proteins including, survivin, c-Myc, Cyclin D1, Wnt1, and SMAD2/3. Furthermore, β-catenin interacting proteins required for active transcriptional complex  and effectors  showed increase in CARF-enriched and decrease in CARF-compromised cells.29, using an oncogenic Ras model of hepatocellular carcinoma (HCC), reported that CARF promotes β-catenin/TCF activation by inhibition of β-catenin and ICAT interaction. He et al.30 reported a small-molecule inhibitor of CARF. Using in vitro and embryonic developmental model of zebra fish, they showed that suppression of CARF led to impaired embryonic development, hematopoietic and caudal fin regeneration. The study showed a direct CARF–Dvl interaction that facilitated TCF/β-catenin transcriptional activity30. In continuation with our earlier studies that demonstrated that CARF is an essential and dual regulator of cell proliferation19. We provide clinical relevance of CARF upregulation in malignant progression of cancer and mechanism of its role in EMT, migration and invasion  amplification is clinically relevant to cancer invasiveness and metastasis, (ii) facilitates EMT and metastatic progression via activation of Wnt/β-catenin signaling and (iii) is a strong therapeutic target for malignant and aggressive cancers.WAF1, and p53 ; β-catenin, Wnt1, E-cadherin, Survivin, Vimentin, Fibronectin, Kinesin, VEGF, MMP2, MMP9, Cyclin D1, hnRNP-K, TGF- β1, and rabbit-/mouse-HRP (Santa Cruz); and mouse/rabbit Alexa Fluor 488- and/or 568-conjugated secondary antibodies and Hoechst 33342 (Invitrogen) additional details provided in Supplementary Information (Table 15. CARF siRNA (Silencer® Select & In vivo Ready Oligos) were purchased from Thermo Fisher. Wnt and β-catenin inhibitors, i.e. IWP2 (Santa Cruz), Pyrvinium pamoate (PP) and iCRT14 (Sigma-Aldrich) were purchased. Tissue microarray slides with embedded clinical tumor  were procured from SuperBioChip  Laboratories.Antibodies purchased from various sources as follows: β-actin-HRP, c-Myc and uPA, (Abcam); N-cadherin, SMAD2/3, p21on Table . Anti-CAhttp://www.cbioportal.org/index.do), Oncomine (https://www.oncomine.org/), and Human Protein Atlas  respectively; while pathway genes-lists were obtained from KEGG (http://www.genome.jp). Detailed procedures of above analyses are provided in supplementary methods.Genomic amplification, transcript and protein expression analysis of CARF in clinical datasets was performed using TCGA  in liquid Nitrogen (LN2) and were regularly revived from the original stocks to avoid genetically instable cells that may arise due to prolong culture. Cells were treated with inhibitors, IWP2 (0.5 μM for U2OS and 5 μM for SKOV-3), PP (2.5 nM for U2OS and 50 nM for SKOV-3), and iCRT14 (1.5 μM for U2OS and 10 nM SKOV-3) for 24 h.Human cancer ); osteosarcoma U2OS, SaOS-2 (JCRB Cell Bank); breast carcinoma—MCF-7, MDA-MB-231 and T47D ; melanoma—G361 and fibrosarcoma-HT-1080 ; hepatocarcinoma—HUH-6, HUH-7, colon carcinoma—DLD-1 and lung carcinoma—H-1299, A549 (JCRB Cell Bank) cell lines were grown in DMEM, while human neuroblastoma  cells cultured in MEM medium. Cultures were maintained as described previouslyNeo, empty vector) and CARF (full length CARF-tagged with GFP)-enriched , viz super-higher expression of CARF) cells were generated and maintained in medium supplemented with G418 (100 μg/mL) as described earlier18. For CARF knockdown, CARF Silencer® Select siRNA was transfected with Lipofectamine® RNAiMAX (Thermo) using standard procedure as previously described28. Optimum siRNA concentration  was determined by viability  assay and the nontoxic doses  were used for transient transfections in U2OS, SKOV-3, MBA-MB-231, and HT-1080 cells.Control  as described earlier32 using CARF-specific primer sets  was performed using Image J software  and represented as percentage of stained area.CARF expression in tissue microarray was examined as described earlier18. Quantitation of signals (performed with the Image J) of three independent blots  was represented as percentage expression.Protein extraction and immunoblotting with specific antibodies (as indicated) was performed as described previously18 using Zeiss Axioplan 2 microscope (with a Zeiss AxioCam HRc camera) and confocal laser scanning microscope . To resolve the subcellular localization of protein, the images were analyzed with the Imaris (Bitplane) software.Immunofluorescence staining was performed as described earlier4 cells in upper chamber of Matrigel (BD BioSciences) coated surface in a 12-well plate. The procedure, staining and image acquisition was performed as earlier described19. Wound-healing assay with different  cells for 48 and 72 h was performed as described earlier18. Phase contrast images (×10 magnification) at different time-points were captured and quantitated for scratched area.Invasion assay was performed by seeding 5 × 106 control  and CARF-enriched  SKOV-3 cells. CARF-targeting adeno-oncolytic virus (RdB-shCARF) was generated19 and employed with lung metastasis model  as described earlier33. Detailed regimes of treatment, doses and tumor growth monitoring are provided in supplementary methods. All observations were performed, updated and duly discussed to avoid blinding on allocation/accessing the experimental outcome. This study was carried out in strict accordance with the recommendations in the Animal Experiment Committee, Safety and Environment Management Division, National Institute of Advanced Industrial Science & Technology (AIST), Japan .Five- to six-weeks-old female nude mice were injected subcutaneously and intravenously with 2.5 × 10t-test or Mann–Whitney U-test (non-parametric), whichever applied, was enrolled to calculate the degree of significance among the control and test group. The statistical significance was expressed as p-value ≤ 0.05; and represented by *<0.05, **<0.01, ***<0.001, while NS marked the insignificant correlation34.All quantitative experiments were performed in triplicate. Data values are represented as mean ± SEM of three individual replicates. The two-tailed Student’s Suppl. Materials


Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules , modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information.Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although  The dysfunction of proteins involved in intercellular adhesion thus causes a variety of pathologies6. Cadherin-based adherens junctions (AJs) connect the actin cytoskeleton between cells and dynamically change the strength of adhesion in response to intercellular tension12. During AJ maturation, α-catenin and vinculin cooperate to remodel the actin cytoskeleton15. α-Catenin participates in the cadherin-catenin complex (CCC), whereas cadherin, β-catenin, and α-catenin serially associate together18. The CCC interacts with actin filaments via α-catenin in the cytoplasmic region of AJs to transmit intercellular tension generated by the actomyosin cytoskeleton20. Under intercellular tension, α-catenin recruits vinculin13, which is another key protein in the architecture of AJs. The C-terminus of vinculin interacts with monomeric and filamentous actin to mediate the link between α-catenin and another actin filament21. α-Catenin and vinculin thereby induce local remodeling of the actin cytoskeleton at AJs to increase the strength of adhesion22, which stably transmits intercellular tension.Adhesive interaction between neighboring cells contributes to the mechanical maintenance of morphogenetic changes in embryos as well as homeostasis in mature tissues24; the vinculin binding site  is embedded in the M1 domain (residues 273–393) of α-catenin, which is further stabilized by the M2-M3 domain (residues 394–634)25. Thus, under tension, it has been suggested that the autoinhibitory interaction between the M1 domain and the M2-M3 domain is disrupted, and the destabilized M1 domain exposes the VBS to recruit vinculin13. Our previous report26 has revealed that, using atomic force microscopy (AFM)-based single-molecule force spectroscopy, the mechanical disruption of the autoinhibitory M1/M2-M3 interaction induces a drastic change in the conformation of α-catenin, together with increased mechanical stability. In addition, another single-molecule study showed that the affinity of α-catenin for the head domain of vinculin increases under tension27. However, although the α-catenin-mediated mechanotransduction is a dynamic molecular process, there is no experimental technique at the molecular level that allows us to observe vinculin recruitment to α-catenin under tension in real time.α-Catenin under tension-free conditions is known to form an autoinhibited conformation against vinculin28 and structural imaging29, have been combined with TIRF-based molecular observation to investigate biomechanical phenomena at the molecular level. Using the new system, we extended α-catenin molecules  using AFM and simultaneously observed their recruitment of full-length vinculin molecules in real time using TIRF. We used full-length vinculin in the experiment to minimize tension-free binding of vinculin to α-catenin, as it forms an autoinhibited conformation against α-catenin32. A previous study based on isothermal titration calorimetry (ITC) reported no detectable interaction between the α-catenin M1-M2 domain, which is an open form fragment without the autoinhibitory M3 domain, and full-length vinculin33. In our study, we hypothesized that the mechanical force drastically destabilizes the M1 domain of α-catenin to allow its interaction with full-length vinculin.Therefore, in the present study, we developed a system combining AFM and total internal reflection fluorescence (TIRF) to directly observe the dynamic recruitment of vinculin to α-catenin. AFM-based techniques, such as DNA cutting/pasting1-M3 domain), modified on coverslips, using AFM and simultaneously observed their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, using TIRF  for the classification was determined by comparison of the results of the control extension experiment by AFM without α-catenin modification onto coverslips  as shown in Fig. ion Fig.  was caus1-M3 domains of α-catenin. The mutated fragment, possibly with an attenuated intra-domain interaction between the M1 and M2-M3 domains, was reported to recruit full-length vinculin in a tension-insensitive manner26. We validated that the labeling of full-length vinculin by Alexa-Fluor 488 dye did not affect its affinity for α-catenin. As a result of the AFM structural imaging, we obtained three-dimensional features of the α-catenin fragments, as shown in Fig. 24. By contrast, the mutated α-catenin  Mnin Fig.  had a di2-M3 domain (residues 385–634), without the M1 domain. This result indicated that vinculin interacted with the M1 domain of α-catenin but did not interact with the M2-M3 domain or any linker components. Thus, using this novel AFM-TIRF combined system, we could directly observe the recruitment of full-length vinculin to α-catenin under tension at the molecular level.The dynamics of full-length vinculin molecules were observed by TIRF simultaneously with measurement of the extension of α-catenin by AFM. In movies obtained in conjunction with successful α-catenin extension, we observed a square pulse in the intensity Fig.  with a pFu, in the retract phase , suggesting that vinculin dissociated from α-catenin under this small amount of extension. Although the isolated head domain of vinculin is reported to dissociate only when the α-catenin M1-M3 domain is almost fully extended27, our result suggested that full-length vinculin dissociates more readily from α-catenin VBS than does the vinculin head domain. We suggested that this difference was caused by the autoinhibitory interaction in the full-length vinculin; the tail domain of vinculin competed with α-catenin VBS to interact with the head domain of vinculin under our conditions, which made the vinculin head domain easier to dissociate from α-catenin VBS under high tension. Our results also suggested that full-length vinculin dissociates easily from α-catenin when the vinculin affinity for α-catenin decreases under attenuated tension, with recovery of the autoinhibited conformation40.From our results, vinculin dissociation events were observed even in cases where the α-catenin was not fully unfolded in the retract phase, i.e. the α-catenin extensions at the rupture events were shorter than its fully extended length of approximately 143 nm. This result suggests that vinculin dissociated from α-catenin at the beginning of the retract phase (from 3.68 s to 4.20 s). This result is consistent with our previous AFM measurement27.The actual extension of α-catenin should also be discussed. We analyzed the tip-coverslip distance by moving the average and deviation in the tip-coverslip distance, which differed by approximately 15 nm in the force curves with and without successful α-catenin extension  and M2-M3 (residues 385–634) were amplified by PCR and cloned into the pGEX6P-3 vector . A DNA fragment of mouse full-length vinculin (residues 1–1066) was cloned into the pET-6b (+) vector (Novagen). These plasmids were verified by DNA sequencing and transformed into Escherichia coli strain BL21Star (DE3) cells (Invitrogen). α-Catenin and vinculin molecules were expressed at 20 °C in Luria-Bertani medium supplemented with 0.1 mM isopropyl-β-d-thiogalactopyranoside. BL21Star cells expressing α-catenin and vinculin were suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl and disrupted by sonication. The supernatant after ultracentrifugation was applied to a Glutathione Sepharose 4B column . Proteins eluted from the column were further purified by anion exchange  and gel filtration  chromatography. N-terminal His6 tags on vinculin molecules were then cleaved using human rhinovirus 3C protease. For AFM structural imaging, GST-tags at N-termini of the wild-type and mutated fragments were cleaved by PreScission Protease .α-Catenin and vinculin were expressed and purified as reported in our previous study26.Coverslips (Matsunami Glass Ind. Ltd.) and silicon nitride AFM tips  were chemically modified for use in AFM  as described in a previous study3-NTA  in 20 mM Tris-HCl buffer for 30 min. The NTA-modified coverslips were then treated with 10 mM NiCl2 /MilliQ water for 30 min and further washed with MilliQ water and 20 mM Tris-HCl buffer. Using NTA-Ni2+-His6 affinity binding, 500 μM α-catenin fragments in 20 mM Tris-HCl buffer were immobilized on the coverslips. The coverslips were finally washed with 20 mM Tris-HCl buffer.To make a chamber to contain the working buffer with vinculin, we placed flat silicon rings on coverslips. The coverslips were cleaned in a plasma cleaner, sonicated in 1 M KOH for 15 min, and then sonicated in ethanol for 15 min. After cleaning, the coverslips were thoroughly washed in MilliQ-treated water . For modification with α-catenin molecules, the coverslips were treated with 2% MPTMS/ethanol for 15 min and then treated with 2 mM maleimide-CThe AFM tips were first cleaned in a plasma cleaner and then treated with 2% APTES/MilliQ for 15 min. The tips were then treated with 6 mM Mal-PEG-NHS ester/20 mM Tris-HCl buffer for 30 min and with 10 mM glutathione/20 mM Tris-HCl buffer for 1 h. The remaining maleimide groups were quenched with 50 mM 2-mercaptoethanol/MilliQ water and washed with 20 mM Tris-HCl buffer.Full-length vinculin molecules were labeled using the Alexa Fluor 488 Protein Labeling Kit , in which the primary amines in vinculin formed covalent bonds with the Alexa 488 dye. Full-length vinculin molecules and the Alexa 488 dye were conjugated at a 1:1 ratio at room temperature. Alexa-488-labeled vinculin molecules were purified using size exclusion chromatography. For each measurement using TIRF-combined AFM, Alexa 488-labeled vinculin molecules were dissolved at a concentration of 10 nM in working buffer . DTT was added to the working buffer to prevent aggregation of vinculin molecules caused by disulfide bonds, and Trolox was used to sustain the fluorescence of the Alexa 488 dye by maintaining reducing conditions in the working buffer.1-M3 fragment (approximately 143 nm). After focusing on the surface of the coverslip, the AFM head unit was brought back to the stage and the AFM tip was directed towards the coverslip surface. The power of the laser was first set at maximum for 30 min to diminish the fluorescence of any vinculin that was nonspecifically attached to the surface, and then the power was decreased to 2.97 W/cm2 using an ND filter to allow for AFM-TIRF measurement under conditions that avoided photo-bleaching. During the AFM force measurement, the fluorescence of vinculin was observed using an EM-CCD camera ; the exposure time was 50 ms and the final frame rate was approximately 14.76 frames/s. To synchronize the force measurement with the vinculin observation, we used the pulse signal output from the AFM controller that then externally controlled the EM-CCD camera. Finally, we analyzed the intensity of fluorescence in the square region (15 × 15 pixels) around the position of the tip.For the AFM-TIRF combined system, we first calibrated the actual force measured by AFM. We adjusted the laser position to the top of cantilever in the working buffer using a non-oil 20x magnification lens and approached the bare coverslip to evaluate the relationship between the change in voltage from the photodiode and the deflection of the cantilever. The deflection was then calibrated to force based on the spring constant of the AFM cantilever. After calibration, we moved the base of the cantilever upward by 1000 nm and then removed the AFM head unit. The objective lens was then switched to a 100x objective lens for TIRF  and the α-catenin-modified coverslip was set on the stage and the space inside the silicon ring was filled with the working buffer containing vinculin. We set up the TIRF condition by controlling the optional axis of the 488-nm laser , so that the penetration depth of the evanescent field was 150 nm, which covered the length of a fully extended αE-catenin MFor the AFM structural imaging, a fresh mica surface was obtained by peeling off the surface layers using tape. Wild-type and mutant α-catenin fragments (1 nM of each) in the working buffer (50 μL) were dropped onto the mica surface and allowed to absorb for 1 h. The solution was then washed using working buffer, and finally fresh working buffer (50 μL) was dropped onto the surface. Silicon AFM tips specialized for structural imaging  were used to obtain the images.Supplementary informationSupplementary Video S1Supplementary Video S2