In vitro, Bisleuconothine A inhibited cell proliferation through induction of apoptosis by increasing the cleavage of caspases in HCT116 and SW480 colorectal cancer cells. Moreover, in vivo, Bisleuconothine A dramatically suppressed tumor growth in HCT116 Xenograft. And further analysis showed that Bisleuconothine A suppressed the Wnt target gene expression in HCT116 Xenograft, which was associated with up-regulation of β-catenin phosphorylation and subsequent Wnt signaling inhibition. Taken together, our study indicated that bisindole alkaloids could be included as a new chemotype of small-molecule Wnt signaling inhibitors, and have great potential to be further developed for anti-tumor agents.Wnt signaling pathway is aberrantly activated in a variety of cancers, especially in colorectal cancer and small molecule antagonists of Wnt/β-catenin signaling are attractive candidates for developing effective therapeutics. In the present study, we identified Bisleuconothine A, a bisindole alkaloid with an eburnane-aspidosperma type skeleton, as a novel and selective Wnt signaling inhibitor by using a cell-based luciferase assay system. Our study found that Bisleuconothine A down-regulated the endogenous Wnt target gene expression through promoting phosphorylation of β-catenin and the subsequent inhibition of its nuclear translocation in HCT116 and SW480 colorectal cancer cells.  Wnt/β-catenin signaling is involved in a multitude of developmental processes and the maintenance of adult tissue homeostasis, as well as maintaining adult stem cells in a pluripotent state . Wnts arAPC and 10% with mutation of CTNNB1, the gene encoding the protein of β-catenin [Mutations or deregulated expression of components of the Wnt pathway can induce diseases, most importantly cancer . Over 94-catenin , such as-catenin , survivi-catenin , Cyclin The broad involvement of Wnt signaling in disease has driven numerous studies to identify novel small molecules inhibitors that target different regulation levels of Wnt signaling. Despite of a couple of inhibitors reported with great potential, the clinical application of therapies to specifically target the Wnt pathway in cancer cells is still in infancy , 10.Melodinus [Leuconotis griffithii, exhibited cell growth inhibitory activity against various human cancer cell lines [Bisindole alkaloids have received considerable attention for their potential biological activity, including anti-tumor , 12, antelodinus –21, in welodinus . In the ll lines . We show50 value of 2.55 ± 0.34 μM. To further confirm the inhibitory effect of Bisleuconothine A on the Wnt/β-catenin signaling, HEK293T cells were transiently transfected with the Wnt/β-catenin signaling reporter ST-Luc, Wnt1 and Renilla, and then treated with Bisleuconothine A for 24 h. As shown in Figure 50 of 4.94 ± 0.10 μM  . The scrM Figure . To inveM Figure , indicatBisleuconothine A is bisindole condensed by (+)-ebumamenine and a tabersonine derivative, and the cytotoxic activity against human cancer cell lines of their analogs have been investigated by our group previously , 17–21. As a well-known anticancer chemotherapeutics, vinorelbine is also a bisindole alkaloid with similar structure with Bisleuconothine A. So we compared the Wnt signaling inhibitory activities of vinorelbine with Bisleuconothine A, and the dual-luciferase reporter assay in HEK293W cells showed that vinorelbine did not exhibit significant Wnt signaling inhibitory activity, indicating a novel anti-tumor mechanism of bisindole alkaloids.50 value was 4.47 ± 0.16 μM and 4.94 ± 0.09 μM in HCT116 and SW480 cells respectively. We next investigated the effect of Bisleuconothine A on the expression of endogenous Wnt target genes Cyclin D1, c-myc, Axin 2 and survivin using Western blot analysis. As shown in Figure To test whether Bisleuconothine A attenuates Wnt/β-catenin signaling in Wnt-dependent cancer cells, HCT116 and SW480 cells were transiently transfected with the Wnt/β-catenin signaling reporter ST-Luc and Renilla. The elevated ST-Luc activity in HCT116 and SW480 cells, due to Ser45 deletion mutation in β-catenin  and APC In the Wnt/β-catenin signaling pathway, β-catenin response transcription is largely dependent on the level of β-catenin, which is regulated by the destruction complex . Thus, tInduction of phosphorylation of β-catenin led to the inhibition of the nuclear translocation of β-catenin, thus, the distribution of β-catenin was analyzed by immunofluorescence assays. As shown in Figure LiCl, an inhibitor of GSK-3β, could down-regulate the level of phosphorylation of β-catenin and subsequently induce nuclear translocation of β-catenin . As show50 values were 2.74 ± 0.22, 3.18 ± 0.08, 1.09 ± 0.06 and 3.05 ± 0.17 μM, which were comparable to those shown to attenuate the activity of Wnt signaling.Given that Wnt signaling is closely associated with cell proliferation and Bisleuconothine A can suppress the Wnt signaling in colorectal cancer cells, we then explored the effect of Bisleuconothine A on cancer cell proliferation with MTS assay. As shown in Figure Cyclin D1 is an important regulator of the cell cycle that is overexpressed in colorectal cancers as a consequence of activated TCF/β-catenin signaling . Our stuin vivo. To increase the water solubility of Bisleuconothine A, the sulfate of Bisleuconothine A    was prep, BLA-S) . As showin vivo test, 7 days after implantation, the BALB/c mice carrying established HCT116 Xenograft were randomly assigned into control and experimental group. The mice were treated daily with 2 mg/kg of Bisleuconothine A sulfate by intraperitoneal injection and injection of normal saline as controls. As shown in Figure p<0.05) compared with controls  and HEK293T were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences . The cells were grown in medium , supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin G sodium and 100 μg/ml streptomycin (HyClone). HEK293W cells  , 8 ng of Renilla reporter plasmid (Promega) and 64 ng mouse Wnt1. In HCT116 and SW480 cells, the cells were transiently transfected with ST-Luc and Renilla. After 3 h incubation, cells were treated with various concentrations of Bisleuconothine A for 24 h and then the cells were lysed. Both luciferase and Renilla activities were measured using the Dual-Luciferase Reporter Assay kit . The luciferase activity was normalized to the Renilla activity.Cells were harvested and lysed in SDS sample buffer . Lysates were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween-20 and incubated at 4°C overnight with the following antibodies: anti-Axin 2, anti-Cyclin D1, anti-c-Myc, anti-Survivin, anti-caspase 3, anti-cleaved caspase 3, anti-caspase 8, anti-PARP-1 , anti-Lamin A/C (Epitomics), anti-β-catenin (BD Biosciences), anti-phopho-β-catenin , and against β-actin , followed by the corresponding horseradish peroxidase-conjugated secondary antibodies. Proteins of interest were visualized and imaged under chemi-luminescent detection using LASmini 4000 .Cells were grown for 12 h before treatment. Drug-treated cells were fixed in 4% paraformaldehyde for 20 min. After being washed with PBS, the slides were treated by 0.1% Triton-X and blocked with 3% BSA in PBS. Then the slides were incubated with anti-β-catenin antibody (BD Biosciences) overnight, washed with PBS, and incubated with corresponding FITC conjugated secondary antibody (Sigma-Aldrich) for 1 h. The cells were then incubated with DAPI  for 10 min and observed under microscopy .2, 0.5 mM DTT, pH 7.9, EDTA-free protease inhibitor cocktail ) and incubated on ice for 10 min. After microcentrifuged, the cells were lysed in buffer A with 0.2% Nonidet P-40 and for 6 minutes on ice. After being microcentrifuged for 5 min at 500 g, the supernatants were collected as cytoplasmic extracts. Pellets were washed with lysis buffer A without NP-40 and resuspended in lysis buffer B , and incubated for 30 min on ice. After being centrifuged at 12,000 g for 10 min, supernatants were collected.Collected cells were re-suspended in lysis buffer A  at the 3′-end of sequence were synthesized at GenePharma . The target sequences were as follows :CTNNB1: 5′-AGCUGAUAUUGAUGGACAG-3′.Scrambled control: 5′-UUCUCCGAACGUGUCACGU-3′.SiRNAs were transfected into SW480 cells using Lipofectamine 2000.3 cells were plated in 96-well plates 12 h before treatment and continuously exposed to test compounds for 48 h. Then MTS (Promega) was added to each well. The samples were incubated at 37°C for 1∼4 h and the optical density (OD) was measured at 490 nm using a microplate reader (Bio-Rad Laboratories). The IC50 values are calculated from appropriate dose-response curves.Cytotoxicity of compounds was determined by MTS method. Briefly, 5×105 cells) were incubated with test compounds for 24 h respectively. Ccells were collected and washed twice with PBS. Cells were fixed with 70% ethanol overnight. Fixed cells were washed with PBS, and then stained with 50 μg/ml propidium iodide  solution containing 50 μg/ml RNase A (Sigma-Aldrich) for 30 min in dark at room temperature. Fluorescence intensity was analyzed by FACSCalibur flow cytometer . The percentages of the distributions in distinct phases of cell cycle were determined using FlowJo V 7.6.1 software.HCT116 and SW480 cells . After incubation for 15 min at room temperature in the dark, the fluorescent intensity was measured using a FACSCalibur flow cytometer .Cell apoptosis was analyzed using the Annexin V-FITC/PI Apoptosis kit  according to the manufacturer's protocols. Cells were seeded in 6-well plates at a density of 2×106 HCT116 cells were injected subcutaneously to the right flank of the BALB/c-nude mice. When the tumors reached a volume of ∼130 mm3, 7 days after implantation, the mice were randomly assigned into two groups (eight mice in each group) and treatments were given daily as follows: (a) intraperitoneal injection of 2 mg/kg Bisleuconothine A sulfate, or (b) intraperitoneal injection of normal saline. Tumors were measured in three dimensions using calipers, and tumor volume (in mm3) was calculated using the following formula: tumor volume (mm3) = (L× W2)/2, where L= length in mm, and W= width in mm. Body weight was recorded regularly during the experiment. After treatments, the mice were sacrificed and the tumors were isolated. Then the tumors were weighed and frozen at −80°C for subsequent western blotting assay.All animal experiments were carried out under an Institutional Animal Care and Use Committee-approved protocol and institutional guidelines for the proper and humaneuse of animals. Three-week-old female BALB/c-nude Mice were purchased from Vital River Laboratory Animal Technology Co. Ltd, and were kept in a pathogen-free environment. For the HCT116 xenograft, 5×10


VM-positive colon cancer samples showed increased Wnt3a expression (p < 0.001) and β-catenin nuclear expression (p < 0.001) compared with the VM-negative samples. In vitro, over-regulated Wnt3a expression in HT29 colon cancer cells promoted the capacity to form tube-like structures in the three-dimensional (3-D) culture together with increased expression of endothelial phenotype-associated proteins such as VEGFR2 and VE-cadherin. The mouse xenograft model showed that Wnt3a-overexpressing cells grew into larger tumor masses and formed more VM than the control cells. In addition, the Wnt/β-catenin signaling antagonist Dickkopf-1(Dkk1) can reverse the capacity to form tube-like structures and can decrease the expressions of VEGFR2 and VE-cadherin in Wnt3a-overexpressing cells. Taken together, our results suggest that Wnt/β-catenin signaling is involved in VM formation in colon cancer and might contribute to the development of more accurate treatment modalities aimed at VM.Our previous study provided evidence that non-canonical Wnt signaling is involved in regulating vasculogenic mimicry (VM) formation. However, the functions of canonical Wnt signaling in VM formation have not yet been explored. In this study, we found the presence of VM was related to colon cancer histological differentiation ( Angiogenesis has a vital function in tumor development because tumors are supplied with oxygen and nutrients and tumor cells are provided with an entry route into circulation. For years, much attention has been focused on the function of sprouting angiogenesis or the recruitment of endothelial cells into tumors from surrounding, pre-existing blood vessels. However, over the last few years, several other tumor microcirculation patterns have been identified, including vessel co-option, recruitment of endothelial precursor cells, intussusception, and vasculogenic mimicry (VM) ,2,3. VM et al. , who fouet al. ,6,7. In et al. ,11,12,13et al. ,15. Theri.e., some Wnt ligands and Frizzled) are overexpressed in colon cancer and are involved in tumor progression [2+-calmodulin kinase or the planar cell polarity pathway; this activation is called non-canonical Wnt signaling. Our previous reports have shown that overexpression of Wnt5a, a representative non-canonical Wnt ligand, may mediate VM formation in ovarian cancer and non-small cell lung cancer [Wnt signaling is involved in a wide range of physiological processes, including embryonic development, cell proliferation, and homeostasis. Activating the mutations of the Wnt-signaling pathway is crucial in tumorigenesis, especially in colorectal cancer. Wnt signaling also has an important function in vascular development and angiogenesis. Previous reports have shown that Wnt signaling contributes to endothelial cell differentiation and vascular remodeling . Both thgression ,20. Addigression ,22. All g cancer ,24. By cin vitro as well as on the VM forming ability in animal xenograft model were studied. In addition, we treated Wnt3a-overexpressing cells with Dkk1, a Wnt/β-catenin pathway antagonist, and determined the tube-structure forming ability and expression of VM-related proteins to further verify the VM- promoting effect of the Wnt/β-catenin signaling pathway.Wnt3a is a representative Wnt protein that signals via Wnt/β-catenin signaling. Previous studies, including our own, have reported that by activating Wnt/β-catenin signaling, Wnt3a can promote epithelial-mesenchymal transition and enhance stemness of cancer cells , both ofUsing H&E staining A,B and Cp < 0.001), TNM stages (p < 0.001), and metastasis/recurrence (p < 0.001). The frequency of VM was significantly higher in poorly differentiated colon cancer  than in well  and moderately  differentiated ones. VM was observed in 33 of 69 patients (47.8%) with advanced stage carcinomas (TNM stages III and IV), in 16 of 148 patients (10.8%) with early-stage carcinomas (TNM stages I and II). A total of 77 (35.5%) colon cancer patients experienced metastasis or recurrence. The patients with VM had a higher rate of metastasis or recurrence  than those ones without VM . No significant correlations were found between VM and patient age or gender, tumor location or size.VM was detected in 39 (19.2%) out of 217 colon cancer cases. The clinical and pathological features of VM in all 217 colon cancer cases are summarized in vs. 38.7%, p < 0.05). Meanwhile, the nuclear expression of β-catenin was more frequently found in the VM group than in the non-VM group . The results indicate that VM is associated with Wnt/β-catenin signaling activation in colon cancer.To assess the relationship between VM and Wnt/β-catenin signaling in colon cancer, we determined the expression of Wnt/β-catenin signaling-associated markers, including Wnt3a and β-catenin. The former is a representative canonical Wnt signaling ligand, and the latter is the key regulator of canonical Wnt signaling. As shown in in vitro at 3-D culture conditions [Our previous study showed that HT29 cells, a better differentiated and less invasive cell line, cannot form vascular networks nditions . Thus, wThree-dimensional culture is used to effectively test not only the vascular behavior of endothelial cells but also the ability of a number of tumor cells to form VM structures. We showed that HT29 cells cannot form tubular structures, whereas Wnt3a-overexpression HT29 cells (clone9 and clone20) formed few tubular structures, thereby indicating that Wnt3a may be an activator for VM formation in HT29 cells A.Moreover, compared with the controls, the Wnt3a-overexpression cells showed increased expression of VE-cadherin and VEGFR2, which are the most representative VM-associated endothelial phenotype proteins. However, we did not observe a significant change in the VEGFR1 expression in the same cells B.p < 0.05)  exhibited a VM structure, whereas all those from control cells showed no VM structures (Wnt3a overexpressing cells grew into larger tumor masses compared with the control cells ( < 0.05) C. The VMructures D, therebDkk1 functions as an antagonist of the Wnt/β-catenin pathway by binding to lipoprotein receptor-related protein 5 or 6 (LRP5/6) and preventing the formation of Wnt-Fz-LRP ternary complexes and downstream signaling transduction. After Dkk1 treatment, Wnt3a-overexpressing cells failed to form vessel-like tube formation A. FurtheVM was first described in melanoma in 1999 , and thiin vitro and increased VM incidence in mouse-transplanted tumors. All these results indicated the VM-inducing effect of Wnt/β-catenin pathway in colon cancer.A growing body of evidence has revealed the importance of Wnt/β-catenin signaling pathways in endothelial cell differentiation and vascular remodeling. During embryonic development, gain-of-function mutation of β-catenin in endothelial leads to vascular remodeling . The actVM repeats the pattern of embryonic vasculogenic networks. Molecular profiling of cells capable of VM showed that a number of highly upregulated genes are involved in angiogenesis and vasculogenesis . When VEin vitro and induced decreased expressions of VEGFR2 and VE-cadherin, thereby indicating that the VM-inducing effect caused by Wnt3a was due to the stimulative effect of Wnt3a on the Wnt/β-catenin pathway.Wnt3a not only activates Wnt/β-catenin signaling but also functions simultaneously with signaling pathways such as FAK or TGFβ ,35. Dkk1In conclusion, this study is the first to report on a previously unrecognized function of Wnt/β-catenin signaling as an important regulatory pathway for VM formation. The results might have a number of implications for clinically useful therapy targets aimed at VM in the future.In the current study, 217 formalin-fixed, paraffin-embedded human colon cancer tissue samples were from Tianjin Medical University Cancer Institute and Hospital from July 2002 to June 2004. All the patients did not receive chemotherapy or radiotherapy before operation. Data of clinical parameters were obtained from patients’ clinical records. The pathological diagnosis of all cases were independently verified by two senior pathologists by observing the H&E sections conserved in Pathology Department of Tianjin Medical University Cancer Institute and Hospital.2. Antibodies for Dkk1, CD34, VEGFR1, VEGFR2 and β-actin were from Santa Cruz . Antibodies for Wnt3a, VE-cadherin and β-catenin were from Abcam . The secondary HRP-conjugated antibodies were from Zhongshan Chemical Co. . Goat anti-mouse IgG-FITC were from Santa Cruz. The eukaryotic expression vector GV230 containing whole coding sequence of Wnt3a was original obtained from Genechem Technology  (The plasmid contains a GFP tag and is neomycin resistant). In this study, the coding sequence of Wnt3a was amplified via PCR using the following primers: forward primer 5ʹ-GAG GAA TTC ATG GCC CCA CTC GGA TAC-3ʹ which contains an EcoR I recognition site, reverse primer 5ʹ-GAC AGC TCG AGCTA CT TGC AGG TGT GCA CGT CGT AG-3ʹ, which contains an Xho I recognition site, and then subcloned into vector pcDNA3.1 (+) eukaryotic expression vector. The recombinant plasmid was verified by DNA sequencing and double enzyme digestion (EcoR I and Xho I). Matrigel was from BD Biosciences . BALB/C nude mice (4 to 6 weeks old) were obtained from Wei Tong Li Hua Experimental Animal Co., Ltd. . Dkk1 recombinant protein was from R&D Systems . For Dkk1 administration in vitro, Dkk1 (1 μg/mL) was added to culture medium. Fifty percent of the medium was replaced with fresh conditioned medium containing Dkk1 every 24 h. After 48 h Dkk1 treatment, cells were harvested and the total cell lysates were collected for measurement by Western blot.Human colon cancer cell line HT29 was obtained from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College . HT29 cells were cultured in DMEM/F12 medium supplemented with 5% fetal calf serum (FCS) at 37 °C and 5% COet al. [The streptavidin-biotin-peroxidase immunohistochemical staining method was used in this study. Briefly, after antigen retrieval for 20 min in citrate buffer  at room temperature, sections were incubated overnight at 4 °C with the primary specific antibody . Then secondary HRP-conjugated antibody was used for 1h at room temperature to immunostained the sections and 3,3ʹ-diaminobenzidine buffer was used as substrate to reveal the signals. The sections were scored blindly by two pathologist using a microscope at 200× magnification. Wnt3a immunohistochemical staining was evaluated by assessing staining intensity and density established by Qi After immunohistochemical staining for CD34 (1:100), the sections were washed with running distilled water for 5 min and incubated with periodic acid for 15 min and Schiff reagent. Finally, all of the sections were counterstained with hematoxylin, dehydrated with ascending grade ethanol and mounted.5 cells/well). After 16 h at approximately 60% confluence, the cells were transfected with Wnt3a plasmid (4 μg/well) in 10 μL of transfection reagent in a final volume of 2 mL of transfection medium. Four hours after transfection, full culture medium without antibiotics was added to the mixture. Twenty-four hours after transfection, neomycin-resistant cells were screened to establish stable HT29 cells that overexpressed Wnt3a.Transfection with plasmid carrying Wnt3a and controlled scrambled plasmid was performed with Lipofectamine according to the manufacturer’s instructions. HT29 cells were seeded in a 6-well plate  was resolved by 10% SDS-PAGE and transferred by electroblotting onto PVDF membrane . Blots were blocked for 1 h and incubated with primary antibodies  overnight at 4 °C. Subsequently, blots were washed in TBS containing 0.1% Tween20 and labeled with goat anti-mouse or goat anti-rabbit IgG-HRP . Immunoreactive bands were visualized on an autoradiography film , and the relative density of bands was analyzed using an Odyssey infrared scanner .4Cl for 10 min and 0.2% triton for 10 min. Then, after blocking with 3% BSA for 1 h, the slips were incubated overnight with anti-β-catenin (1:50) at 4 °C. Slides were washed in PBS and labeled with secondary antibodie for 1 h in the dark at room temperature. After immunolabeling, slides were washed, counterstained with DAPI and viewed with fluorescent microscopy . Colocalization efficiency of DAPI and β-catenin was calculated through Image J software . The nuclear regions dyed with DAPI were selected as the region of interest to quantify the nuclear distribution efficiency of β-catenin. Eighteen images were analyzed.Cells were plated on sterile glass cover slips 1 day prior to staining. Cells were fixed with 10% formalin in PBS for 10 min, quenched with 50 mM NHin vitro. Matrigel (0.1 mL/well) was applied on the twenty-four-well culture plate and incubated at 37 °C for half an hour. The cells were trypsinized and suspended in the complete medium at 2.5 × 105 cells/mL, plated onto the surface of Matrigel at 1 mL/well, and incubated at 37 °C for 48 h.VM-forming ability was tested by using 3-D culture 6 control cells or clone9 cells by subcutaneous injection in the right groin. Tumor volumes were measured every 5 days. All mice were killed on the 30th day post injection. Tumor masses were excised, fixed in formalin, embedded in paraffin, and then were subjected to H&E staining and CD34/PAS double staining.Twenty mice were randomly divided into two groups and received either 3 × 10p < 0.05 were considered statistically significant. The associations between VM and clinicopathologic parameters and the differential expression of Wnt3a and β-catenin between groups were compared using Chi-squared or Fisher’s exact tests in human samples. Differences between groups were assessed by the Mann-Whitney U-test and Student’s t-test.SPSS 16.0 software  was used for data statistical analysis. Wnt/β-catenin signaling is involved in VM formation in colon cancer and might contribute to the development of more accurate treatment modalities aimed at VM.


However, most protocols produce only a limited subset of endogenous MN subtypes. Here we demonstrate that limb-innervating lateral motor column (LMC) MNs can be efficiently generated from mouse and human embryonic stem cells through manipulation of the transcription factor Foxp1. Foxp1-programmed MNs exhibit features of medial and lateral LMC MNs including expression of specific motor pool markers and axon guidance receptors. Importantly, they preferentially project axons towards limb muscle explants in vitro and distal limb muscles in vivo upon transplantation–hallmarks of bona fide LMC MNs. These results present an effective approach for generating specific MN populations from stem cells for studying MN development and disease.Spinal motor neurons (MNs) control diverse motor tasks including respiration, posture and locomotion that are disrupted by neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. Methods directing MN differentiation from stem cells have been developed to enable disease modelling  The differentiation of spinal motor neurons (MNs) from mouse and human embryonic stem cells provides opportunities to model MN development and disease, but most protocols produce only a subset of the MN subtypes found in vivo. Here the authors show that limb projecting lateral motor column MNs can be efficiently generated though the expression of Foxp1. To accurately model development and disease, stem cell-derived populations must fully recapitulate endogenous cell populations—both in the diversity of cell types generated and their functional behaviour. A prime example includes the differentiation of spinal motor neurons (MNs) from mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which has provided an unprecedented opportunity to model the pathogenesis of MN diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy1234In developing mouse embryos, spinal MNs are organized into columns that innervate distinct muscle targets along the length of the body78101112789101112in vivo141516618in vivo is the Forkhead domain transcription factor Foxp1. All LMC and PGC MNs in the developing spinal cord express Foxp1, and Foxp1-mutant animals have severely reduced innervation and motor control of limb muscles1213Most ESC to MN differentiation protocols rely on the use of retinoic acid (RA) and Sonic hedgehog (Shh) or Shh pathway agonists to mimic the natural process of MN formation bona fide LMC MNs in the mouse spinal cord and both mouse and human ESC-derived MNs in vitro. These Foxp1-programmed ESC-derived MNs express molecular markers of LMCm and LMCl motor columns and specific LMC motor pools, indicating conversion from an MMC to LMC fate. Foxp1-programmed MNs express LMC axon guidance receptors and preferentially project axons towards limb muscle explants in vitro, as well as distal limb muscles in chick embryos after transplantation. Last, Foxp1-programmed MNs preferentially form synapses with limb muscles in vitro and in vivo in contrast to control MMC-like MNs, which preferentially innervate axial muscles. Together, these results illustrate the feasibility of generating specific subtypes of MNs from pluripotent stem cells using a transcriptional programming approach, and the importance of MN diversity for achieving functionally distinct behaviours.In this study, we demonstrate that Foxp1 misexpression in differentiating MNs suffices to generate Foxp1 mutants, LMC and PGC MNs transform into MMC and HMC MNs, illustrated by changes in molecular markers, settling position within the ventral horn of the spinal cord and axon projections12Olig2::Cre; Foxp1fl/fl mutants, in which Foxp1 is removed from MN progenitors, are unable to move their forelimb and hindlimb muscles due to the inability of MNs to coalesce into functional motor pools needed to form sensory-motor circuitsHb9::Foxp1 transgenic mice, in which Foxp1 is misexpressed in most spinal MNs under the Hb9 promoter, display an increased generation of LMC and PGC MNs, and a corresponding decrease in MMC and HMC populations12Previous work in our laboratory and others has shown that Foxp1 is necessary and sufficient for the generation and function of LMC and PGC MNs in the mouse spinal cord1213Hb9::Foxp1 transgene could direct LMC MN formation de novo, we interbred Hb9::Foxp1 mice with Foxp1−/− mice and analysed MN formation in different mutant and transgenic allele combinations  blastocysts. Four Hb9::Foxp1; Hb9::GFP (hereafter termed Hb9::Foxp1) ESC lines were generated, and each exhibited characteristic ESC colony appearance and pluripotency marker expression , compared with all Hb9::GFP control MNs. Hb9::Foxp1 ESC-derived MNs also showed a 2.86±0.02-fold increase (mean±s.e.m.) in levels of Foxp1 3′ untranslated region (UTR) mRNA expression compared with Hb9::GFP ESC-derived MNs  7 days after addition of RA and Smoothened agonist (SAG) (Hb9::Foxp1 ESC-derived MNs displayed significantly reduced numbers of Lhx3+ cells (16.5±1.4%) and increased numbers of cells positive for LMCm (Isl1/Foxp1) and LMCl (Hb9/Lhx1) markers   . In conted Isl1) . Interes culture , likely  culture , 13. We  culture .in vivoAldh1a2 mRNA levels were 6.4±1.4-fold higher in Hb9::Foxp1 ESC-derived MNs compared with Hb9::GFP controls  expressed Lhx1, suggesting a slight bias in LMCl formation over LMCm  LMCl and LMCm pools that innervate the latissimus dorsi and cutaneous maximus muscles23+ LMCm pools that innervate the biceps and other forearm flexor muscles; and a Pou3f1+  LMCm pool that innervates the flexor carpi ulnaris muscle of the lower forearm  as LMC MN axons approach the developing limbsFoxp1−/− embryos, there is a complete loss of Etv4+ and Nkx6.1+ LMC motor pools at cervical levels, and a conversion of Pou3f1+ LMC MNs to Pou3f1+ HMC MNs , but did not express Nkx6.1 despite the clear presence of LMCm MNs in the culture   alone or together with the human e (CHAT) . A smalle scarce . Howevere scarce . These Fe scarce , recapit+ embryoid bodies (EBs) in three-dimensional collagen beds between different chick embryo muscle explants and analysed axon outgrowth    or ventral hindlimb muscle  when cultured together with axial muscle explants , indicating that Hb9::Foxp1 motor axons were comparably attracted to both forelimb and hindlimb muscles  . This tral limb) . In contexplants . Similar muscles .Hb9::GFP and Hb9::Foxp1 ESC-derived MNs for expression of known LMC axon guidance receptors. Hb9::Foxp1 MNs expressed increased levels of the EphA4 and c-Ret receptors, which are highly expressed by LMCl MNs and critical for dorsal limb innervation in vivo  to label neuromuscular junctions  of myotubes with GFP+/αBTX+ synapses, compared with limb myotube co-cultures (mean±s.e.m.=40.4±2.3%)   . The opp8% limb) . Further:GFP MNs . HoweverHb9::GFP and Hb9::Foxp1 ESC-derived MNs to assess whether they displayed any differences in functional maturation  on depolarizing current injection as expected for mature MNs, without any disparities in AP frequency or amplitude . These results indicate that Foxp1 misexpression in ESC-derived MNs produces functionally mature MNs with enhanced capacity to innervate limb muscles in vitro.Last, we performed whole-cell patch clamp recordings of the turation . Both HbHb9::GFP MNs have been demonstrated to preferentially project axons towards axial muscle on transplantation into developing chick embryos+ EBs generated from Hb9::GFP and Hb9::Foxp1 ESCs into the neural tube of st.14–17 chick embryos at brachial regions and collected the embryos 3–5 days later , Lhx1+/Foxp1+ (30.6±0.1%) and Foxp1+ only (36.6±1.8%) cells, which matched the distribution of MN subtypes characterized in vitro . By comparison, Hb9::Foxp1 transplants showed significantly more GFP+ axons projecting laterally towards limb muscles   contained GFP+ projections into the forelimbs  of Hb9::Foxp1 transplants also contained GFP+ limb projections  . At lateorelimbs . 75%  had distal projections to the intercostals , suggesting formation of neuromuscular junctions. In contrast, Hb9::Foxp1 axons that innervated axial muscles showed less co-localization of Synaptotagmin 2 and αBTX, indicating fewer synapses + LMCl MNs are generated under these conditions even after the addition of RA + MNs from human pluripotent stem cells+ MNs express Lhx1  followed by human chorionic gonadotropin (50 IU) 48 h later per female. These mice were paired overnight with Hb9::Foxp1 transgenic male mice. E0.5 pre-implantation embryos were isolated the next morning and incubated in hyaluronidase  until cumulus cells were removed from the embryos. About 24 embryos were collected in total and cultured in KSOM media (Millipore) until the blastocyst stage−1; Invivogen), 2-mercaptoethanol (560 nM) and Leukaemia Inhibitory Factor ) plus MEK1 inhibitor . Except as noted, media components were obtained from Invitrogen. About 4 days after plating, 12 blastocysts had hatched and attached to MEFs. Attached inner cell masses were monitored and passaged after 3–4 days of growth. MEK1 inhibitor was removed from the media after the appearance of ES colonies. Mouse ESC colonies appeared within 1 week and were expanded on MEFs until confluentHb9::Foxp1 and Hb9::GFP transgenes and three lines contained only the Hb9::GFP transgene. All mouse ESCs were maintained in mouse ESC media on MEFs.Superovulation was induced in dotropin  IU 48 h Hb9::EGFP mouse ESCs  and newly generated Hb9::GFP and Hb9::Foxp1; Hb9::GFP mouse ESCs were maintained and differentiated into MNs as previously described16−1; Peprotech), Brain-Derived Neurotrophic Factor , and Ciliary Neurotrophic Factor .HBG3 Hb9::GFP and Hb9::Foxp1 EBs were collected 5–7 days after RA and SAG addition. EBs were dissociated using ice-cold Trypsin-EDTA  for 6 min at room temperature, followed by trituration in L-15 Leibovitz media (Thermo Scientific)+ MNs (identified as lacking staining for 7AAD) were sorted and collected using a BD ARIA II FACS machine in the UCLA Broad Stem cell center core facility. Total RNA was isolated from GFP+ MNs using an RNAeasy MiniKit (Qiagen).Foxp1 3′UTR, forward 5′-cagccacgaaagaaacagaag-3′ and reverse 5′-ggtcctggtcacctgattata-3′; mouse Aldh1a2, forward 5′-catggtatcctccgcaatg-3′ and reverse 5′-gcgcatttaaggcattgtaac-3′; mouse EphA4, forward 5′-ctgagccagtaccgaataga-3′ and reverse 5′-gagggcgaagacaaagtaaa-3′; mouse EphB1, forward 5′-gaagaaaggaaggaggtggaa-3′ and reverse 5′-agggaggagtaagaaaggagaa-3′; mouse c-Ret, forward 5′-ctgactgaggtgagagaacaag-3′ and reverse 5′-acacgtaccatagcagcataaa-3′; mouse c-Met, forward 5′-tcagccatcccaatgttctc-3′ and reverse 5′-cgcagatctccatgcttcata-3′; mouse Fgfr1, forward 5′-aggttagccagagggtagaa-3′and reverse 5′-aggagaggaatatgaggagtagg-3′; mouse β-Actin, forward 5′-gccaaccgtgaaaagatgac-3′ and reverse 5′-gaggcatacagggacagcac-3′. Relative mRNA expression levels were determined by normalizing crossing points for each gene to β-Actin and calculating fold changes of Foxp1 misexpression over control Hb9::GFP MNs.Quantitative PCR analysis of MN populations was performed as previously described488-, FITC-, Cy3-, Cy5- and DyLight649-conjugated secondary antibodies (Jackson Immunoresearch) for 1 h at room temperature the following day, and coverslips were mounted with Prolong Gold (Invitrogen). Representative images shown for antibody staining were successfully repeated at least 2–3 times.All embryos and cell cultures were fixed in 4% paraformaldehyde (Fisher). Antibody staining of cryosections and vibratome sections were performed as previously described13212163DIC images were collected using a Zeiss Axioobserver microscope equipped with the Apotome optical imaging system. Fluorescent images were collected using either a Zeiss LSM5 Exciter or Zeiss LSM780 confocal imaging system. Images were processed using Adobe Photoshop and CorelDraw Software. Cell number quantification was performed using the NIH ImageJ software suite with an ITCN add-in for automated cell counting.Hb9::GFP and Hb9::Foxp1 ESC-derived EBs with high levels of GFP expression were manually collected 5–6 days after RA/SAG addition. Axial, forelimb, dorsal hindlimb and ventral hindlimb muscles were dissected from HH stage 35 chick embryos. For adherent muscle co-cultures: dissected muscles were dissociated and plated onto matrigel-coated glass bottom dishes (MatTek Corporation). Hb9::GFP and Hb9::Foxp1 EBs were plated on top of the dissociated muscle and cultured for 3 days. Synapse formation was assayed using Alexa647-conjugated αBTX . For three-dimensional collagen cultures, muscle explants were placed on either side of a single EB in a collagen bed −1; Sigma).Hb9::GFP and Hb9::Foxp1 EBs were dissociated using papain  and plated at low density on Matrigel-coated 35 mm culture dishes (1.2 × 104 cells per dish) in core MN medium with neurotrophic factors and cultured for an additional 2–3 days. Recordings were made from GFP+ cells using standard whole-cell, current-clamp techniques at 20–22 °C. The recording bath solution contained: NaCl (120 mM), KCl (1.9 mM), KH2PO4 (1.2 mM), Na-bicarbonate (20 mM), CaCl2 (2.2 mM), MgCl2 (1.4 mM), D-Glucose (10 mM) and Hepes (7.5 mM). Patch pipettes (resistance 3–5 MΩ) were filled with internal recording solution containing: K-gluconate (140 mM), Hepes (10 mM), EGTA (1 mM), Mg-ATP (4 mM) and Na-GTP (0.3 mM). The pH of both solutions was adjusted to 7.2. Cells with resting potentials <−30 mV were discarded. Patched MNs (maintained in current-clamp mode at approximately −70 mV) were stimulated with 0.1 nA depolarizing current pulses of 250 ms duration. For each MN, this stimulus pulse was applied at least three times . Only data from cells in which the long stimulus pulse elicited the same number of APs were included in this study. Data were collected using Axopatch 2B patch clamp amplifiers with 4-pole Bessel filtering at 5 kHz. Signals were digitized, stored and analysed using pClamp software (Axon Instruments). AP amplitude was measured as the voltage change from the threshold to the peak of the first AP in a train.Hb9::GFP and Hb9::Foxp1 ESC-derived MNs were transplanted into the lumen of the spinal cord, as described previously+ EBs were manually collected 5–6 days after RA and SAG addition and cut into 4–6 pieces. A glass needle was inserted through a small nick in the neural tube of the embryo and a small portion of the neural tube was removed at brachial or thoracic levels. GFP+ EBs were injected into this lesion site. Embryos were allowed to develop for 2–5 days post transplantation and then collected, fixed and prepared for cryosections or vibratome sections.Fertilized chicken eggs (McIntyre Poultry and Fertile Eggs) were incubated at 38 °C for ∼60 h to HH stage 14–17 prior to use. Hb9::GFP and Hb9::Foxp1 ESC-derived MNs were transplanted into the chick spinal cord at brachial levels. About 3 days later embryos with GFP+ cells were identified and used for retrograde tracing experiments. Dextran  was injected into the axial or limb nerve branches. Embryos were cultured at 30 °C for 6–7 h, fixed and prepared for cryosections.−1 of basic fibroblast growth factor). Human ESCs were differentiated to MNs as previously described−1; Sigma), insulin growth factor 1  and NFs ). For immunostaining, hESC-derived MNs were lightly dissociated using 20% TrypLE (Invitrogen) and plated on matrigel-coated glass coverslips in MN maturation media.All human ESC experiments were conducted with prior approval from the UCLA Embryonic Stem Cell Research Oversight (ESCRO) Committee. H9 human ESCs  were maintained on irradiated MEFs in human ESC media  was PCR amplified from the plasmid template HsCD00297105 , and then cloned into the FUCRW lentiviral vector+ cells were identified 3–5 days after infection. About 1–2 weeks after infection, transduced human ESC-derived MNs were fixed in 4% paraformaldehyde for 10 min at room temperature before being processed for immunostaining.The coding sequence of human For analysis of MN populations in E12.5 mouse embryos: mean±s.e.m. values were calculated by pooling at least three sections at both cervical and thoracic levels, collected from at least three embryos of each genotype. For analysis of ESC-derived MNs: 3–5 field images containing ∼100 cells per field were counted for each antibody stain per differentiation batch. Values from three separate differentiation batches were averaged to generate mean±s.e.m. values shown in the figures.in vitro MN-muscle co-cultures and explants was performed blind. Blinding of other experiments and randomization were not performed. Data were not tested for normality and compared using either the Student's t-test, a paired two-tail t-test, or one or two-way analysis of variance with Bonferroni adjustments for multiple comparisons. Variance was similar between groups being compared. The test used for each experiment is noted in the figure legends. All statistics and graphs were generated using Prism6 Graphpad software.No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications13Hb9::Foxp1 transgenic mice. J.A.U. performed the electrophysiology experiments.K.L.A. and B.G.N. conceived and designed the experiments and wrote the manuscript. K.L.A. performed and analysed most of the experiments. D.L.R. generated the How to cite this article: Adams, K. L. et al. Foxp1-mediated programming of limb-innervating motor neurons from mouse and human embryonic stem cells. Nat. Commun. 6:6778 doi: 10.1038/ncomms7778 (2015).Supplementary Figures 1-7


Assessing an animal's body condition may be useful in conservation if condition reflects habitat quality. We found that interpretation of most body condition indices can be confounded by effects of breeding stage, sex and age, and only some vary in ways consistent with the idea that they reflect environmental quality. Neochmia phaeton), a tropical passerine, to describe the variation in seven commonly used condition indices by sex, age, breeding stage, time of day, and year. We found that packed cell volume, haemoglobin, total plasma protein, and scaled mass were all significantly affected by an interaction between sex and breeding stage. Furcular fat varied by sex and breeding stage and also trended by year, scaled mass showed a positive trend with age and varied by time of day, and haemoglobin additionally varied by year. Pectoral muscle scores varied and heterophil to lymphocyte ratio trended only by year. Year effects might reflect a response to annual variation in environmental conditions; therefore, those indices showing year effects may be especially worthy of further investigation of their potential for conservation applications. Pectoral muscle scores and heterophil to lymphocyte ratio may be particularly useful due to the lack of influence of other variables on them. For the other indices, the large variation that can be attributed to individual covariates, such as sex and breeding stage, suggests that one should not interpret the physiological condition of an individual as measured by these indices from their absolute value. Instead, the condition of an individual should be interpreted relative to conspecifics by sex, breeding stage, and possibly age.Body condition indices are increasingly applied in conservation to assess habitat quality, identify stressed populations before they decline, determine effects of disturbances, and understand mechanisms of declines. To employ condition indices in this manner, we need first to understand their baseline variability and sources of variation. Here, we used crimson finches ( Body condition is the physiological state of an individual and reflects how successful that individual is in interacting with its environment. Condition is higher when an individual acquires and assimilates more resources  and inappropriate fire regimens  are restricted to riparian zones throughout the tropical savannas of northern Australia and New Guinea , sedentary passerines that breed during the wet season . The study area was a 2 km stretch of riparian habitat along Annie Creek that is actively managed for conservation. Here we monitored the crimson finch population for breeding activity and sampled the individuals for condition indices over four consecutive breeding seasons .We monitored a population of We captured adult crimson finches in mist-nets during the morning (up to 6 h) to minimize time-of-day effects on condition indices (n = 92). Birds that were in adult plumage upon first capture were aged as being in their first year  if they had a molt limit between their retained, juvenal primary coverts and adjacent newer feathers (n = 39). Together, we refer to these individuals as known-aged birds. In contrast, all other individuals could not be aged because they were first captured as adults, and data on their molt were not collected .We sampled 372 individual birds, which we aged based on known hatch dates, juvenal plumage, and molt limits. We were able to assign an accurate age to the individuals that were originally banded as nestlings or juveniles .Other readily available condition indices include haematological parameters that are a result of an individual's nutritional status, stress level, and immune function. Packed red blood cell volume (PCV) is the proportion of red blood cells to the total volume of blood, and is often considered an aggregate indicator of overall health e.g.  may be cTotal plasma protein is a measure of proteins present in plasma; primarily albumin and globulin. Dehydration causes increases in both albumin and globulin, but an increase in globulin alone suggests inflammation and immune system stimulation , and somHeterophils and lymphocytes are white blood cells primarily involved in the immune response . A high We used all sampled individuals to describe the distribution  of each condition index and to test for pairwise correlations between the indices. If an individual was sampled more than once, we used the average value for that individual for each condition index in these descriptive analyses. We tested whether the continuous variables  were correlated with one another by using Pearson's correlation  since sunrise.After finding significant effects of year and of the interaction between sex and breeding stage on many condition indices (see Results), we examined the parameter estimates and inferred statistical significance from the 95% confidence intervals for those estimates to better understand the effects of these terms on the condition indices. We set statistical significance at α = 0.05. Our figures show least-square means (±SEM) which are consistent with the raw data. We ran all models in JMP 9.0 .n = 149), while the rest were sampled more than once, as follows: twice, 25% (n = 94); three times, 13% (n = 49); four times, 10% (n = 37); five times, 5% (n = 20); six times, 4% (n = 15); and seven times or more, 2% (n = 7). The distributions of observed values  for each condition index are summarized in Table r = 0.74, P < 0.0001; Table We sampled 372 individual birds for some or all of the condition indices during the 4 year study, of which 40% were sampled once  was no longer significant after Bonferroni correction  to interpret differences in condition correctly between individuals and populations.We found that most of the condition indices varied significantly by an interaction between sex and breeding stage. For females, much of this effect is most parsimoniously explained as a consequence of egg laying. Packed cell volume and haemoglobin decrease while total plasma protein and scaled mass increase in egg-laying females. This pattern has been reported previously (packed cell volume, Zonotrichia leucophrys oriantha; Consistent with the result that packed cell volume and haemoglobin are positively and strongly correlated in crimson finches Table  and in bThe interaction between sex and breeding stage may explain the inconsistent results of studies that considered only sex, but did not consider both sex and breeding stage. Some studies found males to have higher PCV values than females (Significant decreases in protein concentration from the pre-breeding to the nestling stage occurred in both sexes, consistent with the findings of Body mass is known to increase with time of day in diurnal birds due to overnight fasting (Rissa tridactyla; Falco sparverius; Locustella luscinioides; Both sexes had lower fat scores while breeding compared with the pre-breeding stage, but the timing of fat loss differed between the sexes, with males losing their fat during the nest-building stage, whereas females maintained high fat levels until the nestling stage. Crimson finch males, and not females, build the nests (The optimal body mass of a bird is influenced by physiological and ecological trade-offs between essential activities. The trade-off between starvation and predation  is an exMost of the condition indices we measured also varied significantly by sex. The mechanism behind the variation due to sex is important for interpreting condition because if, for example, this variation is solely due to hormones during the breeding season then sex may be a less important covariate if birds are sampled during the non-breeding season. Moreover, given that sex also interacts with breeding stage, condition indices should not be collected during the breeding season without detailed knowledge of the reproductive state of the individuals, a suggestion also made by others (Scaled mass was the only condition index to vary with age in crimson finches Table . Lack ofTwo condition indices varied significantly between years in crimson finches, including haemoglobin and muscle scores; H/L ratio and fat scores also varied by year, but these trends were not significant after Bonferroni correction. Others have also found that these and additional condition indices vary by year or season, suggesting an environmental effect on condition (We suspect that the year effects we observed are likely to have resulted from differences in environmental conditions between years, for example variation in food supply mediated by weather. It is this type of relationship between individual condition and environmental conditions that is useful in conservation. If the year effect indeed reflects the environment, then the condition indices that show these year effects, i.e. haemoglobin, pectoral muscle scores, and to a lesser extent, H/L ratio and fat scores, may be the most appropriate ones for conservation applications. We documented considerable variability in condition indices attributable to sex, breeding stage, and the interaction between sex and breeding stage, and to a lesser extent age and time of day. This variation must be accounted for, either statistically or via sampling design, in order to interpret condition indices as indicators of environmental conditions. For example, in interpreting differences between two habitats using haemoglobin as an index, one would need to control for the confounding effects of breeding stage and a sex by stage interaction. In such cases, one should not view an absolute value of a given index as a measure of the physiological condition of an individual, but rather the condition of an individual is relative to conspecifics of the same sex, breeding stage, and possibly age. This has previously been suggested for at least some condition indices (e.g. PCV, If we assume for the moment that the year effects in our analyses do represent responses to annual variation in environmental conditions, then the two condition indices that exhibited no confounding effects such as sex and stage but did exhibit a year effect, i.e. pectoral muscle score and to a lesser extent H/L ratio, represent better choices for conservation applications. Those condition indices that exhibit several confounding effects but no year effect are the poorest choices, because the link between the environment and the condition index is not as direct and is confounded by other variables. However, our assumption about the source of year effects could be incorrect. Although others have demonstrated that condition indices do vary with habitat or environmental quality , we cannUnderlying the use of condition indices in conservation is the often implicit assumption that condition and fitness are positively correlated; that is, even if a condition index exhibits a relationship to habitat or environmental conditions, if this relationship has no consequences for survival or reproduction then it does not inform conservation. Indeed, some condition indices have been found to correlate with primary components of fitness, such as reproductive success (mean corpuscular volume,


Chlamydia pneumoniae. In the current study, using a yeast-two hybrid screen assay, we identified host cell cytoplasmic activation/proliferation-associated protein 2 (Caprin2) as an interacting partner of Cpn1027. The interaction was confirmed and mapped to the C-termini of both Cpn1027 and Caprin2 using co-immunoprecipitation and GST pull-down assays. A RFP-Caprin2 fusion protein was recruited to the chlamydial inclusion and so was the endogenous GSK3β, a critical component of the β-catenin destruction complex in the Wnt signaling pathway. Cpn1027 also co-precipitated GSK3β. Caprin2 is a key regulator of the Wnt signaling pathway by promoting the recruitment of the β-catenin destruction complex to the cytoplasmic membrane in the presence of Wnt signaling while GSK3β is required for priming β-catenin for degradation in the absence of Wnt signaling. The Cpn1027 interactions with Caprin2 and GSK3β may allow C. pneumoniae to actively sequester the β-catenin destruction complex so that β-catenin is maintained even in the absence of extracellular Wnt activation signals. The maintained β-catenin can trans-activate Wnt target genes including Bcl-2, which may contribute to the chlamydial antiapoptotic activity. We found that the C. pneumoniae-infected cells were more resistant to apoptosis induction and the anti-apoptotic activity was dependent on β-catenin. Thus, the current study suggests that the chlamydial inclusion protein Cpn1027 may be able to manipulate host Wnt signaling pathway for enhancing the chlamydial anti-apoptotic activity.We previously identified hypothetical protein Cpn1027 as a novel inclusion membrane protein that is unique to  Chlamydia pneumoniae, a human respiratory pathogen, is associated with a spectrum of diseases such as atherosclerosis and asthma  was amplified from C. pneumoniae AR39 genomic DNA  and cloned into GAL4 DBD ProQuest two-hybrid bait vector pDBLeu, generating the plasmid pDBLeu-Cpn1027(210–527). The MaV203 yeast strain was transformed with pDBLeu-Cpn1027(210–527) as previously described [For yeast two-hybrid screening, the DNA fragment coding for Cpn1027 C-terminal region covering residues 210–527 [designated as Cpn1027(210–527), Cpn1027 was also known as Cp0825 in the escribed  and the escribed . One of escribed .HEK 293T or HeLa cells  were transfected or co-transfected with the following plasmids using the Lipofectamine 2000 transfection reagent following the protocol recommended by the manufacturer (Invitrogen). The pDsRed-Cpn1027, pDsRed-APC4 and pDsRed-Caprin2(245–1128) plasmids were created by cloning the genes of interest into the pDsRed Monomer C1 mammalian expression vector , including full-length Cpn1027 covering residues 1–527 , APC4 , and Caprin2 residues 245–1128 . These genes were expressed as fusion proteins with a red fluorescent protein (RFP) tag fused to their N-termini. The Cpn1027 C-terminal region covering residues 210–527  was cloned into pFLAG-CMV-4 mammalian expression vector  to create the recombinant plasmid pFLAG-Cpn1027(210–527). The Cpn1027(210–527) was expressed as a fusion protein with a FLAG tag fused to its N-terminus.3VO4). The lysates were pre-cleared with protein G Sepharose beads . At the same time, the protein G Sepharose beads from Amersham Biosciences were incubated overnight at 4°C with rabbit anti-RFP polyclonal antibodies . Pre-cleared cell lysates were then incubated overnight at 4°C with the antibody bound Protein G beads. After incubation, Protein G-bound immunocomplexes were washed three times with PBS, resuspended in 2% SDS sample buffer, and subjected to Western blot analysis for protein-protein interactions.To confirm the interaction of Cpn1027 with Caprin2, HEK 293T cells were co-transfected with pFLAG- Cpn1027(210–527) and pDsRed-Caprin2(245–1128), pDsRed-APC4 or pDsRed alone. 24h after transfection, cells were lysed in immunoprecipitation buffer containing  was used to precipitate the cell lysates as described above. The precipitates were used for Western blot detection of RFP.C. pneumoniae AR39 genome as the template while Caprin2 or its fragments using the HeLa cell cDNA library (Invitrogen). Expression of the fusion proteins were induced with IPTG (Invitrogen) and the fusion proteins were extracted by lysing the bacteria via sonication in a Triton X-100 lysis buffer . After high-speed centrifugation to remove debris, the fusion-protein containing supernatants were purified using agarose beads conjugated with glutathione (Amersham Biosciences) following the instructions provided by the manufacturer. After thorough washing, the fusion proteins immobilized onto beads were aliquoted and stored at -20°C. The amount of bead-bound fusion proteins was quantified based on Coomassie blue staining intensity on SDS-PAGE gel and the fusion proteins were adjusted to equivalent amounts for GST pull-down assays.The GST fusion proteins used for the GST pull-down assay were produced by cloning DNA coding for Cpn1027 or its fragments or Caprin2(245–1128) or its fragments into pGEX6p2 vector (Amersham Pharmacia Biotech) as described previously . The pri3VO4. The lysates were incubated overnight at 4°C with bead-immobilized GST-Caprin2(245–1128) beads. To map the interaction domains, HEK 293T cells transfected with pDsRed-Caprin2 for 24h were lysed as described above and the lysates were incubated overnight at 4°C with bead-immobilized GST-Cpn1027(1–209), GST-Cpn1027(210–527), GST-Cpn1027(210–365), GST-Cpn1027(366–527), or GST alone. To identify endogenous proteins, HeLa cell lysates were incubated overnight at 4°C with either bead-immobilized GST-Cpn1027 or GST alone. After incubation, beads were washed three times with PBS. Bound proteins were eluted using 2% SDS sample buffer and subjected to Western blot analyses.To validate Cpn1027 interaction with Caprin2, HEK 293T cells transfected with pDsRed-Cpn1027 or pDsRed alone for 24h were lysed using a lysis buffer containing 20mM Tris-HCl, 130mM NaCl, 10% Glycerol, 1% Triton X100, 2mM EDTA, 1 mM PMSF, 75 units aprotinin per ml, 20 mM leupeptin, and 200mM NaC. pneumoniae infection, siRNA transfection and apoptosis induction as described in sections below were lysed 2% SDS sample buffer and then boiled for 5 min. The boiled samples were resolved in SDS polyacrylamide gel and then transferred to nitrocellulose membranes for labeling with various primary and secondary antibodies. The primary antibodies used include the following: mouse polyclonal antibody against Cpn1027, rabbit polyclonal antibody against RFP/GST, goat polyclonal antibody against GST , mouse monoclonal antibody against β-actin (Sigma), mouse monoclonal antibody against GSK3α/β (BD laboratories), rabbit polyclonal antibody against Axin , mouse monoclonal antibody against β-catenin (BD Laboratories), mouse monoclonal antibody against Bcl-2 (BD laboratories), mouse monoclonal antibody against chlamydial HSP60 (BC7.1), and rabbit polyclonal antibody against CK1α . The primary antibody binding was probed with a HRP (horseradish peroxidase)-conjugated secondary antibody and visualized with an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology). Semi-quantitative densitometry was performed using Bio-Rad Quantity One software.The Western blot assay was carried out as described elsewhere ,37 for dbis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt  by reading the absorbance  at 405 nm using a microplate reader (Molecular Devices Corporation).The interactions of GST fusion proteins with their binding partners were also measured by using a protein array enzyme-linked immunosorbent assay (ELISA) as described elsewhere ,39. The C. pneumoniae AR39 organisms; unpublished data) or, in some cases, a rabbit polyclonal antibody against IncA  dissolved in PBS for 20 min at room temperature followed by permeabilization with 0.1% Triton X-100 (Sigma) for an additional 10 min. After washing and blocking, the cell samples were subjected to antibody and chemical staining. DAPI  was used to visualize nuclear DNA. A rabbit anti-chlamydial organism antibody  (Sigma) for 4 h. The mock treatment cells were incubated in culture medium alone. The cell samples were processed for immunofluorescence assay as described below. The cells fixed in 4% paraformaldehyde (Sigma) were processed for labeling both Chlamydia (green) and DNA (blue) as described above. Cells were evaluated for chromatin condensation [C. pneumoniae infection. For each sample, cells from ten random fields were counted under an Olympus AX-70 fluorescence microscope and the percentage of apoptotic cells was calculated as the number of apoptotic cells divided by the total number of cells counted, ~100 cells for each coverslip.HeLa 229 cells were infected with lsewhere –18. Twenensation ,24,37 anin vitro assays (C. pneumoniae-infected cells but without success. This might be primarily due to the fact that the endogenous Caprin2 was not detectable in cell lysates (data not shown). We also failed to precipitate the endogenous Cpn1027 from C. pneumoniae-infected cell lysates (data not shown), which has been a common challenge for probing the interactions of endogenous Incs with host cell proteins. Despite these technical difficulties, the 3 independent assays above have validated the interactions of C. pneumoniae Cpn1027 with host protein Caprin2. In addition, a RFP-Caprin2 (245–1128) fusion protein but not RFP alone was detected around C. pneumoniae inclusions  of Cpn1027, designated as Cpn1027(210–527), as a bait to search for host cell binding partners from a HeLa cell cDNA library in a yeast two-hybrid screen assay. Our screen yielded several positive cDNA clones (data not shown) but only the clone encoding residues 245–1128 of Caprin2 was found to interact with Cpn1027 reproducibly in various o assays . For exao assays . In a coo assays . The Cpno assays . We alsoclusions , suggestAlthough we have shown that Cpn1027(210–527) interacts with Caprin2(245–1128), it remains unknown what shortest region of each is responsible for the interaction. Using a GST pull-down assay, we first mapped the interacting domain of Cpn1027(210–527). As shown in C. pneumoniae inclusion (C. pneumoniae inclusion membrane. In the presence of Wnt signaling, Caprin2 enhances the interaction of proteins in the β-catenin destruction complex with LRP5/6 to form a signalsome leading to the stabilization of cytosolic β-catenin [C. pneumoniae infected cells. The endogenous GSK3β was detected at the periphery of the C. pneumoniae inclusion (C. pneumoniae inclusion by interacting with Cpn1027.As Cpn1027 is an inclusion membrane-localized protein, interaction with Caprin2 should lead to the accumulation of Caprin2 in the vicinity of the chlamydial inclusion. We found that the ectopically expressed RFP-Caprin2 but not RFP alone was detected around the nclusion , suggestnclusion . Furthernclusion . Vice venclusion . These oC. pneumoniae is known to possess a profound anti-apoptotic activity, we next tested whether knocking down β-catenin in C. pneumoniae-infected cells can inhibit the C. pneumoniae anti-apoptotic activity (C. pneumoniae infection for 24h were transfected with either control siRNA or siRNA targeting β-catenin. 24h after transfection, a portion of the HeLa cell samples were harvested for monitoring the protein levels of β-catenin and Bcl-2. We found that the siRNA targeting β-catenin but not the control siRNA significantly reduced the levels of both β-catenin and Bcl-2 but not β-actin (C. pneumoniae maintained a robust anti-apoptotic activity. However, the siRNA that targets β-catenin blocked the C. pneumoniae anti-apoptotic activity.Since β-catenin plays a critical role in cell survival ,28 and Cactivity . HeLa ce β-actin . The par β-actin . The β-c β-actin . We founChlamydia pneumoniae inclusion membrane protein Cpn1027 can interact with the host Caprin2. First, in a yeast two-hybrid screening, the Caprin2-expressing clone was identified to be positive for interacting with Cpn1027, which was reproduced in a yeast co-transformation experiment. Second, the Cpn1027-Caprin2 interaction was validated in a co-immunoprecipitation assay, in which the RFP-Caprin2 fusion protein but not RFP fusion tag alone or RFP-APC4 fusion protein precipitated FLAG-Cpn1027. Third, a GST-Caprin2 fusion protein but not GST alone pulled down RFP-Cpn1027. Fourth, the interaction between Cpn1027 and Caprin2 was mapped to the C-termini of both molecules using a GST pull-down or ELISA assay. Finally, an RFP-Caprin2 fusion protein was recruited around C. pneumoniae inclusion membrane where Cpn1027 is localized. Since Caprin2 is a regulator in canonical Wnt signaling by facilitating the phosphorylation of the seven-transmembrane-spanning receptor Frizzled/LRP5/6 receptor complex and promoting the sequestration of the β-catenin destruction complex to the cytoplasmic membrane [C. pneumoniae may be able to recruit and sequestrate the β-catenin destruction complex around the inclusion membrane via the Cpn1027-Caprin2 interaction. This hypothesis is further supported by the finding that GSK3β, a key component of β-catenin destruction complex, was also recruited around the C. pneumoniae inclusion.In the current study, we have presented evidence that the membrane , the aboChlamydia pneumoniae inclusion protein Cpn0585 may use its C-terminus to interact with multiple host cell Rab GTPases [Chlamydia pneumoniae inclusion membrane may mimick the function of the Wnt signaling receptor for stabilizing the Caprin2 trimer and recruiting the β-catenin destruction complex around the chlamydial inclusion (The observation that the C-terminal regions in both Cpn1027 and Caprin2 are responsible for their interactions indicates that their C-termini are accessible to each other. All chlamydial inclusion proteins possess a bi-lobed hydrophobic trans-membrane region in either their N- or C-terminal regions ,40,41. T GTPases  while Cp GTPases . The C-t GTPases ,42. By inclusion .C. pneumoniae inclusion. Furthermore, in cells expressing RFP-Cpn1027 (C. pneumoniae inclusions, their close proximity to chlamydial inclusions may be biologically significant.GSK3β, a key component of β-catenin destruction complex, was also localized around the -Cpn1027  or GST-CChlamydia pneumoniae-infected cells, Cpn1027 anchored in the inclusion membrane protein may mimick the function of the Wnt signaling receptor LRP5/6 by interacting with Caprin2 and recruiting β-catenin destruction complex around chlamydial inclusion, resulting in β-catenin accumulation and activation of Wnt signaling pathway-targeted genes as proposed in Caprin2 refers to cytoplasmic activation/proliferation-associated protein 2, which is normally distributed in the host cell cytosol. In response to the activation of Wnt signaling pathway, Caprin2 is recruited to the plasma membrane where it participates in the LRP5/6 signalosome formation by enhancing the interactions between Axin, GSK3β, and LRP5/6, resulting in the sequestration of the β-catenin destruction complex, inactivation of GSK3β, accumulation of β-catenin and translocation of β-catenin to the nucleus for trans-activating Wnt target genes –28. On tC. pneumoniae-infected cells were more resistant to apoptosis induction by staurosporine. β-catenin seemed to play an important role in the C. pneumoniae anti-apoptotic activity since knockdown of β-catenin significantly attenuated the C. pneumoniae anti-apoptotic activity. Thus, we proposed that preserving of β-catenin by sequestrating the β-catenin destruction complex around the chlamydial inclusions might represent an active mechanism for maintaining the C. pneumoniae anti-apoptotic activity. We are aware that inhibition of host cell apoptosis may involve multiple molecular mechanisms since maintaining host cell viability is fundamental for the slow growing C. pneumoniae organisms to complete their growth cycles. Since Cpn1027 is unique to C. pneumoniae, it should be cautious when applying the current finding to C. trachomatis. In C. trachomatis-infected cells, the β-catenin was found to recruit to the chlamydial inclusion [C. pneumoniae anti-apoptotic activity.The Chlamydia-triggered Wnt-like signaling may have multiple effects on the infected cells. In the current study, we found that the nclusion , which m


Furthermore, the erosion resistance of graphene nanosheets-based epoxy resin coating was investigated by electrochemical measurement. The results showed also that the Rrcco (ca. 0.3 mm/year) of graphene nanosheets-based epoxy resin coating was far lower than neat epoxy resin (1.3 mm/year). Thus, this approach provides a novel route for improving erosion resistance and mechanical-thermal stability of polymers coating, which is expected to be used in mechanical-thermal-corrosion coupling environments.The graphene nanosheets-based epoxy resin coating  was prepared by a situ-synthesis method. The effect of polyvinylpyrrolidone/reduced graphene oxide (PVP-rGO) on mechanical and thermal properties of epoxy resin coating was investigated using nanoindentation technique and thermogravimetric analysis, respectively. A significant enhancement ( On the situ-synthesis method. Furthermore, nanoindentation technique was used to measure the mechanical properties of GNS-based epoxy resin coating, in which the Young’s modulus, hardness and plastic index could be obtained. Furthermore, erosion resistance of GNS-based epoxy resin coating was also determined by electrochemical measurements. These results are very important for improving the erosion resistance of polymer coating, which is expected to be used in multi-field coupling environments.In this study, the GNS-based epoxy resin coating was prepared by the −1 were attributed to the location of the carbon-related D peak and the location of the carbon-related graphene peak, indicating the dispersion of the graphene in the epoxy resin matrix . Corrosion current (Icorr) is determined by superimposing the straight line along the linear portion of the cathodic or anodic curve and extrapolating it through Ecorr. Corrosion rate  is calculated from the following equation [EW is the equivalent weight (g), corrI is the corrosion current density (μA/cm2) and ρ is the density (g/cm3).All electrochemical measurements of corrosion potential and corrosion current were performed on a Volta Lab model 21 Potentiostat/Galvanostat in a standard corrosion test cell equipped with a saturated calomel reference electrode (SCE) and a working electrode, and all experimental data were repeated at least three times. The electrolyte was an aqueous solution containing 3.5 wt % sodium chloride. Open circuit potential (OCP) at the equilibrium state of the system was recorded as the corrosion potential [Ecorr (V)equation : (4)Rcorsitu synthesis method, and the graphene modified by PVP could be well dispersed in the epoxy resin matrix and demonstrated good adhesion with epoxy resin matrix. A significant enhancement in the mechanical and thermal properties of epoxy resin coating was obtained with a 0.7 wt % loading of PVP-rGO, such as ca. 213% and 73 °C increase in Young’s modulus and thermal stability, respectively. At the same time, the plasticity index of epoxy resin coating decreased with increases in PVP-rGO content, in which the reduction of this parameter at 0.7 wt % loading was 26.9% compared with that of epoxy resin coating, indicating a significant enhancement in creep properties of GNS-based epoxy resin coating. Furthermore, erosion resistance of GNS-based epoxy resin coating was also investigated by the electrochemical measurements and immersion corrosion experiment in NaCl solution, respectively. It indicated that the GNS-based epoxy resin coating with higher mechanical and thermal properties had better erosion resistance. These results provide a novel route for improving mechanical and thermal properties of inhibited erosion polymer coating, which is expected to be used in thermal-mechanic-corrosion coupled environments.The GNS-based epoxy resin coating was prepared by a


BubR1 or SGO1 results in enhanced chromosomal instability and tumor development in the intestine. Recent studies show that spindle checkpoint proteins also have a role in slowing down the ageing process. Therefore, we want to study whether haplo-insufficiency of both BubR1 and SGO1 accelerates cellular senescence in mice.Spindle assembly checkpoint components BubR1 and Sgo1 play a key role in the maintenance of chromosomal instability during cell division. These proteins function to block the anaphase entry until all condensed chromosomes have been attached by the microtubules emanating from both spindle poles. Haplo-insufficiency of either BubR1 and SGO1 knockout mice and generated primary murine embryonic fibroblasts (MEFs) with mutations in either BubR1, SGO1, or both and analyzed cellular senescence of the MEFs of various genetic backgrounds.We took advantage of the availability of BubR1+/−SGO+/− MEFs had an accelerated cellular senescence characterized by morphological changes and expressed senescence-associated β-galactosidase. In addition, compared with wild-type MEFs or MEFs with a single gene deficiency, BubR1+/−SGO1+/− MEFs expressed enhanced levels of p21 but not p16.We observed that BubR1 and Sgo1 accelerates cellular senescence.Taken together, our observations suggest that combined deficiency of  Senescent cells are characterized as having gradually lost the ability to divide. Senescence is the main risk factor for age-related disorders including diabetes, atherosclerosis, hypertension, osteoarthritis, and Alzheimer’s disease and Parkinson’s disease . There aBubR1 results in embryonic lethality [BubR1 haplo-insufficiency (BubR1+/−) causes enhanced chromosomal instability and tumorigenesis [Spindle checkpoint proteins play a key role in the maintenance of chromosomal instability during cell division . BubR1 iethality . BubR1 higenesis . Extensiigenesis , 8. In cigenesis . TherefoSGO1 also predisposes mice to the development of malignancies [Shugoshin 1 (Sgo1) functions to maintain sister chromatid cohesion, preventing premature cleavage of the cohesin complex by separase , 11. Celgnancies .BubR1 and SGO1 heterozygous mice, we generated primary murine embryonic fibroblasts (MEFs) with mutations in either BuBR1, SGO1, or both. We studied whether deficiency in the checkpoint gene(s) had an impact on cellular senescence. We observed that BubR1+/− and SGO+/− MEFs had an accelerated cellular senescence characterized by the morphological changes and expression of SA-β-Gal. Moreover, double mutant MEFs (BubR1+/−SGO1+/−) exhibited the highest rate of cellular senescence, which was accompanied by early appearance of morphological changes and expression of SA-β-Gal. Compared with wild-type MEFs or MEFs with a single gene deficiency, BubR1+/−SGO1+/− MEFs expressed elevated levels of p21 but not p16.Given the availability of BubR1+/− mice and SGO1+/− mice) were maintained at the animal facility of New York University Langone Medical Center. The maintenance was fully compliant with the general guidelines outlined by the United States Department of Agriculture and the American Association of Laboratory Animal Care under the supervision and specific approval of the Institutional Animal Care and Use Committees. BubR1+/−SGO1+/− compound mutant mice were obtained by crossing BubR1+/− mice with SGO1+/− mice. Primary MEFs of various genotypes were obtained from embryos (embryonic day 14.5) of mice of BubR1+/− and SGO1+/− crossing. MEF cells were cultured at 37 °C under 5 % CO2 in 100-mm plates containing Dulbecco minimum essential medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and antibiotics . Serial cultures were carried out in which cells were trypsinized and replated at a density of 106 cells/100-mm dish every 7 days.Transgenic knockout mice  of 5′ATA AAC CCT CTT GCA GTT GCA TC3′. The reaction conditions included an initial denaturation at 94 °C for 3 min followed by 35 cycles of 94 °C for 45 s, 57 °C for 45 s, and 72 °C for 1 min. The final extension was set at 72 °C for 10 min. PCR products were analyzed by electrophoresis on 2 % agarose gels and detected by ethidium bromide staining.Genotyping was carried out as previously described , 10. Bri4 cells (at passages 3 and 4), 4 × 104 cells (at passage 5), 5 × 104 cells (at passage 6), 6 × 104 cells (at passage 7), and 7 × 104 cells (at passage 8) per well, respectively, on 12-well plates. One day after seeding, MEFs were stained for SA-β-Gal activity according to the manufacturer’s protocol . The percentage of senescent cells was the total number of senescent cells divided by the total number of cells counted under a light microscope.MEF cells were seeded at a density of 3 × 10Western blot analysis was carried out as previously described , 18. Tott test with Welch’s correction for un-equal variances, or with the Mann-Whitney U test. A p value <0.05 was considered statistically significant.Statistical analysis and graphs were generated using the Prism 6 (Graphpad). Data were analyzed for significant differences using the two-tailed, independent BubR1 alleles develop a series of ageing-related pathologies that include shortened life span, growth retardation, fat loss, and impaired wound healing [SGO1 also plays a role in the maintenance of chromosomal stability during cell division and that it remains unclear whether SGO1-deficiency also contributes to the accelerated ageing process, we first obtained primary MEFs that were either haplo-insufficiency of BubR1, SGO1, or both. Morphological examination revealed that SGO1-deficient MEFs appeared to be similar to that of BubR1-deficient ones, which grew at a slightly faster rate than the wild-type MEFs (data not shown). However, compound mutant MEFs (BubR1+/−SGO1+/−) grew at a much slower rate, and a small fraction of these cells exhibited morphologies of senescent cells at early passages  .It is well documented that deficiency in spindle checkpoint control results in chromosomal instability, aneuploidy, and tumorigenesis –21. Rece healing . Given tBubR1+/−SGO1+/− MEFs at very early passages  already expressed SA-β-Gal where few, if any, wild-type MEFs expressed this enzyme  also expressed SA-β-Gal, indicative of their senescent status. However, MEFs with compound mutations displayed a much higher tendency toward senescence. At passage 6, a significant number of BubR1+/− or SGO1+/− MEFs also expressed SA-β-Gal  display of cellular senescence, BubR1+/−SGO1+/− MEFs also had a higher percentage of cells positive for SA-β-Gal at passages 4 and 5. It appears that the highest percentage of SA-β-Gal-positive cells in all genotypes was around 37 %. Thus, there is a synergy of premature ageing between reduced expression of BubR1 and SGO1.We then systematically analyzed SA-β-Gal expression in MEFs of various genotypes at different passages. We observed that a small fraction (around 3 %) of wild-type MEFs started to express SA-β-Gal at passage 4, and there was gradual increase in the percentage of SA-β-Gal-positive cells -deficient and BubR1 (or SGO1)-competent MEFs. These observations suggest that p16 expression may not be responsible for the observed cellular senescence in MEFs that are haplo-insufficiency of BubR1, SGO1, or both.To further confirm accelerated ageing due to the reduced expression of spindle checkpoint genes, we analyzed several molecular markers including p21 and p16 that are frequently linked to cellular senescence. At passage 3, EFs Fig. . ExpressSGO1 also causes increased senescence. Sgo1 appears to play a more important role in suppressing ageing as haplo-insufficiency of SGO1 induces cellular senescence earlier and to a greater extent than haplo-insufficiency of BubR1. At the molecular level, BubR1 and Sgo1 function differently in suppressing premature separation of sister chromatids [In this study, we systematically analyzed cellular senescence in cells deficient in BubR1 and/or Sgo1. It is known that reduced expression of BubR1 induces premature ageing which is associated with enhanced cellular senescence . We haveromatids . BubR1 iromatids , 23, wheWe have shown that enhanced senescence is correlated with elevated expression of p21 but not p16. As it is well known that cellular senescence is associated with permanent cell cycle arrest, it is logical that expression of CDK inhibitors such as p21 is significantly elevated. Although we did not observe that p16 expression is high in MEFs that are undergoing senescence, we cannot exclude the possibility that expression of other inhibitors of the INK family including p15, p17, or p19 is elevated. Although they all function to block cell cycle progression, p21 and p16 act differently in the negative regulation of CDKs. As a member of the INK family, p16 solely acts as an inhibitor that blocks G1 CDKs (CDK4/6). On the other hand, p21 is capable of inhibiting CDKs of several cell cycle transitions including S and G2/M. Given that BubR1 and Sgo1 have primary functions in mitosis, it is logical to speculate that impaired functions of these checkpoint components can trigger cell cycle arrest and senescence by activating p21 expression.BubR1 and SGO1 accelerates cellular senescence which is manifested as morphological changes and enhanced expression of SA-β-Gal.This study is consistent with the notion that chromosomal instability due to reduced expression of spindle assembly checkpoint components contributes to ageing processes. The combined deficiency of


P = 0.022). We tested the functional interaction between STAT3 and Wnt signalling by knocking down STAT3 and β-catenin individually and in combination. Knock-down of β-catenin and STAT3 individually inhibited cell proliferation (P < 0. 001 for each) through G1 arrest. However, simultaneous knock-down of STAT3 and β-catenin had a significantly weaker effect than knock-down of β-catenin alone (P < 0.01). Knock-down of STAT3 and β-catenin, individually and together, inhibited cell motility (P < 0.001) without evidence of interaction. We conclude that STAT3 regulates β-catenin but β-catenin does not regulate STAT3. The STAT3/β-catenin interaction is complex but may reduce the proliferative activity of β-catenin possibly by taking β-catenin protein beyond the optimal level. This may indicate biological differences in tumours where both STAT3 and β-catenin are activated compared to those where only one is activated.Wnt signalling and the signal transducer and activator of transcription 3 (STAT3) are oncogenic signalling pathways which are deregulated in colorectal cancer (CRC). Here we investigated the interaction of these two pathways. Firstly, we investigated biochemical interaction by inhibiting STAT3 and β-catenin (through gene knock-down and dominant-negative TCF4 expression) in nine CRC cell lines. β-catenin inhibition did not affect STAT3 levels, whereas STAT3 knock-down resulted in reduced β-catenin mRNA and protein levels. The reduction in β-catenin protein was not prevented by proteasome inhibition, and IL6-induced STAT3 activation resulted in increased β-catenin mRNA. This suggests that STAT3 positively regulates β-catenin  and evaluation of 44 CRCs by immunostaining supported this by showing an association between nuclear STAT3 expression and nuclear β-catenin ( Colorectal cancer (CRC) represents one of the most common causes of cancer-related death around the world  occur which result in a degradation-resistant but transcriptionally active protein  pathway is found to be activated in around 50–60% of CRCs . The human CRC cell lines SW1222, HT55 and C106 were originally obtained from the European Collection of Cell Cultures (ECACC). These cell lines were a kind gift from Prof Ian Tomlinson . The identity of the cell lines was confirmed prior to commencing the work by mutation profiling as previously described  as previously described  was carried out to quantify mRNA of both STAT3 and β-catenin. Total RNA was extracted from the cells using the RNeasy Mini Kit  following the manufacturer's protocol. cDNA was prepared with the SuperScript III Reverse Transcriptase kit . PCR was performed in a total volume of 25 μl consisting of 1X Brilliant SYBR Green QPCR Master Mix , primers (forward and reverse) at a final concentration of 250 nM, 30 nM ROX (reference dye) and 10 ng of the cDNA template. Samples were tested in triplicate, and no template control and non-reverse transcribed mRNA was included to detect any cDNA or DNA contamination. PCR was performed on an MX3005P QPCR machine (Stratagene) using single thermal cycling profile of (95 °C/10 min) X1; [(95 °C/30 s)/(56 °C/1 min)/(72 °C/1 min)] X40; (72 °C/10 min) X1. Quantification was by the standard curve method using MX3005P QPCR System Software version 4.01, and data were normalized to the HPRT housekeeping gene. The sequence of each primer pair and their products size are detailed in Table S2.Whole cell extracts were prepared using commercially available RIPA lysis buffer . Protein  was loaded on a 10% SDS–PAGE gel and transferred on to PVDF membranes  by semidry transfer. After blocking for 1 h with 5% (w/v) milk powder dissolved in 0.1% TPBS , membranes were incubated overnight at room temperature with the indicated primary antibody. Antibodies and their dilutions were anti- β-actin (Sigma-Aldrich) at 1/2000 dilution, anti STAT3 (Abcam ab50761) at 1/100, anti- c-Myc  at 1/500, anti-β-catenin  at 1/500. After three 10-min washes in 0.1% TPBS, membranes were incubated for 1 h at room temperature with the appropriate horseradish peroxidase-linked secondary antibody. Following three further washes, detection of bound antibody was performed using the enhanced chemiluminescence kit . Bands were visualized using X-ray films .et al. To ascertain the role of proteasomal degradation in the STAT3-mediated regulation of β-catenin, the proteasome inhibitor Z-Leu-Leu-Phe-CHO (Sigma-Aldrich) dissolved in DMSO was added to SW620 cells at a final concentration of 2 μM. Cells were exposed to the proteasomal inhibitor 24 h after transfection with siRNA duplexes. To confirm the effectiveness of the proteasome inhibition (i.e. a positive control), the CRC cell line RKO was treated with the proteasome inhibitor and compared with control cells treated with an equivalent volume of DMSO carrier. The RKO cell line has an intact β-catenin degradation system making β-catenin undetectable by Western blot . A total of 44 anonymized cases of sporadic CRC who underwent surgery between 2004 and 2005 were selected from the archives of the Nottingham University Hospitals Department of Histopathology. One tumour block from each case was selected, and a tissue microarray (TMA) was constructed as previously described  divided by the total. Each experiment was performed in triplicate and repeated on at least three separate occasions.The effect of gene silencing on cell number was evaluated at different time points by the methylene blue assay as previously described . This assay was performed in triplicate on three separate occasions.The effect of gene silencing on the cell cycle was evaluated by flow cytometry. Briefly, the transfected cells were grown in 24 well plates for 48 h before harvesting. Cells were rinsed with PBS and then fixed with 70% ice-cold ethanol for 30 min. Following two washes with 0.1% foetal serum albumin in PBS, the cells were suspended in 1 ml 0.1% FBS containing 10 μl RNase solution  and 50 μl propidium iodide solution  for 20 min before being analysed on a Beckman Coulter flow cytometer . A minimum of 3 × 104 cells were seeded in the top chamber in medium containing 10% foetal calf serum. The lower chamber contained medium which was enriched with 20% foetal calf serum. Migration across the membrane was assessed after 48 h by fixing the cells attached to the bottom surface in 10% methanol for 30 min. These were then stained for 30 min with methylene blue, and the stained cells manually counted.Transwell cell migration was measured using a Boyden chamber containing a polycarbonate filter with an 8 μm pore size (Costar). Cells were serum starved for synchronization, and 5 × 10t-test was used to evaluate data derived from the studies of proliferation, apoptosis and cell motility. A Fisher's exact test was used to test for the association between the expression of nuclear STAT3 and nuclear β-catenin. All tests were two tailed, and a P-value of <0.05 was taken as statistically significant.Statistical analysis was performed using the GraphPad-Prism program . An unpaired Given the mixed published data regarding the nature of the biochemical interaction of STAT3 and β-catenin, we first sought to ascertain whether STAT3 and β-catenin were able to regulate each other. Initially, STAT3 and β-catenin were knocked down individually in six different CRC cell lines that is SW620, HT55, SW837, SW480, SW1222 and SW948, and the effect on protein levels was tested. There was some variation in the level of protein knock-down between cell lines which reflects the variation in transfection efficiency. However, there was robust knock-down of both proteins with the siRNAs used. In every cell line, when β-catenin was knocked down, there was no associated change in the level of STAT3 protein. In contrast, when STAT3 was knocked down, this was associated with a marked reduction in the level of β-catenin protein , whilst STAT3 staining was seen only in the cytoplasm and nuclei. The mean score for all assessable cores from each individual case was used in analysis. For analysis of cytoplasmic staining (of both proteins), the median score was used as a cut-off point to dichotomize into ‘positive’ and ‘negative’. For nuclear staining for STAT3, the median score was used as the cut-off whilst, for β-catenin, any nuclear staining was scored as positive. There was a positive association between nuclear STAT3 expression and nuclear β-catenin expression .To test whether STAT3 altered β-catenin transcription, we measured β-catenin mRNA levels in four cell lines following STAT3 knock-down. In all four cell lines, a fall in STAT3 levels was accompanied by a fall in β-catenin mRNA levels , STAT3 knock-down only (SW620STAT3-), β-catenin knock-down only (SW620βcat−) and knock-down of both STAT3 and β-catenin (SW620STAT3−/β-cat−). The effect of each of these conditions was tested in a variety of functional assays.Having shown that STAT3 can regulate β-catenin, we next sought to ascertain the biological significance of this interaction. STAT3 and β-catenin were knocked down individually and in combination in SW620 to produce four different conditions that is control cells exposed to luciferase siRNA , and β-catenin knock-down alone only caused a reduction in cell number by 56% . However, combined knock-down caused a reduction in cell number of 45% which was significantly less than that caused by β-catenin knock-down alone . These findings were reflected to some degree  when apoptotic cells were evaluated. Numerically, the percentage of apoptotic cells was SW620control = 2%, SW620STAT3−= 5.4%, SW620βcat− = 4.4% and SW620STAT3−/β-cat− =5.7% . As anticipated, β-catenin knock-down alone resulted in a greater proportion of cells in G1 (SW620βcat− = 56%) but this was greater than the combined knock-down (SW620STAT3−/β-cat− = 52% in G1). In all knock-down conditions, the increase in the cells in G1 phase was accompanied by equivalent reduction in the percentage of the cells in S phase with relatively little change in G2.To further investigate the unexpected effects of gene knock-down on cell number, each of the four conditions underwent cell cycle analysis by flow cytometry Figurec. STAT3 STAT3−, SW620βcat− and SW620STAT3−/β-cat−), there was a significant reduction in cell motility compared to the control  but they found no effect of STAT3 on E-cadherin/β-catenin interaction at the cell membrane. These findings are directly contradicted by Rivat et al. . The fall in β-catenin protein level following STAT3 knock-down could not, however, be prevented by a proteasome inhibitor. This would suggest that β-catenin is transcriptionally regulated by STAT3 and is consistent with studies in breast cancer showing direct binding of STAT3 to the β-catenin promoter . This would suggest that, either directly or indirectly, STAT3 has an inhibitory effect on β-catenin-driven proliferative activity. Such an effect has also been described in mouse models where loss of STAT3 promoted proliferation in germline Apc mutant Min mice  showed no difference between β-catenin and combine STAT3/β-catenin knock-down. However, cell cycle analysis raised the possibility that the inhibitory effect of STAT3 may be on progress through G1. Evaluation of cell motility showed more or less an equivalence between all three knock-down conditions with no evidence of interaction. This contradicts the data of Rivat Our data suggest that interaction between STAT3 and β-catenin results in a partial inhibition of cell proliferation but does not affect cell motility. It is highly likely that STAT3 will give a selective advantage, and as STAT3 activation is a late event, one can conjecture that a slight loss of proliferative activity is an acceptable cost for acquisition of other features which may be more relevant to the tumour at that stage. The biological interaction between STAT3 and β-catenin is obviously complex, but it raises the intriguing possibility that tumours in which both these pathways are activated may be biologically different to tumours in which in only of these pathways is activated. Such differences could be exploited to develop further personalized medicine for CRC.In summary, we have investigated the biochemical and biological interaction of the Wnt and STAT3 signalling pathways in CRC. We have shown that STAT3 can regulate β-catenin but not vice versa. Furthermore, the biological interaction between these two pathways is complex, and it is possible that, in advanced CRCs, STAT3 is partially inhibiting β-catenin-driven proliferation.