Hereditary ataxia, or motor incoordination, affects approximately 150,000 Americans and hundreds of thousands of individuals worldwide with onset from as early as mid-childhood. Affected individuals exhibit dysarthria, dysmetria, action tremor, and diadochokinesia. In this review, we consider an array of computational studies derived from experimental observations relevant to human neuropathology. A survey of related studies illustrates the impact of integrating clinical evidence with data from mouse models and computational simulations. Results from these studies may help explain findings in mice, and after extensive laboratory study, may ultimately be translated to ataxic individuals. This inquiry lays a foundation for using computation to understand neurobiochemical and electrophysiological pathophysiology of spinocerebellar ataxias and may contribute to development of therapeutics. The interdisciplinary analysis suggests that computational neurobiology can be an important tool for translational neurology. Results from computational neurobiology can be used to develop additional animal and cellular experiments that may ultimately be translated to clinical practice, i.e., translational neurology. One clinical condition poised to benefit from this marriage is spinocerebellar ataxia (SCA)  Figure . Ataxia ) Figure . In thisThe most common SCAs are spinocerebellar ataxia 1 (SCA1), spinocerebellar ataxia 2 (SCA2), spinocerebellar ataxia 3 (SCA3), and Spinocerebellar Ataxia 6 (SCA6)  also occur in genes directly involved in calcium signaling and plasma membrane excitability, which are critical for cerebellar Purkinje neuron function  signaling upstream of IP3R1 in neuroblastoma cells was developed  in Purkinje neurons that were experimentally verified , and other calcium signaling and glutamatergic proteins  , is sometimes phenotypically confused with SCA if ataxia is prominent  images from several patients, showed that peak wall stress calculated in vivo for abdominal aortic aneurysm (AAA) near the time of rupture was more predictive of rupture than the conventional assessment of aortic diameter (Fillinger et al., There are other computational models that can be used to exemplify the potential of SCA models. Experiment-based computational modeling similar to SCA, including the use of individualized 3D reconstruction of A number of scientific contributions from computational modeling Table  that enhSherry-Ann Brown conceived of, analyzed, designed, drafted, critically revised, approved, and agreed to be accountable for this submitted work. Louise D. McCullough conceived of, critically revised, approved, and agreed to be accountable for this submitted work. Leslie M. Loew analyzed, critically revised, approved, and agreed to be accountable for this submitted work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although cell-in-cell structures (CICs) could be detected in a wide range of human tumors, homotypic CICs formed between tumor cells occur at low rate for most of them. We recently reported that tumor cells lacking expression of E- and P-cadherin were incapable of forming homotypic CICs by entosis, and re-expression of E- or P-cadherin was sufficient to induce CICs formation in these tumor cells. In this work, we found that homotypic CICs formation was impaired in some tumor cells expressing high level of E-cadherin due to loss expression of alpha-catenin (α-catenin), a molecular linker between cadherin-mediated adherens junctions and F-actin. Expression of α-catenin in these tumor cells restored cell-cell adhesion and promoted CICs formation in a ROCK kinase-dependent way. Thus, our work identified α-catenin as another molecule in addition to E- and P-cadherin that were targeted to inactivate homotypic CICs formation in human tumor cells. Majority of internal lumens in our bodies are covered with a layer of epithelial cells, whose integrity is critical for the organs to function properly. The integrity of epithelial tissues depends on intact adherens junctions (AJs), which is a multiple-components complex comprising cadherins, the transmembrane adhesion receptors, and their cytoplasmic binding proteins such as p120-catenin and β-catenin etc.21568CICs refer to the cellular structures formed between viable cells, in which one or more cells exist inside other ones. Early records on CICs could be dated back to last century, when pathologists identified this type of unusual structures in human tumor samples10811Recently, we and others found that formation of homotypic CICs by entosis was dependent on intact AJs and polarized actomyosin contraction8915In our previous work, we found that loss of E- and P-cadherin caused defective CICs formation in a group of human breast cancer cells, such as MDA-MB-231, MDA-MB-453 and SKBR3 and the like, re-expression of E- or P-cadherin alone was sufficient to induce entotic CICs in these cellsp < 0.05). Thus, loss expression of α-catenin is responsible for disrupted cell-cell adhesion mediated by AJs in MDA-MB-468 and H820 cells.To examine a causal role of α-catenin expression in defective cell-cell adhesion of MDA-MB-468 and H820 cells, we expressed mouse α-catenin tagged with EGFP in these cells. As shown in We next examine CICs formation in MDA-MB-468 and H820 cells with restored cell-cell adhesion. As evidenced by western blot  and EGFPTo further confirm the role of α-catenin in CICs formation, we depleted α-catenin expression by RNA interference in MCF10A cells, which display high frequency of CICs. Transfection of three siRNA duplexes efficiently knocked down α-catenin as detected by Western Blot , with siOur previous work showed that cytoskeleton was remodeled during CICs formation induced by E-/P-cadherin expression, we therefore examined the distribution of AJs molecules and cytoskeleton upon α-catenin expression. For both adherent MDA-MB-468 and H820 cells expressing α-catenin, E-cadherin locates neatly at the cell-cell boundary, where α-catenin was also enriched . This paAlthough being extensively reported in human tumors for a long time, the mechanisms underlying CICs formation remained unexplored until recent years, when several research models were established8121888In this work, we report the involvement of α-catenin, a molecular linker between AJs and cytoskeleton, in controlling CICs formation of human tumor cells. Alpha-catenin is well known as a tumor suppressor, whose inactivation by multiple mechanisms contributes to advanced phenotypes of various human tumors242527Different from β-catenin, α-catenin was thought to bind cadherin complex in a dynamic fashion. Alpha-catenin could exist in form of either monomer or homo-dimmer in cells, monomer α-catenin preferentially binds β-catenin to contribute AJs formation, whereas the dimmer binds F-actin, where it inhibits branching and promotes bundling of F-actin299It should be noted that, although α-catenin was not expressed and AJs was compromised, MDA-MB-468 cells could still form CICs at low level after long time induction, suggesting that additional mechanisms existing to promote CICs formation. There are three possibilities, first, other cell-cell adhesions other than AJs could mediate weak cell-cell interaction and induce CICs at low rate, one candidate is Nectin/Afadin adhesion complexCell line MDA-MB-468 was purchased from Beijing Union Cell Resource Center; MCF7 and MCF10A cells were from Dr. Michael Overholtzer ; MDA-MB-436 and ZR75-1 cells were from Dr. Qinong Ye (Beijing Institute of Biotechnology). Breast cancer cells MCF7, MDA-MB-468, MDA-MB-436 and ZR75-1 were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (Gibco). Lung cancer cells H820 and H441 were kindly gifts from Dr. Ji-shuai Zhang in this institute, and were maintained in RPMI-1640 (Hyclone) with 10% fetal bovine serum. MCF10A cells were grown as describedAntibodies were used as follows: anti-E-cadherin ; anti-α-catenin ; β-catenin ; γ-catenin ; α-tubulin ; β-actin ; phalloidin . All of the secondary antibodies (1:3000 for western blot and 1:300 for IF) were purchased from CWBIO, China. ROCK inhibitor Y27632 was purchased from TOCRIS and used at final concentration of 10 uM. Prolong Gold antifade reagent with DAPI was purchased from Life technologies.Age I and Mfe I sites of pQCXIP-EGFP-N1, which was constructed by replacing gene encoding GFP in pQCXIP-GFP-C1 with EGFP gene from pEGFP-N1. pQCXIP-GFP-C1 was gifted by Dr. Alan Hall at MSKCC. Retroviruses were made and used to infect target cells as describedpEGFP-C1-α-catenin was a gift from W. James Nelson from Stanford University. The retroviral construct pQCXIP-EGFP-α-catenin was made by subcloning EGFP-α-catenin fusion gene from pEGFP-C1-α-catenin in between 5 were cultured in 6-well plates for 16 h before transfected with 50 uM siRNAs for α-catenin or control sequence . The knockdown efficiency was measured by Western blot 48 h after transfection.MCF10A cells of 2.5 × 105 MDA-MB-468 or H820 cells were cultured in 6-well plates, which were pre-coated with 0.5% soft agar to prevent cell attachment. After 36 hours, cytospins were made and stained with DAPI, based which cells involved in the formation of cell cluster were quantified. Cells in cluster rate (%) =  ×100. We defined cell cluster as a cell colony which contains more than 6 cells.In order to detect the cell clustering degree, 2.5 × 105 cells were cultured in 6-well plates coated with 0.5% soft agar for 6 hours or longer as indicated. Cytospins were then made by centrifugation at 1400 rpm for 5 min  for immunostaining as describedFor cell-in-cell structures to be induced, about 2.5 × 10Cytospins were first fixed in 10% trichloroacetic acid or 4% paraformaldehyde for 10 min and then washed by PBS for three times, followed by immunostaining with indicated antibodies. Nuclei were counterstained with DAPI when slides were mounted with cover slips by Prolong Gold Antifade reagent for image capture after 24 h air-drying. Images were taken at three channels  by confocal microscopy. Western blot was performed as describedImages of large field were taken by Nikon ECLIPSE Ti-U epi-fluorescence microscope by 10×objectives, and analysed by NIS-Elements F 3.0 software . Besides, images of high resolution were acquired by using OLYMPUS FLUOVIEW FV1000 Confocal Microscope at three channels , and analyzed by FV10-ASW 4.0 Viewer software .P values were calculated by Two-tailed Student’s t-test using SPSS20.0 software (IBM), with statistical significance assumed at P < 0.05.All the experiments were carried out at least three times. Results were displayed as mean ± SD. How to cite this article: Wang, M. et al. Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression. Sci. Rep.5, 12223; doi: 10.1038/srep12223 (2015).


Wolbachia, endosymbionts that reside naturally in up to 40–70% of all insect species, are some of the most prevalent intracellular bacteria. Both Wolbachia wAu, naturally associated with Drosophila simulans, and wMel, native to Drosophila melanogaster, have been previously described to protect their hosts against viral infections. wMel transferred to D. simulans was also shown to have a strong antiviral effect. Here we directly compare one of the most protective wMel variants and wAu in D. melanogaster in the same host genetic background. We conclude that wAu protects better against viral infections, it grows exponentially and significantly shortens the lifespan of D. melanogaster. However, there is no difference between wMel and wAu in the expression of selected antimicrobial peptides. Therefore, neither the difference in anti-viral effect nor the life-shortening could be attributed to the immune stimulation by exogenous Wolbachia. Overall, we prove that stable transinfection with a highly protective Wolbachia is not necessarily associated with general immune activation. Wolbachia, intracellular bacteria inhabiting up to 40–70% of known insect species Wolbachia are maternally transmitted and, in some hosts, provide infected females with a relative fitness advantage by cytoplasmic incompatibility, male killing or other forms of reproductive manipulation. Recently, Wolbachia have been attracting widespread attention due to their ability to protect their hosts against viral infections. This phenomenon has been initially reported in Drosophila melanogaster carrying its natural wMel Wolbachia strain Wolbachia discovered in D. melanogaster that could explain high prevalence of the symbiont in natural populations of fruit flies D. melanogaster in research has placed wMel Wolbachia strain among the most extensively studied insect symbionts. Based on the molecular markers it has been shown that wMel strain consists of five polymorphic variants, namely: wMel, wMel2, wMel3, wMelCS and wMelCS2 wMel phylogenetic analysis wMel-like and wMelCS-like. The wMelCS-like variants reach higher densities in the host and provide more antiviral protection than the wMel-like variants. Moreover, some wMelCS-like variants shorten the lifespan of their hosts wMelPop The ubiquity of wAu is a Wolbachia native to D. simulans that used to be present at low frequencies in Australia and does not induce cytoplasmic incompatibility wMel of D. melanogaster and wAu of D. simulans are closely related and both belong to the Wolbachia supergroup A wAu and one of the most protective Wolbachia wMel variants - wMelCS_b, the two strains used in this study, have been previously described as protective against Drosophila C virus (DCV) and flock house virus (FHV) wMel has been previously transferred from D. melanogaster to D. simulansDrosophila-host association was similar to the protection provided by wAu in its natural host Wolbachia lines were studied in different D. simulans genetic backgrounds, preventing direct comparison of the protective abilities of wAu and wMel.wMelCS_b and wAu in genetically identical D. melanogaster hosts. In mosquitoes recently transinfected with Wolbachia the antiviral effect is frequently associated with activation of the host immune system D. melanogaster – Wolbachia associations antiviral protection is strong but expression of immune genes remains unchanged wMelCS_b and wAu transinfected to D. melanogaster.This study compares the antiviral protection and other phenotypes provided by wAu provides strong protection against viruses in its native D. simulans host Wolbachia endosymbionts of Drosophila melanogaster wMelCS_b is one of the most potent in viral interference Drosophila melanogaster, we used Wolbachia-infected lines in a genetically identical DrosDel w1118 isogenic background wMelCS_b was naturally associated with this background while wAu was introduced from D. simulans to D. melanogasterWolbachia-free line, designated “iso”, was used as a control in all experiments. All flies were virus-free and had homogenized gut microbiota . This effect is almost completely abolished in tetracycline-treated flies derived from Wolbachia-positive stocks . The difference between wAu tet and wMelCS_b tet is very small in the case of DCV infection .To compare antiviral properties of ses: DCV , a natur and FHV , initialwAu carrying flies could be either due to the direct reduction of pathogen load (resistance) or due to neutralization of negative impact of the pathogen on the fly's health without direct influence on the virus titres (tolerance or resilience). To distinguish between these two possibilities we tested the levels of each virus in whole flies either 3 and 6 days post infection (dpi) for DCV or 3 dpi for FHV  and over 13 times more efficient 6 dpi  in reducing the DCV titres than wMelCS_b. Flies carrying wAu have also 5.8 times less FHV 3 days after infection in comparison with wMelCS_b . All these data allow us to conclude that wAu protects better against viral infections than one of the most protective wMel variants and this can be, at least partially, explained by the reduction of the viral titres.The strong inhibition of virus-induced mortality in  for FHV . ConsistwMelCS-like Wolbachia variants Wolbachia infected flies in the absence of viral challenge (wAu shortens the lifespan of flies by 20 days (31% difference in median time to death) in comparison with wMelCS_b  demonstrating that harbouring this protective endosymbiont is associated with a cost in the absence of infection. After elimination of Wolbachia from our fly stocks the flies derived from the wAu line also live shorter, but there is only a 5 days difference (9% in median time to death) between them and wMelCS_b derived flies . This difference and the one we observed for the DCV-infected tetracycline treated wAu and wMelCS_b lines may be due to differences in mitochondria between wAu and wMelCS_b fly stocks , but wAu grows much faster than wMelCS_b . The exponential growth of the symbiont may be the cause of the life-shortening, either by direct tissue damage or by constituting a significant metabolic burden compromising the insect's health. This is reminiscent of host life-shortening by the exponentially growing wMelPop strain The association between lifespan . We obseWolbachia into a new insect species Wolbachia is one of the proposed mechanisms explaining Wolbachia-mediated antiviral protection in mosquitoes Drosophila melanogasterImmune upregulation has been shown to occur after transfer of wAu we examined the expression of genes encoding antimicrobial peptides (AMPs). We chose AMPs that were previously shown to be highly induced by the presence of exogenous WolbachiaDrosophila immune pathways: Toll and Imd . However, this effect was not observed in the previous studies To test if chronic immune activation could be responsible for the high antiviral protection and life-shortening by  and Imd . Quantitosomycin . There iDrosophila - mosquitoes transfers Wolbachia transferred within the same genus, i.e. wAlbB from A. albopictus to A. aegyptiiwAu transferred to D. melanogaster could be explained in various ways. The first possible explanation may be the phylogenetic distances between the source and target host insect species; the most recent common ancestor of A. albopictus and A. aegyptii dates to ∼34–42 million years ago D. melanogaster and D. simulans diverged only 2.3 million years ago wAu could be better pre-adapted to infect D. melanogaster inconspicuously. Another explanation is that D. melanogaster has co-evolved with Wolbachia while A. aegyptii natural populations are not infected with this endosymbiont. Thus, D. melanogaster may have evolved not to respond to Wolbachia infection. This may also explain why A. albopictus has a provisional or no immune response to Wolbachia somatic transient infection wAu and wMel might be so similar that the insect's immune system does not perceive wAu as foreign.Our findings add to previous reports on high AMPs expression not only after wAu and wMelCS explain the different phenotypes. wAu genome is not sequenced, however, several differences between the genome of wAu and wMelCS are described. wAu lacks a 21.86 kb genomic region present in wMelCS, named Octomom, which includes genes from WD0506 to WD0518 Wolbachia effector proteins) and many proteins possibly involved in DNA repair and processing. The amplification of this region has been recently proposed to be responsible for the over-replicative phenotype of wMelPop Wolbachia variant wMel strain genomes and wAu wMelCS). All the above analyses were based on PCR amplification, gene sequencing and DNA hybridization and only the sequencing of the whole wAu genome would allow to complete the comparison.It would be interesting to know which genetic differences between the closely related wAu and one variant of Wolbachia wMel – wMelCS_b – in the same D. melanogaster genetic background and provides a direct comparison of the protective capabilities of the two strains. We conclude that wAu protects better against viral infections – it increases lifespan of virus-infected flies and significantly limits viral replication. Additionally, we have discovered that wAu grows exponentially within this host and significantly shortens its lifespan in the absence of viral infection, demonstrating that harbouring this protective endosymbiont is associated with a fitness cost. Testing the expression of selected antimicrobial peptides showed that there is no difference between wMel and wAu. Therefore, we could not attribute either the difference in anti-viral effect or the lifespan-shortening to the immune activation by exogenous Wolbachia. Our work provides evidence that interspecies Wolbachia transfer is not always associated with general immune up-regulation in the recipient host.Our study uses wMelCS_b in the et al. 2013. All the remaining data, all statistical analysis and all conclusions are original.The data for iso and D. melanogaster with wMelCS_b DrosDel w1118isogenic flies and the matching controls without Wolbachia were described before D. melanogaster with wAu from D. simulans Coffes Harbour (CO) was described before st and 3rd chromosome of the D. melanogaster stock with wAu were replaced with DrosDel w1118isogenic chromosomes using a first and third double balancer line. Next, a second chromosome balancer line was used to replace the 2nd chromosome. As both Wolbachia and mitochondria are maternally transmitted the wAu, wMelCS_b and Wolbachia-free iso control lines may have different mitochondria, despite having the same nuclear genetic background. Cleaning the stocks of possible chronic viral infection and gut flora homogenization were performed as in Drosophila were maintained at a constant temperature of 25°C on standard cornmeal diet. All the experiments were performed on 3–6 days-old male flies.The lifespan of different fly lines was tested at 25°C, with 10 flies per vial, and analysed using Cox hazard models as previously reported Viruses were produced, titrated and used to infect flies as before Wolbachia was extracted using standard phenol-chlorophorm protocol. RNA for assessment of viral titres and gene expression was extracted using Trizol (Invitrogen) with an additional DNAse treatment (Promega) of the AMPs RNA samples prior to cDNA synthesis. cDNA was prepared as described previously Wolbachia, DCV and FHV were previously described Wolbachia variants titre over time was analysed using a linear model (lm) in R DNA for the quantification of Figure S1Statistical analysis of survival curves.  Hazard ratios between either iso Wolbachia-free control or wMelCS_b carrying line and wAu line for: (A) DCV infection, (B) FHV infection, (E) uninfected flies.  Hazard ratios between either iso or wMelCS_b tetracycline-treated line and wAu tetracycline-treated line for: (C) DCV infection, (D) FHV infection, (F) uninfected flies. In all panels error bars represent standard errors of the estimated hazard ratios. The only non-significant differences in Cox hazard ratios are: iso tet vs. wAu tet for DCV infection (C) and both iso tet and wMelCS_b tet vs. wAu tet for FHV infection (D).(TIF)Click here for additional data file.


Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions. S1P1-deficient precursors can differentiate normally within the thymus but are unable to exit the organ 1 expression during differentiation, and therefore mature single-positive (SP) cells expressing higher levels of the receptor are able to respond to S1P gradients In vivo, small concentrations of the natural S1P1 ligand, (S1P) promote lymphocyte migration, whereas therapeutic concentrations of FTY720 (a synthetic S1P agonist) inhibit lymphocyte egress from the thymus and peripheral lymphoid organs, inducing a cell sequestration in those organs and a profound lymphopenia Thymocyte migration is critical for normal T cell development and maturation. From the entrance of precursors into the thymus, to the migration within the organ and finally mature thymocyte egress, several molecules and receptors are implicated, including extracellular matrix (ECM) molecules, chemokines, sphingosine-1-phosfate (S1P) and their respective receptors. ECM proteins such as fibronectin and laminin are present in the thymus in different concentrations depending on the region. They are recognized by integrins constitutively expressed on thymocytes and microenvironmental cells. The ECM-integrin interactions induce cell adhesion and migration, and also mediate cell-cell interactions To add to the complexity of this regulatory network, some molecules initially described in the nervous system are also present in the thymus playing a role in T cell development and function. Neuropilins (NRPs) and semaphorins (SEMAs), initially known as modulators of axon guidance, angiogenesis, and organogenesis Human thymuses were obtained as a by-product of cardiac surgery performed on children aged from one week to 11 years of age at Necker Hospital. Participants and next of kin, caretakers, or guardians on the behalf of the minors/children participants provided their written informed consent to participate in this study, which was approved by the Necker Hospital Ethical Committees for human research and were performed according to the European Union guidelines and the declaration of Helsinki.1 and corresponding neutralizing peptide were from ABR Affinity BioReagents . Rabbit anti-cytokeratin immunoserum was a Dako product  and rhodamine/goat anti-rabbit Ig was from Sigma-Aldrich . Purified anti-SEMA3F was a Chemicon International product . Secondary PE/donkey anti-goat and FITC/donkey anti-rabbit antibodies were from Jackson ImmunoResearch Laboratories Inc. .The following antibodies were used in appropriate dilutions. APC/anti-CD4 and PerCP/anti-CD8 were purchased from Becton-Dickinson . Purified anti-neuropilin-2 and FITC/donkey anti-goat were obtained from Santa Cruz Biotechnology , whereas purified anti-S1PRecombinant human CXCL12 and mouse SEMA3F were from R&D Systems, whereas S1P was a Sigma-Aldrich product.Cells were washed and maintained in PBS for cell counting and subsequent staining. For intracellular staining, a PBS/BSA/saponin solution was used to dilute both primary and secondary antibodies. Cells were then evaluated by flow cytometry in a FACSCalibur or a FACSCanto II device ; analyses were done using a CellQuest or FACSDiva software (Becton-Dickinson).TGCCCAGCTACGACATGGA; NRP2 anti-sense: CAATCTCTCCGGAACGTCCTT; NRP2 probe CCAGATTGTGTTCGAGGGAGTGATAGGG; SEMA3F sense: TCGCCCCAAGCCACTGT; SEMA3F anti-sense: AGCGGTCCTCTGCACGAAT; SEMA3F probe: CAGCGAGATCCTGGTGACCGGC; S1P1 sense: GGCTCTCCGAACGCAACTT; S1P1 anti-sense: CAGGCTTTTTGTGTAGCTTTTCC; S1P1 probe: TTTCCGAGGCCCTCTCCAGCCA; ABL sense: TGGAGATAACACTCTAAGCATAACTAAAGGT; ABL anti-sense: GATGTAGTTGCTTGGGACCCA; ABL probe: CCATTTTTGGTTTGGGCTTCACACCATT. mRNA quality was assessed and normalized by quantification of Abelson (ABL) gene, using guidelines from the Europe Against Cancer program. Samples with an ABL cycle threshold (Ct) higher than 29 were excluded from analysis. Each experiment included two non-template controls for contamination and each sample was performed in duplicate.Following mRNA isolation and cDNA synthesis, RQ-PCR was done with TaqMan Universal PCR Master Mix  in a 7900HT Fast Real Time PCR System and analyzed in SDS 2.3 software (Applied Biosystems). Primers and probes were designed using the Primer Express software (Applied Biosystems). NRP2 sense: Human thymuses were frozen and sectioned (4 µm), and slides were incubated with BSA 1% blocking solution. Samples were subjected to primary antibodies overnight at 4°C or 1 h at room temperature followed by corresponding secondary antibodies for 30 min at room temperature. Stained samples were analyzed by confocal microscopy using a LSM 5 Pascal device (Germany). Negative controls, in which primary antibodies were replaced by unrelated immunoglobulins, or in which the secondary antibody was used alone, did not generate any significant fluorescent signal.6) in 100 µl of migration medium alone (RPMI/BSA 1%) or medium containing SEMA3F  as a chemorepulsive stimulus at 100 ng/ml were added in the upper chambers. After 4 h of incubation at 37°C in 5% CO2 humidified atmosphere, migration was defined by counting the cells that migrated to the lower chambers containing migration medium alone or the medium containing the chemoattractant molecules CXCL12 (200 ng/ml) or S1P (10 nM). Cells were then labeled with appropriate antibodies and analyzed by flow cytometry. In some experiments, cells were first incubated with anti-NRP2 blocking mAb (R&D Systems) before migration.Cell migratory activity was assessed in a transwell system. Inserts bearing 5 µm pore size in transwell plates  were treated with PBS/BSA 1%. Cells , 0.125 mg/ml L-alpha-phosphatidylcholine palmitoyl (Sigma-Aldrich), and 4.5% PFA in PBS. Staining was analyzed by flow cytometry. Mean fluorescence intensity (MFI) values obtained before addition of ligand were arbitrarily set at 100%.Cells were incubated in RPMI 1640 medium/20 mM HEPES. CXCL12 or SEMA3F were then added to the cell suspension. At each indicated time point (15 s to 2 min), an aliquot was taken from the cell suspension and mixed with the labeling buffer, consisting in 10Diagnosis was made on peripheral blood or bone marrow T-cell acute lymphocyte leukemia (T-ALL) samples (n = 136), as previously described . Tumor diagnostic samples from 52 patients with T-cell lymphoblastic lymphoma (T-LBL) were available for molecular analysis. All patients provided informed consent in accordance with the Declaration of Helsinki, and the study was in accordance with local and multicenter research ethical committee approval.t test, one-way ANOVA or the nonparametric Wilcoxon Mann-Whitney test. Differences were considered to be statistically significant when p<0.05 (*), p<0.01 (**) or p<0.001 (***).Results were analyzed by the parametric Student's We first observed that NRP2 and SEMA3F were constitutively expressed in developing human T cells in the thymus. The expression of both NRP2 and SEMA3F was widely observed in the epithelial cells (defined by cytokeratin staining) as well as in non-epithelial components in thymic sections , as well−CD8+ and CD4+CD8− cells as they become CD4highCD8− or CD4−CD8high  thymocytes expressed NRP2, whereas almost all DP cells expressed this receptor . NP2 exp−CD8high . SEMA3F olecules . It is iSEMA3F was first described as being chemorepulsive in the nervous system In contrast, CXCL12, acting through its receptor, CXCR4, is known to reduce axonal responsiveness to several known repulsive molecules, including SEMA3A Pre-treatment of thymocytes with the blocking anti-NRP2 mAb reverted the inhibition of CXCL12 chemoattraction caused by SEMA3F, showing that the response was specifically mediated by NRP2/SEMA3F interactions .Because SEMA3F modulated thymocyte migration, we analyzed the regulation of the cytoskeleton following CXCL12 and SEMA3F stimulus. As shown in 1 on thymocyte migration and egress has been well described in mice. Mouse thymocytes upregulate S1P1 expression as they maturate from DP to SP cells, making them more responsive to S1P, which is present at higher levels in blood allowing their egress from the thymus 1 is a G protein-coupled receptor (thus resembling to CXCR4) we hypothesized that SEMA3F could also block S1P1-induced migration of human thymocytes. We first analyzed S1P1 expression in human thymocytes and we observed higher levels of S1P1 expression on mature SP CD4+CD8− and CD4−CD8+ compared with immature DP CD4+CD8+ thymocytes  and T-cell lymphoblastic lymphoma (T-LBL) samples. T-ALL and LBL are malignant proliferations of T cell precursors whose differentiation is arrested and are thought to originate in the thymus It has been previously demonstrated that CXCR4 is expressed by malignant B and T-ALL, and the expression levels correlate with transmigration towards CXCL12 1. We observed that both neoplasias expressed S1P1 mRNA with no difference between T-ALL and T-LBL samples (1 (1.8 ratio S1P1/control media) and that such migratory response was inhibited 78% when SEMA3F was added  Box plot shows S1P1 mRNA expression analyzed by real time quantitative PCR, compared with the control Abelson (Abl) gene in T-ALL (n =  136) and T-LBL (n =  37) samples. Results were analyzed by the non-parametric Wilcoxon Mann-Whitney test. b) Bars represent migration of a T-ALL sample in a transwell system. Results are represented by migration ratio, and control without stimuli was normalized do 1.0.(TIF)Click here for additional data file.


We join the increasing call to take computational education of life science students a step further, beyond teaching mere programming and employing existing software tools. We describe a new course, focusing on enriching the curriculum of life science students with abstract, algorithmic, and logical thinking, and exposing them to the computational “culture.” The design, structure, and content of our course are influenced by recent efforts in this area, collaborations with life scientists, and our own instructional experience. Specifically, we suggest that an effective course of this nature should: (1) devote time to explicitly reflect upon computational thinking processes, resisting the temptation to drift to purely practical instruction, (2) focus on discrete notions, rather than on continuous ones, and (3) have basic programming as a prerequisite, so students need not be preoccupied with elementary programming issues. We strongly recommend that the mere use of existing bioinformatics tools and packages should not replace hands-on programming. Yet, we suggest that programming will mostly serve as a means to practice computational thinking processes. This paper deals with the challenges and considerations of such computational education for life science students. It also describes a concrete implementation of the course and encourages its use by others. The “cultural gap” between biological and computational sciences has become increasingly evident in recent years. Life sciences are going through a dramatic biotechnological revolution, producing huge amounts of data, which is often placed in public databases. The analysis of these data requires nontrivial computational ideas. Life sciences curricula, however, have hardly been altered to reflect this revolution This gap presumably starts at the classroom, but it lingers later on. Biology in many institutes and labs is still primarily a descriptive science with little computational approaches being used on a daily basis. Computational approaches in this context are not the mere use of tools, but the integration of computational thinking and algorithms to experiments design; to data generation, integration, and analyses; and to modeling. It is often the case that because of the lack of computational background and relevant training, bench biologists employ computational methods as “black boxes” without a deep understanding of the computational concepts, underlying assumptions, and the limitations of such models. The practice of employing computational methods in biology is usually done in one of two flavors: a somewhat “automatic” use of existing bioinformatics tools by biologists or the application of algorithms to biological data by computer scientists and mathematicians. Both modes may result in a misinterpretation of results and in erroneous conclusion making CS5 green” course PLOS Computational Biology is a notable resource for practical tutorials and opinions. Online courses, such as Rosalind , have started to appear, aiming to attract biologists who want to develop programming skills at their own pace. Several books about computational methods, aimed at biologists, have been published recently The majority of biological laboratories would greatly benefit from using computational tools on a daily basis and, consequently, from the presence of an “in-house” expert with a solid computational understanding. Indeed, the need to provide life science students with a wider, deeper computational education, beyond just hands-on skills, is being widely recognized We join the above-mentioned efforts. We urge such an educational revolution in life sciences and propose a novel, stand-alone, concrete educational building block: a non-introductory course, that aims to expose students to the computational “culture” and focuses on developing computational thinking skills http://ca4ls.wikidot.com), is targeted specifically for life science students, both advanced undergraduate and graduate. It is a non-introductory course—basic programming is a pre-requisite (see more details about this choice later). The course's primary goal is:The course we developed, titled “Computational Approaches for Life Scientists”  how relevant the topic is for research and practice in life sciences and (2) to what extent the topic can be harnessed to expose students to the computational “culture” and to practice relevant thinking skills. We tackle a wide spectrum of biological and computational issues, appealing to a fairly broad audience among life science students.Even though this is not a programming course, students are required to solve “real-life” biological problems using code. We introduce the programming language Python at the beginning of the course . It then serves as a vehicle to deliver course topics. While teaching Python, we focus on its practical use, rather than on language syntax and specifications (the latter are more likely to be emphasized in an introductory programming course). Our experience shows that when learning includes concrete, hands-on practice, computational thinking skills are better acquired and underlying concepts are better understood.One important choice in the course's design was to exclusively concentrate on discrete approaches such as finite graphs, strings, digital images (represented as a matrix of discrete elements—pixels), finite state automata, etc. These are highly underrepresented in life science curricula, in which continuous notions, such as derivatives, integrals, and differential equations, are taught more widely We choose a level of formalism that matches students' background. Obviously we do not use the same level of formalism as in “pure” CS courses. Nonetheless, we do insist on taking students out of their “cognitive comfort zone” in the sense that we expect them to handle abstract notions and to formalize their statements and algorithms in a rigorous and logical manner. Still, we leave ample time for classroom discussion and for developing intuition and try not to drift into a too-formal or technical instruction.Upon successful completion of the course, we expect students to:Be familiar with several fundamental concepts and notions in CS, and their applicability to life sciences. Be able to identify problems whose manual solution is not feasible, yet they are amenable to a computational solutionFeel comfortable to communicate with computational biologists/bioinformaticiansBe able to implement basic solutions to simple biological problems they encounter, and to effectively communicate with more experienced programmers for more complex problemsThe course was taught for the first two times in 2013 and 2014 at the Technion, Israel Institute of Technology, Faculty of Biology. In the first round of the course, it was taken for credit by five graduate level and three undergraduate level students. In the second round, it was taken by eight graduate level and nine undergraduate level students. All had elementary programming background in either C, Matlab, or Pascal . Participants were required to submit five home assignments, each including programming tasks and theoretical questions. In the first round, a take-home exam was given at the end, which was replaced in the second round by a final research project: students chose topics that they found interesting among the course subjects, extended them in some manner, and applied them to real biological data. Additional details regarding the projects, and specific project examples, appear in the supplementary To examine the effect of the course on how students view computer science, they were asked to define this discipline before and after the course. Prior to the course, students related the field mostly to the computer as a machine and to software and tools. At the end of the course, however, they tended to relate CS to broader and more abstract terms, such as problem solving and modeling see . We beliObviously, there is more than a single way to expose life sciences students to computational thinking. Yet, based on our experience, and on numerous discussions with life scientists and bioinformaticians, we feel that a single one-semester course, which does not assume a basic programming course as a prerequisite, is likely to miss the goal of teaching computational thinking and computational concepts to life science students. If basic programming is taught from scratch, not enough time will be left for the higher level computational concepts and their relations to biology, so the depth of coverage of computational thinking will be smaller. Alternatively, the use of packages could dominate the hands-on experience, “masking” the computational ideas. On the other hand, having such a basic programming prerequisite, as in our course, enables us to take the students a step further, beyond programming and tool handling. This facilitates exploring abstract computational notions, experimenting “first hand” with coding them, and applying the code to concrete biological tasks. We believe that these days, a basic programming course is a crucial component of every science curricula. This leads to the recommendation that basic programming should be taught separately, prior to a computational thinking course. Such a prerequisite will allow the students to digest programming issues well before, so they need not be preoccupied with technical issues while taking a computational thinking course. Furthermore, we feel that the understanding of computational thinking and the “language” of CS are hard to obtain independently. While a student “speaking” this language can easily educate him/herself in the use of bioinformatics tools, the other direction is far less amenable to self-study. Teachers engaged with computational education for biologists are sometimes tempted to make their course as practical as they can (and many students feel more comfortable staying away from abstract topics). While practical skills are, of course, important and motivating, we believe that time and educational effort must be spent on abstract notions and thinking processes: naming, discussing, and reflecting upon them.Most of these conclusions are supported by the surveys and interviews conducted among course students during the two semesters it was taught. Clearly, a more in-depth evaluation of the course, based on a larger number of participants, is called for. This is planned to take place in future offerings of the course.In our view, an essential part of any course aiming to teach computational thinking to life scientists is the interaction in class, with an able instructor who is knowledgeable in both computer and life sciences. Class interactions in the form of discussions, guided solutions to problems, naming of thinking processes, and exposure of students to alternative (including incorrect) approaches are at the heart of the learning process in this course. Our four-step pipeline instruction model prevents spending too much time on technical aspects since part of the time is explicitly dedicated to reflection and discussion in class.We strongly believe that we have an important message to deliver. We propose a way to take life scientists' computational education a step further. Even small steps in this direction are likely to have substantial consequences in life or medical science practices and research in the long run. Such initiatives can greatly contribute to narrowing the gaps between life sciences and bioinformatics/computational biology and motivate other scientists and science education experts to be involved in similar efforts.Figure S1A “path” through three topics in the pipeline structure.(TIF)Click here for additional data file.Figure S2(A) A microscope slide containing Bacilli anthracis cells and spores (image taken from (TIF)Click here for additional data file.Figure S3Students' attitudes towards home assignments difficulty and effectiveness.(TIF)Click here for additional data file.Text S1A path through three topics in the pipeline structure of the course.(DOCX)Click here for additional data file.Text S2Examples for end of course projects.(DOCX)Click here for additional data file.Text S3Feedback from course students.(DOCX)Click here for additional data file.


CaSm induction decreased gemcitabine-induced cytotoxicity and altered the expression of apoptotic regulation genes, including Bad, E2F1 and Bcl-XL. CaSm-overexpressing Panc-1 cells were twofold more migratory and fourfold more invasive than the driver controls and demonstrated characteristics of epithelial-to-mesenchymal transition such as morphological changes and decreased E-cadherin expression. CaSm induction resulted in changes in RNA expression of metastasis-associated genes such as MMP1, SerpinB5, uPAR and Slug. Using a murine model of metastatic pancreatic cancer, injection of CaSm-induced Panc-1 cells resulted in a higher abundance of hepatic metastatic lesions. Overall, CaSm overexpression contributed to a more aggressive cancer phenotype in Panc-1 cells, further supporting the use of CaSm as a therapeutic target against pancreatic cancer.The cancer-associated Sm-like (CaSm) oncogene is overexpressed in 87% of human pancreatic tumor samples and CaSm knockdown has demonstrated therapeutic efficacy in murine models of pancreatic cancer. Evidence indicates that CaSm modulates messenger RNA degradation; however, its target genes and the mechanisms by which CaSm promotes pancreatic cancer remain largely unknown. Here, we demonstrate that the CaSm overexpression alters several hallmarks of cancer—including transformation, proliferation, chemoresistance and metastasis. Doxycycline-induced CaSm expression enhanced proliferation and both anchorage-dependent and -independent growth of the human Panc-1 cells  Given that most patients present with more advanced disease, and in the face of recent evidence suggesting that dissemination occurs even before primary pancreas tumor formation,7 further research is necessary to investigate what drives metastasis in this disease.Pancreatic adenocarcinoma (PC) is the fourth leading cause of cancer-related deaths in the nation. Owing to its aggressive nature and difficult diagnosis, the majority of patients present with metastatic disease and generally demonstrate poor response to conventional chemotherapy and radiation.8 Also identified as human LSm1, CaSm is a key member in the LSm1–7 complex where it forms a heteromeric ring with six other members of the LSm family. Given its location in cytoplasmic foci along with decapping enzymes Dcp1/2 and exonuclease Xrn1, this LSm complex is hypothesized to facilitate 5′–3′ degradation of cytoplasmic messenger RNA (mRNA).13 Consistent with this hypothesis, previous studies have shown that increased CaSm levels results in multiple genetic alterations—including apoptotic genes, several signaling pathways and cell cycle mediators.15The cancer-associated Sm-like oncogene (CaSm) has been shown to be overexpressed in nearly 90% of human pancreatic tumor samples as well as several other cancer histopathologies.16 Furthermore, decreasing CaSm expression in a murine PC model resulted in decreased in vivo tumor formation and increased survival,17 whereas combinatorial antisense CaSm and gemcitabine therapies additively enhanced survival in a subcutaneous PC mouse model.18 In short, CaSm is a promising target for a difficult cancer that has had few new therapies developed in the past decade.Our laboratory has investigated the potential use of CaSm as a therapeutic target and shown that CaSm expression is necessary for cancer formation in mice.Beyond the LSm1 correlates, relatively little remains known about the role for CaSm in the process of neoplastic transformation or how its downregulation in the setting of established neoplasia leads to decreased tumor growth. To explore this further, we developed models that would allow the induction of CaSm overexpression in a PC cell line intrinsically expressing relatively low CaSm levels. Here, we demonstrate that induced CaSm expression results in increased growth, decreased chemotherapeutic sensitivity and enhanced migration/invasion in PC cells. CaSm upregulation alters the gene expression of critical mediators of apoptosis, metastasis and epithelial-to-mesenchymal transition (EMT), which complements the proposed function for CaSm in mRNA regulation and provides a putative mechanism for CaSm-mediated neoplastic progression.CaSm protein expression was evaluated via western blot analysis in a panel of human PC cell lines to identify a cell line with lower levels of basal expression. CaSm expression was variable among the cell lines, with Panc-1 cells demonstrating the lowest endogenous levels . Owing t18 Reciprocally, induced CaSm expression significantly enhanced cellular growth twofold compared with driver and non-induced controls  mice received subcutaneous dorsal injections of 1 × 105 and 2 × 106 tet-on driver or tet-on CaSm cells and tumor presence was examined biweekly for 8 weeks by palpation. Although nearly all of the mice established tumors during the course of the study, the tet-on CaSm cells formed tumors more readily at both cell concentrations  to characterize and quantify the metastatic hepatic lesions. Histological analysis showed differences in growth patterns of the liver lesions between the two conditions; whereas the driver controls resulted in tight and condensed lesions, the tet-on CaSm cells grew into more diffuse tumors with additional single-cell or small-cell clusters throughout the liver tissue  and the majority of metastatic liver lesions  overexpressing CaSm mRNA compared with normal islet cells.33 Despite this, a direct association linking CaSm overexpression and metastatic pathways was previously unexplored. The data presented here support the possibility that CaSm mediates metastasis through Slug overexpression, which was demonstrated to have a direct role in the migratory capabilities of the CaSm-induced Panc-1 cells. Alterations in MMP1, uPAR and SerpinB5 expression, all of which are associated with invasion and metastasis in PC,39 may also explain the observed CaSm-enhanced invasion and migration. Additional investigation is necessary to determine whether CaSm directly affects the RNA stability of MMP1, Slug, uPAR and SerpinB5 and to further assess the necessity of each of those genes in our observed phenotypes. Also, given that Slug knockdown reduced cellular migration to baseline control levels, it should be investigated whether MMP1, SerpinB5 and uPAR individually contribute to migration and invasion and whether these changes in expression are directly facilitated by CaSm or perhaps downstream of CaSm-mediated Slug overexpression. Provided that Slug expression correlated with CaSm in both PC patient arrays and given the efficacy of Slug siRNA to reduce CaSm-mediated migration, it would also be interesting to investigate using this gene as a therapeutic target to prevent metastatic disease.In addition to building upon previous reports that CaSm is involved in proliferation,15 Interestingly, there is no overlap comparing individual genes between the two studies,15 possibly due to the limited focus of our study or due to differences in expression between breast and pancreatic cancer cells.Given the putative function of CaSm in RNA decapping and stability, it is not surprising that microarray results showed that CaSm induction affected expression within multiple cancer-associated pathways such as apoptosis and invasion. Additional cancer-related pathways that appear to be altered following CaSm induction include transforming growth factor-β, tumor necrosis factor and vascular endothelial growth factor, which are known to contribute to EMT/metastasis, cachexia and angiogenesis, respectively. Further work is needed to validate these genetic alterations and evaluate their relative importance in our functional observations. Previous studies of CaSm upregulation in a breast cell line demonstrated similar results with altered expression of genes associated with apoptosis, mitogen-activated protein kinase and transforming growth factor-β signaling pathways.28 Studies in MCF10a and Sum44 human mammary cell lines further support that CaSm overexpression promotes proliferation and transformation, potentially through modification of cancer-related gene expression.15 In addition to this cell-based data, human pancreatic cancer microarray data indicate that CaSm expression correlates with several of the genes discussed here, notably Slug, MMP1 and uPAR signaling pathways. It is interesting to note that in both patient-based arrays, CaSm expression correlated with SerpinB5; whereas this contradicts the inverse relation identified through the microarray, SerpinB5 is commonly upregulated in pancreatic cancers,40 although known to be absent or downregulated in human breast, prostate and gastric cancers,41 indicating that, in addition to CaSm, other cancer-related pathways are likely involved in regulating expression of SerpinB5 in human pancreatic cancer.CaSm was ubiquitously expressed in all of our human PC cell lines, attesting to its importance in PC development while limiting our experiments to the Panc-1 cells, which comparatively expressed the lowest amounts of CaSm protein. This work builds on previous studies investigating the effect of CaSm knockdown in human (AsPC-1 and Capan-1) and murine (Panc02) pancreatic cancer cell lines, in addition to studies in prostate and lung cancer cell lines.in vivo, demonstrating that CaSm overexpression, believed to be an early event in PC progression, contributes to the highly metastatic and deadly nature of the disease. In addition, mice injected with tet-on CaSm cells presented with significant weight loss compared with mice injected with driver controls, further indicating that CaSm contributes to metastatic development, as cachexia indicates advanced metastatic disease and poorer prognosis in PC.43Importantly, we were able to confirm that the molecular changes associated with upregulation of CaSm significantly contributed to tumor formation and dissemination of the Panc-1 cells These studies provide important insight on the role of CaSm-mediated neoplastic progression and present potential pathways for the use of CaSm as a therapeutic agent. The pluripotent changes resulting from CaSm overexpression are consistent with current mechanistic evidence, which implies that CaSm is involved in facilitating mRNA decapping and degradation, with altered CaSm expression levels having the ability to lead to alterations in mRNA expression. Taken together, this information may be used to improve CaSm knockdown gene therapy or to find alternative measures to combat CaSm-mediated tumor progression in pancreatic cancer.2. Cells were regularly screened for mycoplasma contamination using the MycoAlert Detection Kit .Human PC cells were obtained from ATCC  and maintained in RPMI  or Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  at 37 °C in 5% COThe full-length human CaSm DNA sequence was cloned into pRetro-X-Tight-Pur plasmid . Panc-1 cells were transfected with the pRetro-X Tet-on-Advanced plasmid (Clontech) using Lipofectamine2000  according to the manufacturer's protocol. Stable clones and pools were selected by G418 antibiotic resistance  in complete media and then tested for doxycycline inducibility following a transient transfection with the pRetro-X-Luc control vector. The pool with the highest induction was used as the driver control cell line. A subset of this pool was co-transfected with the pRetro-X-Tight-Pur-CaSm plasmid and a stable pool obtained by G418 and puromycin  antibiotic selection. CaSm expression was confirmed using real-time PCR and western blot, demonstrating low basal levels and efficient induction of the pool. Tet-on driver Panc-1 cells were maintained in DMEM media supplemented with 10% tetracycline-screened FBS (Hyclone), 1 mg/ml G418, and 1 μg/ml doxycycline . Tet-on CaSm Panc-1 cells were propagated in DMEM media supplemented with 10% tetracycline-screened FBS, 1 mg/ml G418, 1 μg/ml puromycin and 1 μg/ml doxycycline. Unless otherwise noted, experiments were initiated using cells grown in doxycycline-containing media for 4–9 weeks.28 anti-E-cadherin  and anti-N-cadherin . Anti-GAPDH  was used as loading control. The membrane was incubated in horseradish peroxidase-conjugated secondary antibodies for 1 h. Goat-anti-IgY  was used as secondary antibody against the CaSm antibody, horse-anti-mouse IgG  against the E- and N-cadherin antibodies, and goat-anti-rabbit-IgG  against the GAPDH antibody. West Pico Chemiluminescent reagent  was used to develop the membrane that was then was exposed to CL-Xposure film (ThermoScientific).Whole-cell lysates were obtained using RIPA buffer  and protease inhibitors (Sigma). Protein (25–30 μg) was separated on a 12.5% polyacrylamide gel before being transferred to a Hybond ECL nitrocellulose membrane . The following primary antibodies were used for overnight incubations, diluted in 5% non-fat milk: anti-CaSm IgY ,For cell growth assays, 100 000 tet-on driver and tet-on CaSm Panc-1 cells were plated in triplicate in six-well plates on day 0. On days 1, 3 and 4, cells were trypsinsized, neutralized with FBS-containing media, stained with 0.4% Trypan blue solution (Sigma) and quantified by hemocytometer (with at least 95% viability).Fifty or 100 tet-on driver and CaSm Panc-1 cells were plated in single-cell suspension in triplicate in six-well plates. Media was refreshed twice weekly. After 14 days in culture, cells were fixed in 10% buffered formalin and stained with 2.5 mg/ml crystal violet in 2% ethanol. The number of colonies with >50 cells was recorded.A 0.6% agar base was applied to six-well plates and allowed to solidify. Tet-on driver and tet-on CaSm Panc-1 cells were suspended in 0.4% agar in DMEM media supplemented with 1 μg/ml doxycycline and appropriate selection antibiotics and overlaid atop the base layer. Cells were plated at concentrations of either 500 or 1000 cells per well and fed weekly with 0.4% agar containing media and appropriate selection antibiotics. After 5 weeks, cells were stained with 2.5 mg/ml crystal violet in 2% ethanol for 1 h and destained with water. Colonies >0.1 mm were counted in triplicate wells.M gemcitabine  with replicates six wells per condition. Ninety-six hours after treatment, cells were fixed with 5% trichloroacetic acid (Sigma) for an hour, stained with 0.4% SRB (Sigma) for 30 min and dissolved in 10 mM Tris Base before absorbance was read at 560–564 nm. Percent survival was determined by calculating the absorbance of treated cells to respective untreated controls.Tet-on driver or CaSm Panc-1 cells were seeded in each well of a 96-well plate (5000 cells per well) and incubated overnight. Cells were then treated with 0–100 μM gemcitabine. Cells were harvested on days 0, 1, 3 and 5 and fixed in ice-cold 70% ethanol overnight before resuspension in 0.1 μg/μl propidium iodide (Sigma) and 0.2 μg/μl RNase A (Sigma) followed by flow cytometry cell cycle analysis with the LSRFortessa . Apoptosis was determined as the percentage of cells with sub-G1 content.Tet-on driver and tet-on CaSm Panc-1 cells were seeded overnight in duplicate in a six-well plate (100 000 cells per well) before being treated with 200 n2 First Strand kit and RT2 SYBR Green qPCR master mix was used for the microarray master mix reagent according to the manufacturer's manual . The Cancer PathwayFinder PCR array (SABiosciences) was run on the Roche 480 Lightcycler  with two plates run for each tet-on driver+doxycycline, tet-on CaSm and tet-on CaSm+doxycycline. The plates included controls for genomic DNA contamination, reverse transcription and positive controls. Analysis was performed using SABioscience's Free PCR Array Data Analysis Software (ΔΔCt method) with B2M, HPRT1 and GAPDH as pooled housekeeping genes. The raw and normalized data are available on the GEO Omnibus repository (accession code GSE74319).Tet-on driver and tet-on CaSm Panc-1 cells were grown with and without doxycycline (1 μg/ml) for 4 weeks. Levels of CaSm expression were confirmed using reverse transcriptase–PCR . RNA isoRNA was isolated with the RNeasy Plus kit (Qiagen) and complementary DNA synthesis performed using iScript cDNA synthesis kit  with 1 μg of RNA template. Real-time PCR was performed with 2.5 μl of a 1:10 dilution of complementary DNA using 5 μl Lightcycler 480 Probes Master mix (Roche), 0.1 μl fluorescent probe (Roche) and 0.5 μM forward and reverse primers (Sigma). The following cycling conditions were used: pre-incubation at 95 °C for 10 min, 40 cycles of denaturing at 95 °C for 10 s, annealing at 55° for 10 s and extension at 72 °C for 30 s. A single data acquisition occurred at the end of each extension period. Relative expression was calculated using ΔΔCt, with values normalized to GAPDH.Twenty-four-well cell culture inserts with 8.0 um pores (BD Biosciences) were coated with 15 μg/ml fibronectin (ThermoScientific) overnight at 4 °C. Tet-on driver and tet-on CaSm Panc-1 cells were plated in serum-free DMEM onto the top chamber of the fibronectin-coated chambers (20 000 cells per well); DMEM supplemented with 10% FBS was used as the chemoattractant in the bottom wells. The migration chambers were incubated at 37 °C for 4 h. At this point, non-migrated cells were removed by gently wiping the top chamber three times with a clean cotton swab. Diff-Quick kit  was used to fix and stain the migrated cells. Cells were plated in triplicate and counted at × 40 magnification with at least five fields counted per chamber.Invasion assays were performed using 24-well BioCoat Matrigel Invasion Chambers (BD) according to the manufacturer's instructions. Matrigel chambers were rehydrated as directed before plating 15 000 tet-on driver or tet-on CaSm Panc-1 cells in serum-free DMEM onto the top chamber the inserts; DMEM supplemented with 10% FBS was used as the chemoattractant in the bottom of the wells. After 48-h incubation at 37 °C, non-invading cells on the top of the insert membrane were gently scrubbed away before invasive cells were stained with Diff-Quick and counted at × 40 magnification. Cells were plated in triplicate with three fields counted per chamber.Tet-on driver and CaSm cells were plated in six-well plates and allowed to reach 40% confluence. Tet-on CaSm cells were either left untreated or transiently transfected with 5 μg scramble  or Slug siRNA (Invitrogen HSS185949) using Lipofectamine RNAiMAX (Invitrogen). Seventy-two hours after transfection, cells were trypsinized for real-time PCR and migration assays as described above.Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from Jackson Labs  and maintained in DLAR facilities with food and water provided ad libitum. All murine experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina.Six-to-eight-week old NOD.Cg-5 (n=10 for both tet-on driver and tet-on CaSm) or 2 × 106 (n=9 tet-on driver and n=10 tet-on CaSm) cells in 200 μl phosphate-buffered saline on day 0. Animals were randomly assigned to groups during the time of cell injections. Doxycycline feed  was provided to all animals ad libitum beginning on day 0. Tumor presence was evaluated by palpation twice a week, beginning on day 7. All cells were expanded in the presence of 1 μg/ml doxycycline for 2–3 weeks before injection. No animals were excluded over the course of the study.NSG mice received dorsal subcutaneous injections of 1 × 106 cells in 0.05 ml phosphate-buffered saline (Hyclone)) were injected into the exteriorized spleen using a sterile 28-gauge needle and hypodermic syringe. Animals were randomized into groups at the time of injection (n=6 for each tet-on driver and tet-on CaSm Panc-1 cells). Cells had previously been maintained in 1 μg/ml doxycycline for 3 weeks before injection. Doxycycline feed  was provided ad libitum to all animals beginning on day 0. Animal weights were taken at the time of tumor injection and weekly beginning at week 3; no animals were excluded over the course of the study. Six weeks after injection, mice were killed and livers and spleens resected. Liver tissue was fixed overnight in 10% buffered formalin, set in paraffin blocks and cut into 8 μm thick sections , with one section cut every 500 μm for five total sections per sample. Sections were stained with standard hematoxylin and eosin staining by the Tissue Biorepository Core facility. Hematoxylin and eosin images were analyzed at × 40 magnification with at least five fields per section. Tumor area was quantified on the images using ImageJ software . The number of hepatic lesions/image was evaluated and normalized to the total tissue area of the image by a blinded investigator.NSG mice were anesthetized using 2% isofluorane  in 1.5 l/min oxygen and received a single, preemptive analgesic of 3 mg/kg carprofen . The left flank was shaved and sterilized with Betadine . Once appropriate anesthesia was achieved, 1 cm left subcostal incision was made through the abdominal skin and peritoneum. Tet-on driver or tet-on CaSm Panc-1 cells in . For the metastatic model, at least four animals per group were needed to achieve 80% power, assuming that the tet-on CaSm cells would form twice the number of liver metastases as the tet-on driver controls (4.0±1.0 mets/100 mm2 compared with 2.0±1.0 mets/100 mm2). In all studies, P-values <0.05 were considered significant.Statistical analysis was performed using GraphPad Prism 5 software . Proliferation and SRB assays were analyzed using two-way analysis of variance, and tumor-free survival was analyzed using the log-rank Mantel Cox test. Two-tailed unpaired Student's


ErgcEC-KO) in mice causes embryonic lethality with vascular defects. Inducible endothelial deletion of ERG (ErgiEC-KO) results in defective physiological and pathological angiogenesis in the postnatal retina and tumors, with decreased vascular stability. ERG controls the Wnt/β-catenin pathway by promoting β-catenin stability, through signals mediated by VE-cadherin and the Wnt receptor Frizzled-4. Wnt signaling is decreased in ERG-deficient endothelial cells; activation of Wnt signaling with lithium chloride, which stabilizes β-catenin levels, corrects vascular defects in ErgcEC-KO embryos. Finally, overexpression of ERG in vivo reduces permeability and increases stability of VEGF-induced blood vessels. These data demonstrate that ERG is an essential regulator of angiogenesis and vascular stability through Wnt signaling.Blood vessel stability is essential for embryonic development; in the adult, many diseases are associated with loss of vascular integrity. The ETS transcription factor ERG drives expression of VE-cadherin and controls junctional integrity. We show that constitutive endothelial deletion of ERG (  •Inducible deletion of endothelial ERG in mice causes defective angiogenesis•ERG controls vascular stability through Wnt/β-catenin signaling•β-catenin activation rescues the angiogenic defect in vivo in ERG-deficient mice•Overexpression of ERG in vivo stabilizes VEGF-induced angiogenesis Birdsey, Shah et al. show that the endothelial ETS factor ERG controls Wnt/β-catenin signaling by promoting β-catenin stability, through pathways mediated by VE-cadherin and the Wnt receptor Frizzled-4. In vivo, ERG overexpression stabilizes VEGF-dependent angiogenesis. Thus, ERG is an essential regulator of angiogenesis and vascular stability through Wnt signaling. Angiogenesis is essential during embryogenesis and is a critical component of many diseases. Coordination of growth and stability signals is required for effective angiogenesis . DiseaseMultiple interactions at endothelial cell-cell junctions control vascular integrity. Crucial among these are the adhesion molecule vascular endothelial (VE)-cadherin and its intracellular partner β-catenin, an essential component of the canonical Wnt pathway -dependent angiogenesis and promotes stability of VEGF-induced new blood vessels.Cre/loxP strategy. The floxed allele of Erg was obtained by inserting two loxP sites flanking exon 6  resulted in embryonic lethality between embryonic day (E)10.5 and E11.5, with no live offspring -iCreERT2) (Erg was confirmed by PCR in Ergfl/fl/Cdh5(PAC)-iCreERT2 mice (henceforth referred to as ErgiEC-KO) (Ergfl/fl), ERG was strongly expressed in endothelial cells (EC) from all regions of the vascular plexus, including tip and stalk cells, arteries, veins, and capillaries  . FollowigiEC-KO) H. The regiEC-KO) I and S1Jillaries K, in linillaries . Deletiocoverage C and dencoverage D in the coverage E. Next, coverage . At day controls F–1H. TheErgiEC-KO mice compared to controls, indicating increased vessel regression. Pericyte recruitment, measured by staining with neuron-glial antigen 2 (NG2) and desmin, was significantly decreased along all vessels in the vascular plexus, including veins and arteries in ErgiEC-KO mice   A showed cEC-het) B, as expAt the junctions, VE-cadherin binds β-catenin, protecting it from degradation; this interaction is required for the control of junction stabilization . β-catenErgcEC-het mice  to compare the data set from transcriptome profiling of ERG-deficient HUVEC  with theSince ERG inhibition decreases β-catenin protein, but not mRNA levels, we tested whether ERG regulates β-catenin degradation. β-catenin protein expression was restored in ERG-deficient EC in the presence of the proteosomal degradation inhibitor MG132 A. We invErgcEC-het mice  region upstream of the Fzd4 transcription start site F and S4DWnt/β-catenin signaling can promote EC proliferation  and induTo test the functional relevance of Wnt signaling in ERG-dependent angiogenesis, we used an in vitro sprouting assay . ERG-defErgcEC-KO mutants following LiCl treatment  or Lacz (Ad.Lacz) adenovirus; basic fibroblast growth factor (bFGF), which can induce stable new vessels in this model , caused embryonic lethality at a later stage (E13.5)   and did Using the inducible endothelial specific Cdh5(PAC)-iCreERT2 line, we show that ERG is required for angiogenesis in the developing retina of newborn mice and for tumor blood vessel growth in adult mice. ERG deficiency results in vessel regression and reduced pericyte recruitment, demonstrating that ERG controls vascular stability. Interestingly, ERG overexpression in the in vivo Matrigel plug model resulted in increased pericyte recruitment to vessels. This suggests that ERG may promote stabilization of angiogenesis in part through control of pericyte recruitment. A recent paper has described a role for ETS factors (including ERG) in arterial specification and reported increased ERG expression in arterial-derived EC in vitro . HoweverErgcEC-KO embryos are reminiscent of those associated with deletion of endothelial β-catenin. Endothelial deletion of ERG causes embryonic lethality earlier than the E12.5 reported for endothelial deletion of β-catenin -CreERT2  by intraperitoneal injection (IP) at postnatal (P) day 1, P2 and P3. Retinas were collected at P6 and processed as described .Ergfl/fl and ErgiEC-KO mice as described  was injected IP into pregnant female mice at E8.5 and E9.5. Embryos were harvested at E10.5 and yolk sac vasculature was analyzed by light microscopy and immunostaining.165 (Peprotech) containing 109 plaque-forming unit adenovirus expressing either Lacz or ERG. After 3, 7, or 10 days, 100 μl of a 1:1 mixture of 10 mg/ml Dextran:FITC (2×106 MW) and Dextran:TRITC (4.4×104 MW) was injected intravenously 15 min prior to harvesting plugs. Plugs were imaged whole-mount using confocal microscopy. Volocity software (Perkin Elmer) was used to reconstruct 3D images of the vessels from serial Z-sections. The extent of TRITC-dextran tracer extravasation was quantified by subtracting the signal corresponding to the FITC-dextran tracer (intravascular) from the signal corresponding to the TRITC-dextran tracer (intravascular + extravascular).C57BL/6 mice received a subcutaneous injection of Matrigel (BD Biosciences), as described . MatrigeErgfl/fl and ErgcEC-het mice as described (Primary mouse lung EC were isolated from control escribed . Rat APCERG expression was inhibited using either GeneBloc antisense oligonucleotides (Silence Therapeutics) (ERG (Hs_ERG_7); both denoted as siERG in the text. Control GeneBloc antisense or AllStars Negative Control siRNA (QIAGEN) are denoted as siCtrl.Primary HUVEC were harvested from umbilical cords . Human Epeutics)  or siRNAChIP was performed using ChIP-IT express (Active Motif) as previously described .For Fzd4 transactivation assays, a 1,010-bp region of the Fzd4 promoter  was cloned into the pGL4 Luciferase Reporter Vector (Promega). TCF reporter constructs TOPFLASH and FOPFLASH were used to measure the transcriptional activity of β-catenin/TCF . In someCell proliferation was determined in vitro using a BrdU proliferation ELISA kit (Roche) according to the manufacturer’s instructions.The 3D in vitro model of angiogenesis was performed as described previously .Values are presented as means ± SEM. Statistical significance was determined by using unpaired two-tailed Student’s t test. Differences were considered significant with a p value < 0.05.G.M.B. designed, carried out, and supervised in vivo and in vitro experiments, analyzed and interpreted results, and wrote the manuscript. A.V.S. designed and carried out in vitro experiments, analyzed, interpreted, and conceptualized results, and wrote the manuscript. N.D. designed and performed in vivo experiments, analyzed, and interpreted results. B.G. provided advice on bioinformatic analysis and interpretation and contributed to scientific discussion; Y.Y. performed bioinformatic analysis and interpretation; and L.R. and K.H.D. performed and supervised the tumor angiogenesis experiments, analyzed results, and contributed to scientific discussion. L.O.A. and S.T.K. performed experiments and analyzed results. I.M.A. provided advice on retina isolation and optimized ERG retinal staining; E.D. provided reagents and contributed to scientific discussion; J.C.M. contributed to scientific discussion; H.G. provided advice and contributed to scientific discussion; R.H.A. assisted in the design of the transgenic mice, provided reagents, advice, and contributed to scientific discussion; and A.M.R. provided funding, conceived, designed, and supervised the study, interpreted results, and wrote the manuscript.


BAMBI) is regarded as an essential regulator of cell proliferation and differentiation that represses transforming growth factor-β and enhances Wnt/β-catenin signaling in various cell types. However, its role in skeletal muscle remains largely unknown. In the current study, we found that the expression level of BAMBI peaked in the early differentiation phase of the C2C12 rodent myoblast cell line. Knockdown of BAMBI via siRNA inhibited C2C12 differentiation, indicated by repressed MyoD, MyoG, and MyHC expression as well as reductions in the differentiation and fusion indices. BAMBI knockdown reduced the activity of Wnt/β-catenin signaling, as characterized by the decreased nuclear translocation of β-catenin and the lowered transcription of Axin2, which is a well-documented target gene of the Wnt/β-catenin signaling pathway. Furthermore, treatment with LiCl, an activator of Wnt/β-catenin signaling, rescued the reduction in C2C12 differentiation caused by BAMBI siRNA. Taken together, our data suggest that BAMBI is required for normal C2C12 differentiation, and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.Bone morphogenic protein and activin membrane-bound inhibitor ( BAMBI) is a transmembrane protein composed of 261 amino acids in mice, which shares high similarity with the transforming growth factor-β (TGF-β) family type I receptor in their extracellular domains, while lacking an intracellular kinase domain [BAMBI has been reported to promote the invasion, migration, and proliferation of cancer cells [BAMBI significantly inhibits the expression of carcinoma-associated fibroblast markers in human bone marrow mesenchymal stem cells without affecting their stem cell and tumor-tropic properties [Bone morphogenic protein and activin membrane-bound inhibitor  transcriptional complex . Wnt3a cem cells . MyoD isactivity .BAMBI expression significantly inhibited the nuclear translocation of β-catenin and the transcription of Axin2, two indicators of Wnt/β-catenin activity [It is well known that BAMBI exerts its functions through several signaling pathways, such as blocking TGF-β/Smad  and actiactivity ,23,24. Sactivity ,6. The iactivity ,26. ThesBAMBI expression following the treatment of C2C12 cells with LiCl, in accordance with previous results in Simpson–Golabi–Behmel syndrome preadipocytes [BAMBI was previously identified as a downstream target of Wnt/β-catenin signaling in several other cell types [Notably, we did not observe any change in ipocytes , althougll types ,27. ThesTo further elucidate the mechanisms of BAMBI’s functions in regulating myogenic differentiation, direct knockdown of TCFs or specific inhibitors against the physical interaction between β-catenin and TCFs may be among the ideal methods to use in future research. Wnt signaling represents a gigantic regulatory network that consists of more than 40 components, and the relationship between myogenesis and Wnt signaling is far from being fully elucidated ,16. The v/v) fetal bovine serum , 2 mM L-glutamine, and 1% (v/v) penicillin/streptomycin solution at 37 °C and 5% CO2. When the cell density reached 90%, the growth medium was replaced with differentiation medium, which was DMEM medium containing 2% (v/v) horse serum (Invitrogen), 2 mM L-glutamine, and 1% (v/v) penicillin/streptomycin solution. All the used cells were at similar passages (P5–7). Media were changed every two days. LiCl  was diluted to 1 M using ultrapure water.C2C12 cells   were designed and purchased from Invitrogen (® I reduced serum medium (Invitrogen) for 4 h prior to transfection. Lipofectamine® 2000 transfection reagent (Invitrogen) and siRNA or NC were gently mixed according to the manufacturer’s protocol, and then added to the culture supernatant. After 6 h of incubation, Opti-MEM I medium was replaced with fresh DM.vitrogen . When th® reagent  was applied to extract cellular total RNA. The RNA quality and concentration were estimated using agarose gel electrophoresis and spectrophotometry , respectively. The total RNA was processed into single stranded cDNA using a reverse transcription kit (TaKaRa Bio). Real-time quantitative PCR was performed using a SYBR® green kit (TaKaRa Bio) on a Bio-Rad iQTM5 system . GAPDH was used as a housekeeping gene for normalizing the expression of other genes. The 2Ct−∆∆ algorithm was employed to estimate the relative expression level of each gene. The sequences of primers can be found in Trizolw/v) defatted milk at room temperature for 2 h, and then incubated at 4 °C overnight with primary antibodies, including MyoD , sc-760, Santa Cruz Biotechnology, Dallas, TX, USA), BAMBI, MyHC , β-catenin , laminB1 , and GAPDH . The next day, the membranes were washed with TBST and incubated with goat anti-mouse or anti-rabbit secondary antibodys  at room temperature for 2 h. The membranes were washed three times with TBST and then exposed using a ChemiDoc XRS imaging system (Bio-Rad). The captured images were analyzed by Quantity One 4.6.3 software (Bio-Rad). The protein level of whole cell lysates was normalized against the expression of GAPDH, whereas the protein level of nuclear lysates was normalized against the expression of laminB1.Total and nuclear proteins of C2C12 cells were extracted using kits from Vazyme, Nanjing, China. The protein concentration was determined using the bicinchoninic acid assay kit (Vazyme), and then 5× protein loading buffer was added to the lysates prior to full denaturation in boiled water for 10 min. A total of 20 µg of protein was electrophoresed on a 12% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane . The membrane was blocked in 5% (w/v) paraformaldehyde for 15 min. 0.5% (v/v) Triton™ X-100 was used for permeabilization. The cells were then blocked in 0.5% (w/v) bovine serum albumin diluted in PBS. After blocking, the cells were incubated first with anti-MyHC antibody  at 37 °C for 2 h, and then with red fluorescence-labeled secondary antibody  at 37 °C for 1 h. The nuclei were stained with DAPI  for 10 min. Images were captured using a Nikon TE2000 microscope at 100× magnification . The numbers of myotubes with 1–3, 4–6, and >6 nuclei were counted and averaged among three images per treatment at 100× magnification. The differentiation index was determined as the percentage of MyHC-positive nuclei among total nuclei, and the myotube fusion index was determined as the distribution of the nucleus number in total myotubes [Four days post myogenic differentiation, C2C12 cells were washed with cold PBS and fixed with 4%  using the Student Our studies identify that the BAMBI is an essential and positive regulator of C2C12 differentiation, and Wnt/β-catenin signaling mediates its role during myogenic differentiation.


Activation of the wnt signaling pathway is a major cause of colon cancer development. Tankyrase inhibitors (TNKSi) have recently been developed to block the wnt pathway by increasing axin levels to promote degradation of the wnt-regulator β-catenin. TNKSi bind to the PARP (poly(ADP)ribose polymerase) catalytic region of tankyrases (TNKS), preventing the PARylation of TNKS and axin that normally control axin levels through ubiquitination and degradation. TNKSi treatment of APC-mutant SW480 colorectal cancer cells can induce axin puncta which act as sites for assembly of β-catenin degradation complexes, however this process is poorly understood. Using this model system, we found that siRNA knockdown of TNKSs 1 and 2 actually blocked the ability of TNKSi drugs to induce axin puncta, revealing that puncta formation requires both the expression and the inactivation of TNKS. Immunoprecipitation assays showed that treatment of cells with TNKSi caused a strong increase in the formation of axin-TNKS complexes, correlating with an increase in insoluble or aggregated forms of TNKS/axin. The efficacy of TNKSi was antagonized by proteasome inhibitors, which stabilized the PARylated form of TNKS1 and reduced TNKSi-mediated assembly of axin-TNKS complexes and puncta. We hypothesise that TNKSi act to stimulate TNKS oligomerization and assembly of the TNKS-axin scaffold that form puncta. These new insights may help in optimising the future application of TNKSi in anticancer drug design. This pathway is important for development and its aberrant activation is a major cause of colorectal cancer (CRC) , 2. β-caTankyrases (TNKSs) 1 and 2 are poly(ADP)ribose polymerases (PARPs) which have gained attention in recent years as promising drug targets due to their role in down-regulating axin and stabilizing β-catenin. Indeed, TNKS inhibitors (TNKSi) have been developed that can disrupt the wnt pathway by causing an increase in axin and subsequent decrease in β-catenin levels . CertainThe addition of poly(ADP)ribose (PAR) groups to axin by TNKS label it for degradation by the proteasome –12. TNKShttps://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/antibiotics/penicillin-streptomycin-mixtures.html at 37°C in a 5% CO2-humidified incubator. Cell lines were treated with different proteasome inhibitors: 20 μM MG132  for 6 h, if not stated differently, and 10 μM Bortezomib  for 4 h. Cells were treated with 2.5 μM XAV939 and 5 μM IWR-1  and 5 μM G007-LK  to inhibit TNKS activity. Transfections were carried out using Lipofectamine 2000  according to manufacturer instructions. We transfected cells with the plasmid pβ-catenin-WT-GFP [The primary cell line used here are human SW480 colorectal cancer cells (APC protein truncated at amino acid 1337), purchased from the commercial cell repository CellBank Australia (Sydney) in August 2014. The cells were confirmed to express key markers unique to this cell line (ie. nuclear beta-catenin and mutant truncated APC) and were verified mycoplasma negative. Some control experiments were performed using human embryonic kidney HEK293T cells and mouse NIH 3T3 fibroblasts . Cells n-WT-GFP . A combiCells were lysed using RIPA buffer containing protease inhibitor cocktail Complete  and 0.1 M of sodium orthovanadate . Samples were boiled for 5 min at 95°C and loaded on to 7.5% or 10% SDS-acrylamide gels. Gels were blotted on nitrocellulose membranes  and blocked for 1 h at room temperature with 3% skim milk in 0.2% TBST. Primary antibodies were incubated overnight at 4°C. Anti-mouse/rabbit/goat horseradish peroxidase conjugated (HRP) secondary antibodies  were incubated for 1 h at room temperature. Western Bright ECL Western Blotting Kit  was used to detect chemiluminescence, and blots were imaged using the ChemiDoc MP Imaging system . Band intensities were measured using Image Lab software from Bio-Rad.2, 10 mM EGTA), and MT-fixation buffer . Cells were washed twice with MT-stabilizing buffer, then incubated with MT-stabilizing buffer plus 0.2% Triton X-100 for 5 min at 37°C to extract soluble proteins. Cells were then washed twice with MT-stabilizing buffer and fixed with MT-fixation buffer for 30 min at 37°C. Cells were then stained for microscopy as outlined below.The following buffers were pre-warmed at 37°C before start; MT-stabilizing buffer  secondary antibodies and Hoechst dye  nuclear stain. The following primary antibodies were used for western blotting and immunofluorescence: Axin , axin , β-catenin , β-actin , TNKS (H-350) , GSK3β (L-17) , APC (H-290) , phosho β-catenin , non-phosho β-catenin antibodies  and α-tubulin .Immunofluorescence microscopy cell imaging acquisition was performed with an Olympus DeltaVision core deconvolution microscope with SoftworxResolve 3D software. Images were taken for every field using a 40x objective and deconvolution was used to process the images.Cells were lysed using the IP lysis buffer . Protein extracts were quantified and 1 mg of protein was incubated overnight under constant rotation with 2 μg of the required antibody . 30 μl of protein-G agarose  beads were added to the samples and incubated for a further 3 h at 4°C under constant rotation. Beads were washed three times with 400 μl of the IP lysis buffer to remove non-specific binding. Then the beads were incubated with 30 μl of SDS sample buffer and boiled for 5 min at 95°C. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes.6 cells were treated with 200 ml of ice-cold 0.1% Triton X-100 in CSK buffer for 10 min at 4o C. The cells were pelleted by low-speed centrifugation (5 min at X1000 g) and the supernatant represents the soluble fraction. The pellet with insoluble protein was solubilized in 90 ml of RIPA buffer for 20 min and the protein suspension centrifuged at maximum speed to obtain the insoluble fraction pellet analyzed by SDS-PAGE.To obtain cell fractions enriched for soluble and insoluble protein, two methods were employed. Cells (untreated or treated) were trypsinized and lysed normally in RIPA buffer to obtain a soluble lysate, or an enriched insoluble protein pool was obtained by lysing and extracting cells on the plate with SDS-PAGE (Laemmli) sample buffer for 15 min and collecting the extracted cell material using a cell scraper. All lysates were analyzed by SDS-PAGE. Alternatively (see Supplementary data), 1 x 10Data analysis was performed in GraphPad Prism 5 and where appropriate included use of Student’s t-test and one-way or two-way ANOVA. Differences were considered to be statistically significant if P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).TNKSi have previously been shown to disrupt wnt signaling by inducing β-catenin turnover, even in APC-mutant SW480 CRC cells where β-catenin is over-expressed , 9. HereSince the TNKS inhibitors, which block the poly(ADP)ribose catalytic domain, can induce formation of axin puncta we asked whether the loss of expression of either TNKS1, TNKS2 or both, also drives axin puncta assembly in SW480 cells. We knocked down TNKS1 and TNKS2 expression by siRNA treatment -ribose groups both to themselves and to axin , 8, 13. When considered together, our findings are consistent with a model in which TNKSi induce axin puncta/β-catenin degradation complexes at least partly through the de-PARylation of TNKS, which in turn leads to the promotion of TNKS oligomerization and the formation of axin-TNKS-β-catenin complexes. This is likely to contribute to the assembly of large axin/TNKS macromolecular complexes that generate puncta. This model is supported by previous literature showing that the PAR modification can inhibit TNKS oligomer formation and maintain the TNKSs in a more soluble state , 8. It iThe mechanism of axin puncta formation and their role in coordinating the β-catenin degradasome complex  is an imAn interesting aspect to this study was the impact of proteasome inhibition, which effectively blocked the action of TNKSi on axin puncta formation at different stages. This is a novel result. Other studies have used MG132 for shorter time periods with overexpressed wnt components and observed some increase in N-terminal phosphorylated β-catenin at the puncta , 29, butS1 FigSW480 cells were treated for 24 h with the TNKSi 2.5 μM XAV939 and 5 μM IWR-1. Immunofluorescence microscopy of endogenous axin in green shows the formation of cytoplasmic axin puncta after TNKS inhibition.(JPG)Click here for additional data file.S2 FigNIH 3T3 control cells (left panel) or pβ-catenin-GFP (green) transfected cells (right panel) were treated with TNKSi for 24h (5 μM G007-LK) and then stained with antibodies against different β-catenin degradation complex components and analysed by microscopy. The control cells had very efficient turnover of β-catenin sometimes making detection of β-catenin degradation components difficult at axin puncta. Only after expressing ectopic β-catenin-GFP, was β-catenin, APC and GSK3β then frequently detected at axin puncta.(JPG)Click here for additional data file.S3 FigA. HEK293T cells were untreated or treated for 6 h and 24 h with 5 μM G007-LK (+/- 6 h with 20 μM MG132) and cell extracts were then harvested and subjected to immunoprecipitation (IP) and analysed as in B. SW480 cells were untreated or treated for 6 h and 24 h with 2.5 μM XAV939 (+/- 6 h with 20 μM MG132) and cell extracts were then harvested and subjected to immunoprecipitation (IP). These results are similar to those observed using the other TNKi in (JPG)Click here for additional data file.S4 FigSW480 cells were treated simultaneously with 20 μM of MG132 and 5 μM of G007-LK for up to 18h. Cells were fixed and fluorescently stained for axin (green). Under these conditions, the co-treatment of MG132 completely blocked formation of TNKSi-induced axin puncta and instead promoted the translocation of axin to the nucleus.(JPG)Click here for additional data file.S5 FigSW480 cells were treated with single or combined doses of tankyrase inhibitors (2.5 μM XAV939 and 5 μM IWR-1) and proteasome inhibitors (20 μM MG132 or 10 μM Bortezomib). The proteasome inhibitors were added for 6 h (MG132) or 4h (Bortezomib) toward the end of the 24 h TNKSi treatment. The data confirmed the MG132 results described in (JPG)Click here for additional data file.S6 FigTo confirm the data shown in (JPG)Click here for additional data file.