Typically, refractory high-entropy alloys (RHEAs), comprising a two-phase ordered B2 + BCC microstructure, exhibit extraordinarily high yield strengths, but poor ductility at room temperature, limiting their engineering application. The poor ductility is attributed to the continuous matrix being the ordered B2 phase in these alloys. This paper presents a novel approach to microstructural engineering of RHEAs to form an “inverted” BCC + B2 microstructure with discrete B2 precipitates dispersed within a continuous BCC matrix, resulting in improved room temperature compressive ductility, while maintaining high yield strength at both room and elevated temperature. These alloys have recently received much attention because of their attractive combination of properties not achievable by other classes of metallic alloys. For example, the first two RHEAs, NbMoTaW and NbMoTaVW, showed weak temperature dependence of yield strength in the temperature range from 600 °C to 1600 °C, with yield strength above 400 MPa at 1600 °C5. Unfortunately, these two alloys had high density (>12 g/cm3) and further developments aimed at reducing the alloy density while keeping superior high-temperature properties. In the second generation RHEAs, high-density Ta and W were replaced with lower density refractory elements, such as Cr, Mo, Nb, V and Zr, and low density Al and Ti were added13. This resulted in RHEAs with lower densities and high-temperature strengths better than the properties of Ni-based superalloys and Fe-based steels14. With the exception of the equimolar HfNbTaTiZr19 and some of its derived compositions21 most of the reported RHEAs have poor room temperature compressive plasticity, which makes them difficult to process and limits their engineering applications. First-principles calculations showed that alloying intrinsically brittle Mo and W with subgroup IV or V transition metals can make them intrinsically ductile22. This composition-induced brittle to ductile transition was explained by changes in the electronic structure, which induced Jahn-Teller distortions and transitioned the elastic instability mode from tensile to shear failure22. Extending this theory to RHEAs, Sheikh, et al.21 found that single-phase BCC RHEAs consisting of subgroup IV, V and VI metals are intrinsically ductile if the valence electron concentration (VEC) is less than 4.5 and intrinsically brittle if VEC ≥4.6.Refractory high-entropy alloys (RHEAs) are a relatively new class of multicomponent materials that are based on several refractory metals, but may also contain other, generally low density, elements and typically have a body centered cubic (BCC) crystal structureet al.23 and Lilensten, et al.24 used a “metastability-engineering” approach, which is similar to well-known transformation-induced plasticity (TRIP), to improve tensile ductility of BCC RHEAs by tailoring the stability of constituent phases. Transformation-induced increase in tensile strain and work-hardening capability were successfully achieved by inducing formation of a stronger HCP phase in a strain-localization region of tensile-tested Ta0.5HfZrTi23 or α” martensite in HfNb0.18Ta0.18Ti1.27Zr24. Strain-induced precipitation of the second phase inside the metastable BCC matrix caused strain hardening and slowed strain localization, thus increasing elongation. Similar approaches to enhance uniform tensile ductility resulting from deformation-induced phase transformation were discussed in steels26, BCC based titanium alloys28, and FCC-based HEAs29. Unfortunately, this method is only applicable to intrinsically ductile RHEAs that show low tensile ductility due to rapid strain localization and necking. It cannot be applied to inherently brittle RHEAs that fracture without strain localization/necking and, often, without any macroscopic strain.Huang, 31 to have a characteristic superalloy-like microstructure, consisting of cuboidal BCC nano-scale precipitates within a coherent B2 matrix, resembling the γ(fcc) + γ’(ordered L12 precipitates) microstructure exhibited by many currently used nickel base superalloys. Although they showed exceptionally good strength at both room and elevated temperatures, substantially exceeding those of single-phase BCC RHEAs, these novel two-phase RHEAs have very limited room temperature compressive ductility, which can be explained by the inherent brittleness of the ordered B2 matrix phase32. Unfortunately, the approaches for improving ductility discussed above cannot be applied to this class of Al-containing RHEAs.Recently, several Al-containing RHEAs were reported0.5NbTa0.8Ti1.5V0.2Zr was selected, based on its low density (7.4 g/cm3) and previous reports of excellent room and high temperature yield strength9. This alloy was cast, hot isostatically pressed (HIPed) and then homogenized at 1200 °C for 24hrs followed by slow cooling (10 °C/min) to room temperature. The resultant microstructure consists of two BCC phases  with very similar lattice parameters that form a very fine, inter-woven baskteweave-like, nano-phase structure9. Subsequently, this will be referred as Condition (1). The alloy has a room-temperature yield strength of 2035 MPa but only 4.5% compression strain before fracture in Condition (1). The present study focuses on improving the ductility of this alloy by tuning the microstructure, while maintaining its high yield strength.The present work is the first demonstration of enhancing the ductility of high-strength BCC + B2 two-phase RHEAs by controlling their microstructure. For this, AlThe alloy in Condition (1) was solutionized at 1400 °C for 20 min followed by water quenching to achieve a single-phase microstructure. This will be referred to as Condition (2). The alloy in Condition (2) was then annealed at 600 °C for 120 hrs and water-quenched (subsequently referred to as Condition (3)) to possibly develop a two-phase BCC + B2 microstructure.9) as seen in the backscattered electron (BSE) SEM image in Fig. Condition (1) was studied here in greater detail using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atom probe tomography (APT). The Condition (1) microstructure is summarized in Fig. 33. These profiles are constructed by delineating the B2/BCC interface using an iso-concentration surface of Al = 10.5 at%. The B2 phase (highlighted in red in the reconstruction map) is rich in Al and Zr whereas the BCC phase is rich in Nb and Ta  distribution of the B2 and BCC phases, and the elemental partitioning across the interface, was studied using atom probe tomography (APT). An example of the reconstructed APT dataset is shown in Fig.  Ta Fig. . The appBCC zone axis. Additionally, careful analysis of the <011>BCC zone axis  can be described as highly refined, nanometer-scale mixture of ordered B2 regions within a BCC matrix.The microstructure of the alloy in Condition (2) was substantially different from Condition (1). The average grain size in this condition is ~150 µm. Figure xis Fig.  revealedxis Fig. . This weBCC zone axis  exhibits a typical superalloy type microstructure as seen in the backscatter SEM image in Fig. xis Fig. , revealease Fig. , indicatThe average Vickers microhardness values with standard deviations, of Conditions (1), (2) and (3), are 562 ± 16 HV, 405 ± 11 HV and 475 ± 12 HV, respectively. The highest hardness was found in Condition (1) due to its fine-scale B2 (hard) + BCC decomposed microstructure, as compared to the single-phase weakly ordered BCC microstructure in Condition (2). Intermediate hardness was observed in Condition (3) due to the presence of discrete B2 precipitates within the continuous BCC matrix, since the scale of the two-phase BCC + B2 microstructure in Condition (3) is substantially coarser than Condition (1).σ0.2 = 2032 MPa, true peak stress σp = 2035 MPa) but showed a true compressive fracture strain εf of only 4.7%. In Condition (2), the alloy was very ductile at room temperature and did not show any evidence of fracture even after a true strain of 60%, with a noticeably lower yield stress (σ0.2 = 1065 MPa). The alloy in Condition (2) showed continuous strengthening after yielding at a strain hardening rate dσ/dε ≈ 118 MPa. The brittle nature of Condition (1) is due to the presence of continuous B2 channels, whereas Condition (2) is largely a single-phase BCC alloy with a small degree compositional fluctuations, and thus has more ductility. Condition (3) provided an intermediate room temperature yield stress (σ0.2 = 1345 MPa) and showed noticeable hardening, with the average hardening rate of dσ/dε ≈ 1220 MPa, until σp = 1772 MPa was reached at the peak true compressive strain εp = 16.9%. This hardening rate is considerably higher than that observed in Condition (2). After that, a strain softening stage was observed  did not occur abruptly, as in Condition (1), but progressively, and deformation was stopped at a true stress of 665 MPa and ε = 60%.Compression properties (true stress vs. true strain behavior) of this RHEA were strongly dependent on the microstructure Fig. , Table 1ved Fig. . First c2) Ni-base superalloy. However, Condition (1) is stronger (but less ductile) than Condition (3) since the harder B2 phase is continuous in Condition (1) while the softer BCC phase is continuous in Condition (3). Further, the scale of the two-phase BCC + B2 microstructure in Condition (3) is substantially coarser than Condition (1).Microstructure showed the same influence on strength as for hardness. Condition (2) was the weakest, as it is best characterized as a single-phase solid solution with no strengthening precipitates. Conditions (1) and (3) are both characterized as precipitation–hardened alloys resembling the microstructure of a γ (fcc) + γ’ (ordered L1σ0.2 = 1774 MPa) but low compressive plasticity (εf = 3.8%), while the yield stress was lower in Condition (3) (σ0.2 = 1423 MPa) but the compressive plasticity was substantially higher (εf = 16.2%). In both conditions, fracture occurred catastrophically. Condition (2) exhibited the lowest yield stress, σ0.2 = 975 MPa, but very high compressive plasticity with εf > 60%. Condition (3) had a slightly higher yield stress and lower compressive plasticity at 600 °C than at 25 °C. The reasons for this unusual behavior have important implications for the development of this alloy family for high temperature applications and are discussed in the following section.High temperature compression testing was carried out at 600 °C for all three conditions, and the results are shown in Fig. Overall, Condition (1) has the highest strength; Condition (2) has the highest compressive ductility; while Condition (3) provides a combination of high yield stress and good ductility.Important details of the phase stabilities and phase transformations in this alloy remain uncertain. Based on the results, it can be assumed here that the alloy is a disordered BCC single-phase structure at 1400 °C, and that the fine-scale composition modulations observed in APT Fig.  develop The experimental results presented in this paper clearly show that Condition (1) has the highest strength; Condition (2) has the highest compressive ductility; while Condition (3) provides a combination of high yield stress and good ductility. These differences in compressive properties can be broadly rationalized based on the respective microstructures and phases observed in these three conditions. Thus, in case of Condition (1), the “superalloy-like” microstructure consists of a continuous ordered B2 matrix, interspersed with a regular array of disordered cuboidal BCC precipitates. While the B2 matrix provides a very high strength to this condition, it suffers from rather poor ductility due to its ordered nature. Condition (2) exhibits a microstructure consisting of a highly refined, nanometer-scale mixture of ordered B2 regions within a BCC matrix. Consequently, this condition exhibits a very high compressive ductility while maintaining a reasonably high strength (>1 GPa), since the matrix is disordered BCC and is strengthened by the refined B2 precipitates. Condition (3) consists of a disordered continuous BCC matrix with a higher phase fraction of coarser B2 precipitates, as compared to Condition (1). While the higher B2 phase fraction is beneficial for strength, the continuous BCC matrix provides good compressive ductility. Despite the higher B2 phase fraction in Condition (3) as compared to Condition (1), the change in the continuous phase from BCC in Condition (3) to B2 in Condition (1) is responsible for the higher compressive strength in Condition (1).34 provide guidance on material parameters that must be quantified to predict the level of strengthening in the present alloys, and more recent models specifically address the influence of coherent B2 precipitates within a disordered BCC matrix in HEAs35 and in maraging steels36. However, the application of these precipitation strengthening models requires knowledge of a number of parameters, including: the degree of short-range ordering as a function of thermal treatment; the size and spacing of the short-range ordered regions; the lattice misfit between the BCC and B2 phases (requiring high resolution X-ray diffraction measurements); the elastic properties of the BCC and B2 phases; interfacial and fault energies; and the volume fraction of the B2 precipitates. While simple approximations may suffice for some of these parameters in less complicated alloys, these parameters can vary a great deal in the current RHEA, and the approximations are likely to give large uncertainties. Additional scientific studies are therefore required to establish the influence of concentrated alloy additions  on the multitude of parameters that can influence precipitation strengthening in these complex RHEAs.The main goal of the present work was to improve the room temperature ductility of an initially brittle RHEA through microstructure modifications by thermal treatment. The goal was successfully achieved and an improved balance of high strength and good compressive ductility was demonstrated by “inverting” the microstructure from a continuous B2 matrix with discrete BCC precipitates to one consisting of a continuous BCC matrix with discrete B2 precipitates. The details of the strengthening mechanisms in these microstructures are beyond of the scope of the present study. However, it can be speculated that strong element bonding in the B2 matrix is responsible for high strength  of Condition (1); while in the case of Conditions (2) and (3), precipitation strengthening from B2 precipitates and dislocation mobility in BCC matrix are likely to play a substantial role. Classical precipitation hardening modelsThe strength of metals almost always decreases with increasing temperature, and so the observation in the present work that the yield strength is higher at 600 °C than at RT for Condition (3) is unusual. The magnitude of the increase shown in the present work is about 6%, which is significantly larger than a typical sample-to-sample experimental uncertainty of ± 1%.3Al, where cross-slip occurs between two competing glide systems, <011>{111} and <011>{100). Sessile kinks produced by this cross-slip strengthen the alloy with increasing temperature, until a temperature is reached where the sessile segments become glissile and strength then decreases with further increases in temperature. Such a mechanism is conceivable in the present alloy, since the B2 phase has many competing slip systems, including <001>{110} and <111>{112}37. Anomalous strengthening is not observed in binary B2 alloys, since the critical resolved shear stresses between competing systems are so different, and only one slip mechanism is usually observed. However, the unusual compositional complexity of the B2 phase in the present study may enable two or more different slip systems to compete. It is a further speculation that sessile dislocation debris may be formed by the transition between these different slip systems.The data needed to establish the mechanism responsible for the anomalous strengthening measured for Condition (3) are difficult to obtain and are beyond the scope of the present work. Nevertheless, it is possible to speculate on the potential mechanisms. The best-known example of anomalous strengthening is for the long-range ordered intermetallic compound, Ni38. Both of these mechanisms can occur in materials with similar dislocation structures, where deformation is controlled by the motion of screw dislocations. Initial studies show that screw dislocations are also responsible for plasticity in the RHEA40, and so either of these mechanisms are conceivable in the present case.Other mechanisms for anomalous strengthening involve the influence of cross-kinks or the non-conservative ‘dragging’ of jogsThe discussion offered here is highly speculative. The details of the deformation mechanisms at room temperature and at 600 °C for this RHEA require further investigation, and is recommended for future study. Additional studies are also suggested at higher temperatures and under tensile loading.0.5NbTa0.8Ti1.5V0.2Zr was carried out in the present study using advanced TEM and APT techniques. The results reveal that the homogenized and slow cooled (~10 °C/min) microstructure (Condition (1)) has an ordered B2 phase as the continuous matrix, interspersed with discrete cuboidal-like pockets of a disordered BCC phase. While this microstructure morphologically resembles the γ (fcc) + γ’ (ordered L12) type microstructure observed in many Ni-base superalloys, it is “inverted” in the sense that the continuous matrix phase is the ordered B2 phase, resulting in limited compressive ductility. The ductility of this alloy was substantially increased via microstructural engineering involving controlled annealing treatments, resulting in a phase inversion. Consequently, the hard, ordered B2 compound that forms the continuous phase in Condition (1) is replaced by the more ductile, disordered, BCC phase in Condition (3). The resultant microstructurally-engineered RHEA not only exhibits high yield strength at room temperature (~1345 MPa) and 600 °C (~1423 MPa), but also substantial compressive ductility at room temperature (>20%). The microstructurally engineered Condition (3) also shows higher strength and ductility at 600 °C relative to room temperature – very few systems provide this capability. This opens up the door for the possible use of these RHEAs in real engineering applications. Additional studies are required to further explore these features.In summary, careful microstructural examination of Al0.5NbTa0.8Ti1.5V0.2Zr alloy was prepared by vacuum/argon arc melting using high purity (99.9% or higher) elements. Details of the alloy preparation are given elsewhere9. The actual composition of the alloy is given in Table The AlTM and transmission electron microscopy (TEM) using an FEI Technai G2 TF20TM operating at 200 kV. Chemical composition of the alloy, phases and local regions was determined using energy dispersive spectroscopy (SEM-EDS) and atom probe tomography (APT). Atom probe experiments were performed using a local electrode atom probe (LEAP 3000x HR®) from Cameca Inc. The TEM foils and the atom probe tips were prepared using Focused Ion Beam (FIB) (FEI Nova 200 NanoLab). All atom probe experiments were conducted in the temperature range of 40–60K, using a voltage evaporation mode with 0.5–1.0% evaporation rate and pulsing voltage maintained to 20% of the steady-state voltage at a frequency of 200 kHz. Raw data from the atom probe experiments were analyzed using Cameca’s IVAS 3.6.8® .Microstructural characterization was performed using back-scattered electron (BSE) imaging in FEI Nova-NanoSEM 230Vickers microhardness was measured on polished cross-section surfaces by applying a load of 4.9 N for 15 seconds, and an average of 10 indents were taken into account and the standard deviation in these values has been included in the reported error bars.41 and ASM recommendations43.The compression samples were extracted from heat treated blanks and surface-polished with 600-grit sandpaper. The final sample dimensions were 4.25 mm × 4.25 mm in the cross-section and 6.8 mm in height, providing the height-to-width aspect ratio 1.6, in agreement with the ASTM standard−6 torr, and this or higher vacuum was maintained during testing. The sample was then heated to the test temperature at 50 °C/min, soaked at the temperature for 15 min under 20 N force control, and then compressed at a constant ram speed that provided an initial strain rate of 0.001 s−1. Room temperature compression tests were conducted in air using a servo-hydraulic MTS machine at the same strain rate conditions. Thin (~50 μm) Teflon foil was used as a lubricating material between the dies and sample contacting surfaces. Displacement in all tests was monitored and synced with load output from the test frames using Image Correlation software. The compression behavior was analyzed in terms of true stresses and true strains, which take into account a continuous increase in the sample cross-section during compression deformation.Compression tests at 600 °C were conducted using a computer controlled Instron mechanical testing machine outfitted with a Brew vacuum furnace. Silicon carbide dies were coated with boron nitride powder to reduce friction between samples and die contact surfaces. Prior to each test, the furnace chamber was evacuated to 10Supplementary Figure


To examine signal transmission through sub-cellular compartments and its effect on transcription levels in individual cells within a population, we used the Wnt/β-catenin signaling pathway as a model system. Wnt signaling orchestrates a response through nuclear accumulation of β-catenin in the cell population. However, quantitative live-cell measurements in individual cells showed variability in nuclear β-catenin accumulation, which could occur in two waves, followed by slow clearance. Nuclear accumulation dynamics were initially rapid, cell cycle independent and differed substantially from LiCl stimulation, presumed to mimic Wnt signaling. β-catenin levels increased simultaneously at adherens junctions and the centrosome, and a membrane-centrosome transport system was revealed. Correlating β-catenin nuclear dynamics to DOI:http://dx.doi.org/10.7554/eLife.16748.001 Cells in an animal’s body must communicate with one another to coordinate many processes that are essential to life. One way that cells do this is by releasing molecules that bind to receptors located on the surface of others cells; this binding then triggers a signaling pathway in the receiving cell that passes information from the surface of the cell to its interior. The last stage of these pathways typically involves specific genes being activated, which changes the cell’s overall activity.cyclin D1, controls cell division. However it was not understood how β-catenin builds up in response to a Wnt signal and influences the activity of genes.Wnt is one protein that animal cells release to control how nearby cells grow and divide. One arm of the Wnt signaling pathway involves a protein called β-catenin. In the absence of a Wnt signal, there is little β-catenin in the cell. When Wnt binds to its receptor, β-catenin accumulates and enters the cell’s nucleus to activate its target genes. One of these genes, called cyclin D1 gene change in living human cells. These analyses were initially performed in a population of cells, and confirmed that β-catenin rapidly accumulates after a Wnt signal and that the cyclin D1 gene becomes activated.Using microscopy, Kafri et al. have now examined how the activities of β-catenin and the Individual cells in a population can respond differently to signaling events. To assess whether human cells differ in their responses to Wnt, Kafri et al. examined the dynamics of β-catenin in single cells in real time. In most cells, β-catenin accumulated after Wnt activation. However, the time taken to accumulate β-catenin, and this protein’s levels, varied between individual cells. Most cells showed the “average” response, with one major wave of accumulation that peaked about two hours after the Wnt signal. Notably, in some cells, β-catenin accumulated in the cell’s nucleus in two waves; in other words, the levels in this compartment of the cell increased, dropped slightly and then increased again.cyclin D1. Instead, cells appeared to sense how quickly the amount of β-catenin in the nucleus changes over time, and this rate influences the activation of cyclin D1.So how does β-catenin in the nucleus activate target genes? Kafri et al. saw that the absolute number of β-catenin molecules in the nucleus did not affect the activity of Importantly, problems with Wnt signaling have been linked to diseases in humans; and different components of the Wnt signaling pathway are mutated in many cancers. An important next challenge will be to uncover how the dynamics of this pathway change during disease. Furthermore, a better understanding of Wnt signaling may in future help efforts to develop new drugs that can target the altered pathway in cancer cells.DOI:http://dx.doi.org/10.7554/eLife.16748.002 While tCCND1 gene was integrated as a single copy allele into human HEK293 cells using Flp-In recombination  showed that the accumulation was comprised of two phases; an initial rapid one, in which the peak of the change in accumulation was reached 60 min after induction, and a second accumulation phase in which cellular β-catenin continued to amass but at a declining rate up until 180 min . SubsequTo examine whether the dynamics of nuclear entry of β-catenin were modified during Wnt activation and how they compared to β-catenin shuttling out of the nucleus, we used fluorescence recovery after photobleaching (FRAP). Nuclei of cells showing nuclear β-catenin, either after 2 hr of Wnt3a activation or transiently overexpressing β-catenin, were photobleached, and nuclear import of β-catenin was monitored over time  top. TheThe averaged population data obtained from living cells presented above  are in aThe similar dynamics of decline in β-catenin levels in the nucleus and the cytoplasm suggests that β-catenin is not simply shuttling in and out of the nucleus, but rather reflects an enhanced activity of the degradation arm controlling β-catenin levels. To test this, we added lithium chloride , a glycogen synthase kinase-3β (GSK3β) inhibitor that mimics Wnt signaling . Indeed,Since the maximum levels of β-catenin accumulation differed between cells in the population , and we Cluster analysis of the dynamic behavior of β-catenin in individual living cells, shows the dramatic difference between Wnt signaling activation by Wnt3a compared to LiCl  and ~20%To examine the cell cycle and Wnt response more closely in a large population of living cells we used the Fucci system  and 7, wβ-catenin is normally present in the adherens junctions proximal to the cell membrane, and is bound to E-cadherin in the membrane and to α-catenin, which mediates the connection between the adherens junction and the actin cytoskeleton . Not mucTo examine if Wnt signaling changed the dynamics of β-catenin at the membrane we performed FRAP experiments on this region and found that the recovery dynamics were slow and indicative of slow exchange of β-catenin molecules at the membrane. Yet, similar recovery in unactivated, Wnt3a-treated and LiCl-treated cells was observed, meaning that there was no change in the dynamics of protein exchange but rather an increase in the number of β-catenin molecules in the membrane-bound fraction .In many of the Wnt-induced cells that were followed in the live-cell movies we noticed the appearance of β-catenin in a single prominent dot , Video 3The accumulation dynamics of β-catenin at the centrosome occurred in parallel to the accumulation seen in the nucleus, cytoplasm and adherens junctions. However, centrosomal levels were significantly higher, five-fold higher compared to the initial state . The rat1/2) of 1.9 s, similar to other centrosomal components . We counted the number of cellular and nascent CCND1-MS2 mRNAs in Wnt3a-treated cells  and compSignaling factors that translocate into the nucleus following signal transduction do so via different modes of shuttling. For instance, some factors display continuous nucleo-cytoplasmic oscillations  , while sCCND1 to Wnt. Even though Wnt/β-catenin signaling has been highly studied, the basic propagation dynamics of this signal in single living cells have not been characterized. This is due to the lack of an appropriate system that would allow analysis of the behavior of a fluorescent β-catenin protein that resembles the endogenous protein . Indeed, measurements of CCND1 activity in living cells following Wnt activation showed a positive change in several parameters relating to gene activation; not only did the frequency of CCND1 activation in the cell population rise and the time to activate CCND1 shorten, but the levels of CCND1 transcriptional output increased, the timeframe of gene activity became substantially longer, and gene resting periods were shortened. Overall, this means that Wnt signaling increases the number of CCND1 mRNAs generated, by increasing the frequency of RNA polymerase II recruitment to the promoter and by lengthening the time of promoter responsiveness. Interestingly, even when an already active CCND1 gene received the Wnt signal, the levels of gene activity increased.However, the fact that CCND1 transcriptional activity with the measured dynamics of β-catenin nuclear accumulation from the separate experiments, we found that the rate of change of nuclear β-catenin correlated well with transcription induction  and mock CM, L-Wnt-3A and L- mouse fibroblast cells were grown in DMEM and 10% FBS, and CM was prepared according to the American Tissue Culture Collection (ATCC) instructions . Wnt actThe Fucci system  was used for cell cycle phase detection. For G1 phase detection, the pRetroX-G1-Red vector (mCherry-hCdt1) was used, and for S/G2/M phase the pRetroX-SG2M-Cyan vector (AmCyan-hGeminin). The Fucci system, being a viral-based system first required the introduction of the mouse ecotropic retroviral receptor on the membrane surface of HEK293 CCND1-MS2 cells expressing YFP-β-catenin. Transient transfection was performed 24 hr prior to infection using PolyJet transfection with the pBABE ecotropic receptor plasmid . This step was performed twice for each infection. After mCherry-hCdt1 infection, mCherry positive cells were collected by FACS and maintained in medium containing puromycin . Cells were then transfected with the pBABE ecotropic receptor plasmid and 24 hr post-transfection, the cells infected with AmCyan-hGeminin. Positive cells were collected by FACS and maintained in medium containing neomycin (500 μg/ml) and puromycin (1 μg/ml). For infections, HEK293T cells were maintained in DMEM containing 10% FBS and used to package the Fucci retroviruses, which were collected over a period of three days before infecting the ecotropic HEK293 cells.SDS-PAGE and Western blotting were performed as previously described . PrimaryRenilla luciferase construct using PolyJet transfection. 50 ng of each plasmid were used. A luciferase assay was performed after 24 hr using the Dual-Glo Luciferase assay system . After standardization with Renilla luciferase activity, a relative luciferase activity was obtained and the mean and standard deviation from triplicate wells was calculated. Each experiment was performed three times. YFP-β-catenin (HEK293 CCND1-MS2 cells were co-transfected with the cyclin D1 promoter −1745CD1LUC Firefly luciefarse construct  and eith-catenin  was obtaCells were harvested and DNA quantification was performed using 5 μg/ml DAPI solution (Sigma). The BD FACSAria III cell sorter was used. For quantifying DNA in fixed cells, we used a 405 nm laser for excitation and a 450/40 nm bandpass filter for detection. Data were processed and analyzed using FlowJo software. The average quantification of 3 repeated experiments is presented (mean ± sd).Cells were grown on coverslips coated by Cell-Tak (BD Biosciences), washed with PBS and fixed for 20 min in 4% PFA. Cells were then permeabilized in 0.5% Triton X-100 for 3 min. After blocking, cells were immunostained for 1 hr with a primary antibody, and after subsequent washes the cells were incubated for 1 hr with secondary fluorescent antibodies. Primary antibodies: mouse anti-β-catenin and rabbit anti-pericentrin . Secondary antibodies: Alexa488-labeled goat anti-mouse IgG and Alexa594-labeled goat anti-rabbit . Nuclei were counterstained with Hoechst 33342 (Sigma) and coverslips were mounted in mounting medium.CCND1-MS2 cells were grown on coverslips coated by Cell-Tak (BD Biosciences) and fixed for 20 min in 4% paraformaldehyde, and overnight with 70% ethanol at 4°C. The next day cells were washed with 1x PBS and treated for 2.5 min with 0.5% Triton X-100. Cells were washed with 1x PBS and incubated for 10 min in 40% formamide . Cells were hybridized overnight at 37°C in 40% formamide with a specific fluorescently-labeled Cy3 DNA probe . The next day, cells were washed twice with 40% formamide for 15 min and then washed for two hours with 1X PBS. Nuclei were counterstained with Hoechst 33342 and coverslips were mounted in mounting medium. The probe for the MS2 binding site was:CTAGGCAATTAGGTACCTTAGGATCTAATGAACCCGGGAATACTGCAGAC.3D stacks  of the total volume of the cells were collected from fixed CCND1-MS2 cells. The 3D stacks were deconvolved and the specific signals of mRNAs were identified . mRNA identification was performed in comparison to deconvolved stacks from cells not containing the MS2 integration, which therefore served as background levels of nonspecific fluorescence. No mRNAs were identified in control cells. The sum of intensity for each mRNA particle and active alleles was measured in the same cells using Imaris, as previously described , 2013. T2 control . For long-term imaging, several cell positions were chosen and recorded by a motorized stage . In these experiments, HEK293 Flp-in CCND1-MS2 expressing MS2-GFP cells were imaged in 3D (26 planes per time point) every 15 min, at 0.26 µm steps for 6 hr. HEK293 Flp-in CCND1-MS2 cells expressing YFP-β-catenin were imaged in 3D (15 planes per time point) at 0.7 µm steps, every 15 min, up to 18 hr. For presentation of the movies, the 4D image sequences were transformed into a time sequence using the maximum or sum projection options or manually selecting the in-focus plane using the ImageJ software. Time-lapse data were collected from single cells in several fields and on several days until reaching an appropriate sample size, and then all single-cell data were pooled and either averaged and presented as plots, or presented as single cell data.Wide-field fluorescence images were obtained using the Cell^R system based on an Olympus IX81 fully motorized inverted microscope  fitted with an Orca-AG CCD camera (Hamamatsu) driven by the Cell^R software. Live-cell imaging was carried out using the Cell^R system with rapid wavelength switching. For time-lapse imaging, cells were plated on glass-bottomed tissue culture plates  coated by Cell-Tak (BD Biosciences) in medium containing 10% FBS at 37°C. The microscope is equipped with an incubator that includes temperature and COIs). Background from another location in the nucleus (In) was subtracted for each frame, and the final intensity was calculated using: I = Is(t) − In(t) and then normalized to the initial intensity.The intensity of the active transcription sites labeled with MS2-GFP fluorescence in time-lapse movies was corrected for photobleaching using ImageJ, and the 3D movies were transformed to 2D by choosing the in-focus plane in which the intensity of the transcription site is the highest. Movies were manually tracked and the intensity measured for each frame (Ic) was multiplied by the area occupied by the centrosome (Ac): I = Ic(t) *Ac(t). For membrane intensity, a sum projection of the 3D movies was used.Measuring the intensity of the YFP-β-catenin signal in the subcellular compartments was performed manually using ImageJ, and background was subtracted from all measurements. When YFP-β-catenin levels were low, DIC images that were acquired in parallel were used for nucleus detection. For measurements of centrosome intensity, the intensity of the centrosome in each frame  ratio of YFP-β-catenin were obtained by division of the YFP-β-catenin intensity levels measured. Correlation coefficient values were calculated by comparing the intensity of β-catenin over time between all possible pairs of sub-cellular compartments, from Wnt activation onset. Values of rate of change (2 using a live-cell chamber system (Tokai). For each acquisition, YFP-β-catenin was bleached using the 491 nm laser. Six pre-bleach images were acquired. In FRAP, post-bleach images were acquired every 0.8 s for 80 s in the cytoplasm and the nucleus, every 1 s for 2 min in adherens junctions, every 0.4 s for 40 s at the centrosome, and every 1.5 s for 8 min to measure nuclear import and export rates. In FLIP, images were acquired every 1.9 s for 280 s in the cytoplasm and the nucleus. The experiments were analyzed using ImageJ macros previously described (FRAP and FLIP experiments were performed using a 3D-FRAP system (Photometrics) built on an Olympus IX81 microscope  equipped with an EM-CCD , 491 nm laser, Lambda DG-4 light source (Sutter), XY and Z stages (Prior), and driven by MetaMorph (Molecular Devices). Experiments were performed at 37°C with 5% COescribed . Data frWhere We used a simple model for describing β-catenin concentration  is indeed an important goal.Essential revisions:1) Combine the two reporter systems in the same cell. This is essential because there is no single-cell measurement of how β-catenin dynamics and transcription are related, which is the whole premise of the paper.2) Quantify the kinetics of nuclear import/export e.g. by measuring the rate of change in nuclear-to-cytoplasmic ratio (or difference).3) Simplify the title to better reflect the main findings of the manuscript.4) The manuscript should be simplified, especially the Introduction.5) Demonstrate at the single cell level whether in a cell nucleus accumulating YFP-β-catenin, an increased CCND1 transcription can be observed at the same time and to same extent.6) Use a cell line in which the FP tag is knocked in to the wild type β-catenin locus. At minimum, the two-fold total amount of β-catenin in the cells has to be clearly stated.7) Reviewer #1:The authors investigate the intracellular dynamics of β-catenin, and the transcriptional response of a downstream promoter, following Wnt signaling. Both aspects are studied in individual live cells, using a YFP‐tagged version of the protein and an MS2-GFP labeled version of the RNA. Mapping the "signaling-to-gene-induction dynamics" (as promised in the title) is indeed an important goal. I was therefore excited about reading the paper. Unfortunately, I ended up rather disappointed, since I felt that the promise was not fulfilled:1) Most conspicuously, the authors never actually combine the two reporter systems in the same cell. Thus, there is no single-cell measurement of how β-catenin dynamics and transcription are related, which a-priori seemed like the whole premise of the paper. Even in the Discussion, the authors still state that their approach "combines the real‐time measurements of the signaling protein factor together with gene activity readout in individual living cells". How so?2) There are other instances where the data doesn't match up to the verbal statements. For example, the authors repeatedly mention "the kinetics of nuclear clearance", "shuttling in and out of the nucleus", or the "very slow egress" of the protein, but the kinetics of nuclear import/export are never quantified, e.g. by measuring the rate of change in nuclear-to-cytoplasmic ratio (or difference). Why is that?3) The manuscript is also overly long. The Introduction, for example, reads like part of a review article-it broadly surveys the field rather than leading the narrative to the specific questions that will be addressed in the work. Elsewhere, there are multiple occurrences of ambiguous statements. Even the title, to me, is ambiguous unless some hyphens and commas are added. Thus, significant re-writing should be done if the manuscript is accepted.Reviewer #2:1) The title needs to be simplified and it should better reflect the main findings of the manuscript.2) The manuscript would benefit from simplification. It contains too much data that will prevent the general reader from following the flow of the results and their importance. For example is 3) The authors aim is to understand by which mechanism a signalling pathway acting at the cell population level is creating variability in responses at the single cell level. However, this question is not fully answered in this manuscript .4) 5) Along the same lines the authors write, "YFP-catenin is equal to endogenous levels". As mentioned above, this means that there is a 2x fold increase in the total amount of β-catenin protein that can influence the results obtained with YFP-β-catenin and especially by those studying the target gene activation. At present the authors give the impression that the study is performed in a system in which the total β-catenin is at endogenous levels. It would be better to use a cell line in which the FP tag is knocked in to the wild type β-catenin locus. At minimum, the two-fold total amount of β-catenin in the cells has to be clearly stated.6) 7) eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Quantifying β-catenin subcellular dynamics and gene expression kinetics during Wnt signaling in single living cells" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.While both reviewers agree that the manuscript is improved, in the ensuing discussion they both also agree that the two reporters in the same cell is a requirement not met. One raises the possibility to modify your conclusions to accommodate this omitted data, the other feels that it is necessary but would agree to oversee a modified manuscript that does not depend on this experiment.We all feel the manuscript would be much stronger with the experiment than without, but it's up to you how you want to handle this revision.Reviewer #1:I do not think that the authors have improved the paper sufficiently to warrant publication. Specifically:1) The main request, to "combine the two reporter systems in the same cell", was not fully met. What the authors did was simultaneously measure β-catenin (protein) and CCND1 (RNA) in FIXED cells. While this measurement can potentially provide useful information, it falls short of the goal of detecting, and correlating, both species in real-time in live cells.The way the authors analyze the protein/RNA data also seems strange. They count the total number of CCND1-MS2 RNA molecules per cell and correlate it with the nuclear level of β-catenin . However2) The text needs to be edited and proofread. Proofreading by an English speaker would have helped in some instances , but in other instances this is a matter of ambiguous and confusing statements. The editorial team will have to address this problem if the manuscript is chosen for publication. To give just one example, the title, "Quantifying β-catenin subcellular dynamics and gene expression kinetics" implies that both the subcellular dynamics and the gene expression kinetics are those of β-catenin, but this is not actually the case.Reviewer #2:The authors answered (more or less) my comments and made most of the suggested improvements. The manuscript is considerably improved. Essential revisions:1) Combine the two reporter systems in the same cell. This is essential because there is no single-cell measurement of how β-catenin dynamics and transcription are related, which is the whole premise of the paper.This point was raised by both reviewers , namely, to show that β-catenin levels lead to an increase in the CCND1 transcript population in the same cell. In order to demonstrate in single cells within a population that β-catenin nuclear accumulation correlates with high CCND1 mRNA levels, we counted single mRNA molecules in single cells expressing the YFP-β-catenin protein using RNA FISH, after Wnt addition for 2 hours. We show that cells in the population that have high YFP-β-catenin nuclear accumulation also have significantly higher numbers of the mRNA. We also perform correlation analysis showing the connection between YFP-β-catenin and mRNA numbers. This now appears in a new 2) Quantify the kinetics of nuclear import/export e.g. by measuring the rate of change in nuclear-to-cytoplasmic ratio (or difference).This suggestion was useful since we now can show the difference between YFP-β-catenin import and export in living cells. The import FRAP experiment  now also3) Simplify the title to better reflect the main findings of the manuscript.Following remarks by both reviewers we changed the title to:“Quantifying β-catenin subcellular dynamics and gene expression kinetics during Wnt signaling in single living cells”. We acknowledge that the previous title was grammatically problematic as mentioned by reviewer 1, but that was due to the 120-character cap. We hope the new title gives a better impression of the study as a whole.4) The manuscript should be simplified, especially the Introduction.We have made large efforts to cut down and tighten the text. The Introduction has been simplified and extensively shortened, leaving only the relevant background for the study. We have also focused the Discussion section and removed less important issues. We removed one supplementary figure  and also5) Demonstrate at the single cell level whether in a cell nucleus accumulating YFP-β-catenin, an increased CCND1 transcription can be observed at the same time and to same extent.This point was raised by both reviewers , namely, to show that β-catenin levels lead to an increase in the CCND1 transcript population in the same cell. In order to demonstrate in single cells within a population that β-catenin nuclear accumulation correlates with high CCND1 mRNA levels, we counted single mRNA molecules in single cells expressing the YFP-β-catenin protein using RNA FISH, after Wnt addition for 2 hours. We show that cells in the population that have high YFP-β-catenin nuclear accumulation also have significantly higher numbers of the mRNA. We also perform correlation analysis showing the connection between YFP-β-catenin and mRNA numbers. This now appears in a new 6) Use a cell line in which the FP tag is knocked in to the wild type β-catenin locus. At minimum, the two-fold total amount of β-catenin in the cells has to be clearly stated.We do not have a cell line with the FP knocked in. We started this project some years before CRISPR appeared on the scene. We now mention specifically that YFP-β-catenin is overexpressed above the background of the endogenous protein. We also quantified this according to the Western blots in order to give an actual number, and find that YFP-β-catenin protein levels are 0.8x fold  over the endogenous levels of β-catenin protein. This is mentioned twice, in the Results and in the Discussion. 7) That was a good idea since the data are contained in the movie. We therefore analyzed the data for the cytoplasm as suggested and indeed the cytoplasmic fluorescence decreases in correlation to the increase in nuclear accumulation.Reviewer #1:The authors investigate the intracellular dynamics of β-catenin, and the transcriptional response of a downstream promoter, following Wnt signaling. Both aspects are studied in individual live cells, using a YFP‐tagged version of the protein and an MS2-GFP labeled version of the RNA. Mapping the "signaling-to-gene-induction dynamics" (as promised in the title) is indeed an important goal. I was therefore excited about reading the paper. Unfortunately, I ended up rather disappointed, since I felt that the promise was not fulfilled:1) Most conspicuously, the authors never actually combine the two reporter systems in the same cell. Thus, there is no single-cell measurement of how β-catenin dynamics and transcription are related, which a-priori seemed like the whole premise of the paper. Even in the Discussion, the authors still state that their approach "combines the real‐time measurements of the signaling protein factor together with gene activity readout in individual living cells". How so?This point was raised by both reviewers, namely, to show that β-catenin levels lead to an increase in the CCND1 transcript population in the same cell. In order to demonstrate in single cells within a population that β-catenin nuclear accumulation correlates with high CCND1 mRNA levels, we counted single mRNA molecules in single cells expressing the YFP-β-catenin protein using RNA FISH, after Wnt addition for 2 hours. We show that cells in the population that have high YFP-β-catenin nuclear accumulation also have significantly higher numbers of the mRNA. We also perform correlation analysis showing the connection between YFP-β-catenin and mRNA numbers. This now appears in a new 2) There are other instances where the data doesn't match up to the verbal statements. For example, the authors repeatedly mention "the kinetics of nuclear clearance", "shuttling in and out of the nucleus", or the "very slow egress" of the protein, but the kinetics of nuclear import/export are never quantified, e.g. by measuring the rate of change in nuclear-to-cytoplasmic ratio (or difference). Why is that?First, we have tried to be more careful in our statements so that they better describe the actual data. With regard to issues of dynamics of import and export of β-catenin we have made the following changes and added new data. The import FRAP experiment  now also3) The manuscript is also overly long. The Introduction, for example, reads like part of a review article-it broadly surveys the field rather than leading the narrative to the specific questions that will be addressed in the work. Elsewhere, there are multiple occurrences of ambiguous statements. Even the title, to me, is ambiguous unless some hyphens and commas are added. Thus, significant re-writing should be done if the manuscript is accepted.We have made large efforts to cut down and tighten the text. The Introduction has been simplified and extensively shortened, leaving only the relevant background for the study. We have also focused the Discussion section and removed less important issues. We removed one supplementary figure  and alsoFollowing remarks by both reviewers we changed the title to: “Quantifying β-catenin subcellular dynamics and gene expression kinetics during Wnt signaling in single living cells”. We acknowledge that the previous title was grammatically problematic as mentioned by reviewer 1, but that was due to the 120-character cap. We hope the new title gives a better impression of the study as a whole.Reviewer #2:1) The title needs to be simplified and it should better reflect the main findings of the manuscript.See previous response.2) The manuscript would benefit from simplification. It contains too much data that will prevent the general reader from following the flow of the results and their importance. For example is We have made large efforts to cut down and tighten the text. The Introduction has been simplified and extensively shortened, leaving only the relevant background for the study. We have also focused the Discussion section and removed less important issues. We removed one supplementary figure  and also3) The authors aim is to understand by which mechanism a signalling pathway acting at the cell population level is creating variability in responses at the single cell level. However, this question is not fully answered in this manuscript .We address this point below, in comment 7.4) We checked this, and there is no significant difference between endogenous and YFP-β-catenin in the LiCl columns nor between the Wnt columns, in Following these experiments the authors conclude that the exogenously expressed YFP-β-catenin can be used in the further experiments as it behaves similarly to the endogenous protein. Nevertheless, in these YFP-β-catenin cells the total amount of β-catenin is the double than that in wild type cells and this β-catenin overexpression could lead to misinterpretations.We answer this below, as the issue of the amount of exogenous β-catenin is also raised in the next comment.5) Along the same lines the authors write, "YFP-catenin is equal to endogenous levels". As mentioned above, this means that there is a 2x fold increase in the total amount of β-catenin protein that can influence the results obtained with YFP-β-catenin and especially by those studying the target gene activation. At present the authors give the impression that the study is performed in a system in which the total β-catenin is at endogenous levels. It would be better to use a cell line in which the FP tag is knocked in to the wild type β-catenin locus. At minimum, the two-fold total amount of β-catenin in the cells has to be clearly stated.We do not have a cell line with the FP knocked in. We started this project some years before CRISPR appeared on the scene. We now mention specifically that YFP-β-catenin is overexpressed above the background of the endogenous protein. We also quantified this according to the Western blots in order to give an actual number, and find that YFP-β-catenin protein levels are 0.8x fold  over the endogenous levels of β-catenin protein. This is mentioned twice, in the Results and in the Discussion. 6) That was a good idea since the data are contained in the movie. We therefore analyzed the data for the cytoplasm as suggested and indeed the cytoplasmic fluorescence decreases in correlation to the increase in nuclear accumulation.7) This point was raised by both reviewers, namely, to show that β-catenin levels lead to an increase in the CCND1 transcript population in the same cell. In order to demonstrate in single cells within a population that β-catenin nuclear accumulation correlates with high CCND1 mRNA levels, we counted single mRNA molecules in single cells expressing the YFP-β-catenin protein using RNA FISH, after Wnt addition for 2 hours. We show that cells in the population that have high YFP-β-catenin nuclear accumulation also have significantly higher numbers of the mRNA. We also perform correlation analysis showing the connection between YFP-β-catenin and mRNA numbers. This now appears in a new We therefore suggest that the balance between accumulation and degradation is going to affect the outcome in each cell. The Kirschner group has shown (Science 2012) that Wnt does not completely abolish the activity of destruction complex. So we suggest that if the total levels of accumulation are similar in most cells  then is should be the level of inhibition of the destruction complex that varies in each cell and that determines the response.Reviewer #1:I do not think that the authors have improved the paper sufficiently to warrant publication. Specifically:1) The main request, to "combine the two reporter systems in the same cell", was not fully met. What the authors did was simultaneously measure β-catenin (protein) and CCND1 (RNA) in FIXED cells. While this measurement can potentially provide useful information, it falls short of the goal of detecting, and correlating, both species in real-time in live cells.The way the authors analyze the protein/RNA data also seems strange. They count the total number of CCND1-MS2 RNA molecules per cell and correlate it with the nuclear level of β-catenin . HoweverThe issue raised in both revisions was the request to demonstrate β-catenin dynamics and cyclin D1 transcription in the same cell, preferably in a living cell. Obviously, we have tried repeatedly over the years to get this to work in living cells in two colors, but to no avail. We attempted different approaches, and decided to remain with the cell clone expressing the YFP-β-catenin protein so not to begin characterizing another cell clone, but this meant that we could not co-express the GFP-MS2 protein in this clone together with YFP-β-catenin due to the GFP/YFP overlap. Following the request during the first revision, we increased our efforts and began purchasing many types of transfection reagents, and started cloning new versions of either CFP or mCherry MS2-CP, in order to obtain a stable clone with two colors. However, we did not obtain any stable clones by the end of the first revision. Transient transfections did not produce any useful data either because of inefficiency of transfection under these conditions and typical overexpression of the MS2-CP, which masked the transcription site of the single gene. Therefore, to obtain reliable and comparable data we required a stable clone. We have continued trying to generate stable clones with additional types of transfection reagents we purchased during then second revision cycle. We also obtained other MS2-CP plasmids, but all these efforts have been unsuccessful. Perhaps the cells are not very receptacle to the stable integration of additional constructs, because they have already been grown with 3 types of antibiotics. The bottom line is that despite many efforts and financial investment, we have not been able to establish this analysis in two colors in living cells.But we have performed the analysis of β-catenin and CCND1 mRNA together in fixed cells , and feeFinally, we have made sure not to imply in the manuscript that the two color analysis was performed in living cells, and have: a) not mentioned β-catenin in the part that describes cyclin D1 transcription in living cells, as not give the wrong impression; b) mentioned explicitly that the RNA quantification in correlation to β-catenin accumulation was done in fixed cells; and c) added a sentence in the Discussion that says it outright: “Although we were unable to examine YFP-β-catenin dynamics and CCND1-MS2 transcription activity simultaneously in living cells, by integrating the measurements of CCND1 transcriptional activity with the measured dynamics of β-catenin nuclear accumulation from the separate experiments, we found that…”.2) The text needs to be edited and proofread. Proofreading by an English speaker would have helped in some instances , but in other instances this is a matter of ambiguous and confusing statements. The editorial team will have to address this problem if the manuscript is chosen for publication. To give just one example, the title, "Quantifying β-catenin subcellular dynamics and gene expression kinetics" implies that both the subcellular dynamics and the gene expression kinetics are those of β-catenin, but this is not actually the case.Grammar – the text has been reviewed and corrected by an academic.Our revised manuscript is now titled “Quantifying β-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells”, following reviewer 1’s comments.


Autism spectrum disorder (ASD) is a neurodevelopmental disease which is characterized by a deficit in social interactions and communication with repetitive and restrictive behavior. In altered cells, metabolic enzymes are modified by the dysregulation of the canonical WNT/β-catenin pathway. In ASD, the canonical WNT/β-catenin pathway is upregulated. We focus this review on the hypothesis of Warburg effect stimulated by the overexpression of the canonical WNT/β-catenin pathway in ASD. Upregulation of WNT/β-catenin pathway induces aerobic glycolysis, named Warburg effect, through activation of glucose transporter (Glut), pyruvate kinase M2 (PKM2), pyruvate dehydrogenase kinase 1(PDK1), monocarboxylate lactate transporter 1 (MCT-1), lactate dehydrogenase kinase-A (LDH-A) and inactivation of pyruvate dehydrogenase complex (PDH). The aerobic glycolysis consists to a supply of a large part of glucose into lactate regardless of oxygen. Aerobic glycolysis is less efficient in terms of ATP production than oxidative phosphorylation because of the shunt of the TCA cycle. Dysregulation of energetic metabolism might promote cell deregulation and progression of ASD. Warburg effect regulation could be an attractive target for developing therapeutic interventions in ASD. Autism spectrum disorders (ASD) is a neurodevelopmental disease which is characterized by a deficit in social interactions and communication with repetitive and restrictive behaviors , poor eyDysregulation of the core neurodevelopmental pathways is associated with the clinical presentation of ASD, and one of the major pathways involved in developmental cognitive disorders is the canonical WNT/β-catenin pathway . SeveralMetabolic enzymes are modified by the dysregulation of the canonical WNT/β-catenin pathway. Upregulation of WNT/β-catenin signaling leads to activation of pyruvate dehydrogenase kinase-1 (PDK-1), which decreases the activity of the pyruvate dehydrogenase complex (PDH). Upregulation of WNT/β-catenin signaling also activates monocarboxylate lactate transporter-1 (MCT-1) . This doMitochondrial deregulation is one of the main metabolic abnormalities observed in ASD physiopathology –17. SeveThere is some common denominator between these metabolic abnormalities, which strongly suggests the reprogramming of cellular energy metabolism with increase lactate production induced by over-expressed canonical WNT/β-catenin pathway in ASD.We focus this review on the hypothesis of Warburg effect induced by over-expressed canonical WNT/β-catenin pathway in ASD.Wingless and integration site  pathway is a cascade of several signaling implicated in development, growth, and metabolism . WNT sigDysregulation of the canonical WNT/β-catenin pathway is observed in numerous diseases , such as2+ pathway.WNT family genes are 19 members which are classified as canonical and non-canonical WNT pathway. Canonical WNT ligands are seven, as WNT1, WNT2, WNT3, WNT8a, WNT8b, WNT10a and WNT10b). They are activators of the WNT/β-catenin pathway. Canonical WNT ligands are secreted by neurons and immune cells in the CNS . The nonWNT extracellular ligands bind low density lipoprotein receptor-related protein 5 and 6 (LRP 5/6), Frizzled (FZD) receptors, and then disheveled (DSH), resulting in β-catenin accumulation and nuclear translocation. Thus, N-nuclear β-catenin bind T-cell factor/lymphoid enhancer factor (TCF/LEF) . The comThe absence of binding between membrane receptors and WNT extracellular ligands characterizes the downregulation of WNT/β-catenin pathway. The β-catenin complex destruction is formed by adenomatous polyposis coli (APC), AXIN and glycogen synthase kinase-3β (GSK-3 β). This complex binds β-catenin to degrade it into the proteasome . ActivatPhosphatidylinositol 3-kinase/serine/threonine kinase (protein kinase B)/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is implicated in proliferation, growth, protein synthesis and metabolism –47. WNT/Several studies have shown the major role of activated WNT/β-catenin pathway in ASD –60. NumeSeveral studies has shown a main role of numerous compounds of the WNT/β-catenin pathway in ASD, such as WNT1 , WNT2 6, WNT3 6, WNT7A , APC 7070–72, β-The knockout of the gene encoding phosphatase and tensin homolog protein (PTEN), a cytoplasmic protein suppressor of WNT/β-catenin pathway, has been identified as a high-risk ASD susceptibility gene –80. PTENValproate  is an anti-convulsing agent discovered in 1963 and used for treatment of bipolar disorders or migraine , 87. VPAIn neural stem cells of the CNS, VPA can increase WNT3a expression and β-catenin accumulation . In rat The Warburg effect  consists to a conversion of a large part of glucose into lactate regardless of oxygen . ActivatSeveral studies have shown that aerobic glycolysis is induced by overactivation of the WNT/β-catenin pathway through a direct activation of PDK1 and MCT-1 , 96, 97.Increase rate of glucose metabolism is associated with the overactivation of PI3K/Akt pathway . ActivatGlut-1 and Glut-3 are mainly important for the insulin-sensitive homeostasis of glucose transport . Then, tActivated c-Myc also activates glutaminolysis and tends to nucleotide synthesis  by activUp to now, few studies have described the expression of the different glycolytic enzymes in ASD. However, several studies have shown elevated lactate levels in ASD patients , 108–110The canonical WNT/β-catenin pathway is upregulated in ASD, and is one of the major pathways involved in developmental cognitive disorders. In the present review, we examine accumulating evidence of the reprogramming of cellular energy metabolism induced by over-expressed canonical WNT/β-catenin pathway for a shift in energy production from mitochondrial oxidative phosphorylation to aerobic glycolysis as the alternative of ATP despite the availability of oxygen; a phenomenon called Warburg effect. Over-activation of the WNT/β-catenin pathway induces the transduction of WNT/β-catenin target genes, c-Myc and cyclin D1, and activates PI3K/Akt pathway, leading to HIF-1α stabilization. Both transcription of WNT-responsive genes and HIF-1α stabilization induce the transactivation of genes encoding aerobic glycolysis enzymes c-Myc, PDK, LDH-A, and MCT-1, which might explain the decreased glucose entry into the TCA cycle in mitochondria, and the conversion of a large part of glucose into lactate in cytosol, observed in the ASD. Dysregulation of cellular energy metabolism induced by over-expressed canonical WNT/β-catenin pathway might promote dysregulation and progression of the core neurodevelopmental pathways associated with the clinical presentation of ASD. Warburg effect regulation might be an innovative mechanism for therapeutic development in ASD, through the canonical WNT/β-catenin pathway as potential therapeutic target.


In genitourinary system, bladder cancer (BC) is the most common and lethal malignant tumor, which most common type is bladder urothelial carcinoma (BUC). Long non-coding RNA (lncRNA) Taurine Up-Regulated 1 (TUG1) gene is high-expressed in several malignant tumors, including BC. In this study, over-expression of TUG1 was found in BUC tissues and cell line resistant to doxorubicin (Dox). Knockdown of TUG1 inhibited the Dox resistance and promoted the cytotoxicity induced by Dox in T24/Dox cells. TUG1 knockdown also depressed the Wnt/β-catenin pathway, and the activation the Wnt/β-catenin pathway partly reversed the inhibitory effects of TUG1 knockdown on Dox resistance in T24/Dox cells. In conclusion, up-regulation of lncRNA TUG1 was related with the poor response of BUC patients to Dox chemotherapy, knockdown of TUG1 inhibited the Dox resistance of BUC cells via Wnt/β-catenin pathway. These findings might assist in the discovery of novel potential diagnostic and therapeutic target for BUC, thereby improve the effects of clinical treatment in patients. In genitourinary system, bladder cancer (BC) is the most common and lethal malignant tumor, which most common type is bladder urothelial carcinoma (BUC). Surgical operation is the preferred and key preference for BUC patients, systemic and intravesical chemotherapy may decrease BUC cell metastasis and improve patient survival . AlthougAccumulating evidences showed that the long non-coding RNAs (lncRNAs) might play important roles in carcinogenesis , 4. LncRYoung TL et al. at 2005 [Taurine Up-Regulated 1 (TUG1) gene, a novel lncRNA mapping to 22q12.2, was originally identified in retinal development by  at 2005 . Recent  at 2005 –15. TUG1 at 2005 , 17. TheIn this study, we emphasized the pivotal roles and regulatory mechanism of TUG1 on chemoresistance in BUC, which might helpful to improve the effects of clinical treatment in BUC.To screen the lncRNAs associated with Dox resistance in BUC, the 4 Dox (−) and 4 Dox (+) BUC samples were examined with LncRNA array. In the array, TUG1 showed an over seven-fold up-regulation in Dox (−) BUC samples compared with Dox (+) BUC samples Figure . In addiP < 0.05). And, the expression of TUG1 in T24/Dox cells was much higher than that in T24 cells .The half maximal inhibitory concentration (IC50) of T24 cells and T24/Dox cells to Dox was 0.85 ± 0.13 mg/L to 3.26 ± 0.28 mg/L. T24/Dox cells showed more powerful resistance to DOX than T24 cells, which resistance index (RI) was 3.84 , which demonstrated that knockdown of TUG1 depressed Dox resistance in T24/Dox cells.The CCK8 assay showed knockdown of TUG1 inhibited the IC50 of T24/Dox cells to Dox from 3.08 ± 0.23 mg/L to 1.14 ± 0.17 mg/L . Secondly, single label flow cytometry indicated T24/Dox cells with TUG1 knockdown showed a significant S phase block . Thirdly, TUG1 knockdown advanced cell apoptosis of T24/Dox cells by double label flow cytometry .Furthermore, the T24/Dox cells were treated with Dox (0.5 mg/L), and the impacts of TUG1 knockdown on the Dox-induced cytotoxicity were detected. Firstly, knockdown of TUG1 restrained cell viability of T24/Dox cells by CCK8 assay  analysis of lncRNAs differentially expressed between Dox (−) and Dox (+) BUC tissues indicated the major enriched processes were cell cycle and apoptosis. And Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed the most enriched pathway was Wnt/β-catenin pathway. Therefore, Wnt/β-catenin pathway was chosen as targets to highlight the TUG1 associated Dox resistance in BUC.r = 0.1137 and P = 0.0189  in 426 BUC samples. Secondly, TUG1 knockdown decreased the expression of β-catenin protein in T24/Dox cells . Thirdly, the TOP/FOP Flash luciferase reporter assay showed TUG1 knockdown depressed the TOP/FOP ratio in T24/Dox cells significantly , which represented the transcriptional activity of Wnt/β-catenin pathway.Firstly, the co-expression patterns  between TUG1 and β-catenin (CTNNB1) in TCGA Pan-Cancer (PANCAN) showed that the expression of TUG1 was positively related to the expression of β-catenin Figure , with a Together, the data demonstrated TUG1 knockdown restrained the activity of Wnt/β-catenin pathway.P < 0.05).The expression vector for β-catenin (pE-β-catenin) was transfected into T24/DR cells to activate Wnt/β-catenin signaling which restrained by knockdown of TUG1. Western blotting and TOP/FOP Flash luciferase reporter assays indicated the Wnt/β-catenin pathway was activated significantly . In addition, under Dox treatment (0.5 mg/L), β-catenin enhancement promoted cell viability and cell division as well as suppressed cell apoptosis in T24/Dox cells . However, ss-TUG1 and pE-β-catenin could not restore the Dox resistance to the original level of pE-β-catenin group.While combining use ss-TUG1 and pE-β-catenin, T24/Dox showed significantly increased Dox resistance when compared with ss-TUG1 group. The IC50 of Dox in T24/Dox cells was increased from 0.98 ± 0.16 mg/L to 1.89 ± 0.21 mg/L , and might be a potential prognostic biomarker and therapeutic target for HCC ; lncRNA In our lncRNA array, TUG1 showed an over seven-fold up-regulation in Dox (−) BUC samples compared with Dox (+) BUC samples. And, the following qRT-PCR confirmed the up-regulation of TUG1 in BUC tissues and cell line resistant to Dox. The results revealed that the up-regulation of TUG1 was correlated with the poor response of BUC patients to Dox chemotherapy, which provided initial evidence that TUG1 played an important role in the Dox resistance of BUC.Recent studies reported TUG1 was up-regulated in colorectal cancer resistant to methotrexate, knockdown of TUG1 re-sensitized the colorectal cancer cells to methotrexate ; high TUIn order to validate the vital roles of TUG1 on Dox resistance, loss of function assays were applied to clarify the impacts of TUG1 on Dox resistance in BUC. Knockdown of TUG1 restrained the IC50 of T24/Dox cells to Dox, and advanced the Dox-induced cytotoxicity, which demonstrated that TUG1 knockdown inhibited Dox resistance in T24/Dox cells. However, the underlying mechanism remain unclear.The data of microarray assay was analyzed by bioinformatics, and KEGG pathway analysis showed Wnt/β-catenin pathway was the most enriched pathway. And, TCGA Pan-Cancer (PANCAN) showed that the expression of TUG1 was positively related to the expression of β-catenin in BUC. Together with the published references , Wnt/β-cBased on above findings, we put forward hypothesis that knockdown of lncRNA TUG1 might inhibit Dox resistance of BUC via Wnt/β-catenin pathway. Subsequent series of experiments confirmed that activating Wnt/β-catenin pathway by upregulating β-catenin reversed the effects of TUG1 knockdown on Dox resistance in T24/Dox cells, Wnt/β-catenin pathway partially mediated effecting induced by TUG1 knockdown in 24/DR cells.In our previous study, knockdown of PVT1 gene could downregulate the expression of multidrug resistance protein 1 (MDR1) and multidrug resistance associated protein 1 (MRP1) expression in BUC cells through Wnt/β-catenin pathway, and then participate in the formation of chemoresistance to Dox and cisplatin . Zhang ZIn conclusion, high-expression of lncRNA TUG1 was related with the poor response of BUC patients to Dox chemotherapy, knockdown of TUG1 inhibited Dox resistance of BUC via Wnt/β-catenin pathway. Our findings elucidate a potential mechanism of BUC chemoresistance, and indicate TUG1 might be a novel potential therapeutic target in BUC.2. The DOX resistant T24/Dox cell line was established in our laboratory previously via co-culture with continuously increasing DOX  concentrations, which showed resistance to DOX [Human BUC T24 cell lines were purchased from the American Type Culture Collection . T24 cells were cultured Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum , and maintained at 37°C in a humidified incubator with 5% COe to DOX .82 BUC specimens were gathered from the Department of Urinary surgery of Shengjing Hospital of China Medical University from Mar 2014 to Apr 2016 through cystoscope. All BUC patients were treated by chemotherapy with DOX, and the chemotherapy effects were evaluated in accordance with RECIST Response Evaluation Criteria. Patients resistant to DOX (−) were 51 cases, and patients sensitive to DOX (+) were 31 cases. This study was approved by the Ethics Committees of China Medical University, and the permissions of surgical patients were achieved.® Green Master Mix Kit . The primers of TUG1 were 5′- CAAGAAACAGCAACACCAGAAG -3′ (forward) and 5′- TAAGGTCCCCATTCAAGTCAGT -3′ (reverse) [BUC tissues and cells were treated with TRIzol reagent  to extract total RNA. According to manufacturer’s instructions, the expression of TUG1 was detected by SYBRreverse) . The relThe smart silencer for TUG1 (ss-TUG1) and negative control vector (ss-NC) were constructed by the Ribobio Corporation . pEGFP-N1-β-catenin (pE-β-catenin) was constructed by cloning the full sequence of β-catenin into pEGFP-N1 vector, and empty pEGFP-N1was used as negative control (pE-NC).According to manufacturer’s instructions, the T24/Dox cells were transfected with Lipofectamine 3000™ , and qRT-PCR was applied to examine the transfected efficiency. The stable cell line was established by treating with G418 .The Cell Counting Kit-8 (CCK8) method was used to examine the cell proliferation ability. In details, T24/Dox cells were seeded in 96-well plate. Each well was added with 10 μl Cell Counting Kit-8 , and incubated for 2 hours at 37°C. Absorbance at 450 nm was recorded using the SpectraMax M5 microplate reader  .6 cells were harvested and following the cells were fixed in 70% cold ethanol, pelleted and washed once with PBS and re-suspended in propidium iodide (PI) solution (50 μg/mL) for 30 minutes in the dark. Then, the cell cycles were determined by the FACScan Canto II flow cytometry  within 1 hour and the results were analyzed through the Diva 8.0 software.The cell cycle was analyzed by flow cytometry. In brief, 1 × 106 /ml cells were incubated with 2.5 μl FITC-Annexin V and 2.5 μl propridium iodide (PI) for 10 minutes in the dark at room temperature, and 400 μl binding buffer was added to terminate the reaction. The data was analyzed by FACSCanto II using the Diva 8.0 software . Cells at the right lower quadrant (FITC-Annexin V-positive and PI-negative) were defined as apoptosis.The double label flow cytometry was used to detect the apoptosis rate with Annexin V-FITC apoptosis detection kit  according to manufacturer’s instructions. 1 × 10Cells were treated with DOX at various concentrations  24 hours later. The cell viability was detected by CCK8 method 48 hours later. Based on the data, we charted the dose-response curve and calculated the half maximal inhibitory concentration (IC50) , 32.Protein (30 μg) extracted from T24/Dox cells were subjected to 10 % sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and transferred to a 0.22 μm polyvinylidene difluoride (PVDF) membrane . The PVDF membrane was blocked and incubated with β-catenin antibodies  at 4°C overnight. Then, the membrane was incubated with second antibody and treated with BeyoECL Plus reagent . The image was scanned and analyzed with Image J  by normalized to the inference gene of β-actin.The TOP Flash and FOP FLASH luciferase reporter vectors which contain wild-type or mutant TCF binding sites were purchased from Biovector NTCC Ltd . T24/Dox cells were transiently co-transfected with luciferase plasmid and ss-TUG1 vector using Lipofectamine™ 3000. 48 hours later, dual-lucy assay kit from Vigorous Biotech  was used to quantified the activity of Wnt/β-catenin pathway according to manufacturer’s instructions . The firt-test and one-way ANOVA. A P-value of less than 0.05 was considered to be statistically significant and indicated by (* and #).All data were showed as means ± SD of five independent experiments and analyzed with GraphPad Prism 5.0 . The data were analyzed using Student’s


Vascular calcification was previously considered as an advanced phase of atherosclerosis; however, recent studies have indicated that such calcification can appear in different situations. Nevertheless, there has been a lack of mechanistic insight to explain the difference. For example, the roles of nuclear factor‐κB, a major regulator of inflammation, in vascular calcification are poorly explored, although its roles in atherosclerosis were well documented. Herein, we investigated the roles of nuclear factor‐κB signaling in vascular calcification.IKKβ, an essential kinase for nuclear factor‐κB activation, in vascular smooth muscle cells  and subjected them to the CaCl2‐induced aorta injury model. Unexpectedly, KO mice showed more calcification of the aorta than their wild‐type littermates, despite the former's suppressed nuclear factor‐κB activity. Cultured VSMCs from the aorta of KO mice also showed significant calcification in vitro. In the molecular analysis, we found that Runt‐related transcription factor 2, a transcriptional factor accelerating bone formation, was upregulated in cultured VSMCs from KO mice, and its regulator β‐catenin was more activated with suppressed ubiquitination in KO VSMCs. Furthermore, we examined VSMCs from mice in which kinase‐active or kinase‐dead IKKβ was overexpressed in VSMCs. We found that kinase‐independent function of IKKβ is involved in suppression of calcification via inactivation of β‐catenin, which leads to suppression of Runt‐related transcription factor 2 and osteoblast marker genes.We produced mice with deletion of IKKβ negatively regulates VSMC calcification through β‐catenin–Runt‐related transcription factor 2 signaling, which revealed a novel function of IKKβ on vascular calcification. Inhibition of IKKβ, an essential kinase for nuclear factor‐κB activation, in vascular smooth muscle cells, augments vascular calcification.IKKβ has a kinase‐independent function, and it is involved in the suppression of vascular calcification.IKKβ is the target of aspirin, and statins suppressed the activity of nuclear factor‐κB, but both are reported to have no suppressing effect. They even accelerate vascular calcification.This study could account for the mechanism, at least in part.Vascular calcification was previously considered as a degenerative process seen in advanced atherosclerosis lesions, but recent studies have indicated that such calcification can appear in a different manner. Calcification not only accompanies with atherosclerosis but also accompanies diabetes mellitus or renal failure in the absence of atherosclerosis.NF‐κB is a family of transcription factors that plays critical roles in inflammation and atherosclerosis, and reports have indicated that it is a target of statins.2‐mediated aortic injury, which is an established animal model for vascular calcification.In this study, we produced mice with a deletion of IKKβ in vascular smooth muscle cells (VSMCs), a cell type contributing to calcification,The data, analytic methods, and study materials will not be made available to other researchers for purposes of reproducing the results or replicating the procedure because some materials are used for other unpublished projects.+ mice  to generate IKKβ flox/wt Sm22α‐Cre+/− mice, which were mated with IKKβ flox/flox mice to generate IKKβ flox/flox Sm22α‐Cre+/− (KO) mice or IKKβ flox/flox Sm22α‐Cre−/− (wild‐type [WT]) mice. To generate transgenic mice with constitutively active IKKβ kinase (KA)+/− mice to generate IKKβ‐KA flox/wt Sm22α‐Cre+/− (KA) mice or IKKβ‐KA flox/wt Sm22α‐Cre−/− (WT) mice. To produce transgenic mice with kinase‐dead IKKβ (KD) in smooth muscle cells, IKKβ‐KD flox/flox mice were mated with IKKβ flox/flox Sm22α‐Cre+/− (KO) to generate IKKβ‐KD flox/flox IKKβ flox/flox Sm22α‐Cre+/− (KD) mice. These KD mice have complete knockout of endogenous IKKβ from VSMCs, followed by knockin of kinase defective form of IKKβ. In the mating protocol for all kinds of mice, we could have WT mice within littermate; therefore, we used the littermate WT as control mice in all experiments.All animal experiments were performed in accordance with the institutional guidelines of the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University . For this study, transgenic mice were crossed onto a C57BL/6J background >10 times. To block smooth muscle cell NF‐κB signaling, we used IKKβ knockout selectively in smooth muscle cells of mice. IKKβ flox/flox mice were generously given by Professor Michael Karin and mated with Sm22α, a protein that is specifically expressed in smooth muscle cells, and CreTo generate conditional IKKβ (K44A) expressing mice, a CAG promoter, a loxP flanked STOP cassette, andIKKβ (K44A) cDNA were inserted into the mouse Gt(Rosa)26Sor locus by homologous recombination. A 3.4‐kb upstream DNA fragment and a 4.3‐kb downstream DNA fragment from the unique XbaI site in intron 1 of Gt(Rosa)26Sor locus were amplified by polymerase chain reaction (PCR) from RENKA ES cell genomic DNA and used as the homology arms for the targeting vector. To construct the expression unit, a chemically synthesized DNA sequence containing a DYKDDDDK‐tagged mouse IKKβ (K44A), a rabbit β‐globin polyadenylation signal, and a VloxP sequence were combined with an frt‐flanked PGK_neo cassette (phosphoglycerate kinase 1 promoter‐driven neomycin‐resistant gene). At the 5′ region of this expression unit, a CAG promoter and a loxP flanked STOP cassette containing chloramphenicol acetyltransferase cDNA and Vlox43L were inserted. Then, the promoter‐expression unit was subcloned between 2 homology arms. As a negative selection marker, an MC1 promoter‐driven diphtheria toxin A cassette was placed at the 5′ region of the homology arm. The resulting knock‐in targeting vector contains MC1 promoter‐driven diphtheria toxin A, a 3.4‐kb 5′ homologous arm, a CAG promoter, the first loxP site, chloramphenicol acetyltransferase cDNA, polyA, Vlox43L, an frt‐flanked PGK_neo cassette, the second loxP site, a DYKDDDDK tag, IKK2 (K44A) cDNA, bGH polyA, VloxP, and a 4.3‐kb 3′ homology arm. This vector was linearized and introduced into RENKA ES cells (C57BL/6 genetic background) by electroporation. After selection using geneticin, the resistant clones were isolated and their DNA was analyzed to identify homologous recombinants by PCR using the following primer sets: sc_R10603, 5′‐GTTACTCCACTTTCAAGTTCCTTATAAGC‐3′; neo_108r, 5′‐CCTCAGAAGAACTCGTCAAGAAG‐3′; sc_5AF3, 5′‐ATCCTAAGGTCACTTTTAAATTGAGG‐3′; and neo_G02, 5′‐ATCAGGACATAGCGTTGGCTAC‐3′. Genomic Southern hybridization was probed with a neomycin resistance gene. Homologous recombinant ES cell clones were aggregated with 8‐cell embryos of ICR mice to generate chimeric mice. Chimeric mice with a high contribution from the RENKA background were bred with C57BL/6 mice. Germline transmitted mice were mated to B6;D2‐Tg(CAG‐FLP)18Imeg mice to eliminate the PGK_neo cassette from the genome through Flp/frt‐mediated excision. The knock‐in allele was identified by PCR with the following primers: 5AF1, 5′‐CCATTGGCTCGTGTTCGTG‐3′; and mutant_R, 5′‐CCGGGGGATCCACTAGTA‐3′. The WT allele was identified by PCR with the following primers: 5AF1 and wild_R, 5′‐ACTCCCGCCCATCTTCTAGA‐3′.2, as described previously,2 treatment, but too much dilatation and calcification seen in WT aorta interfere with the evaluation of the difference between WT and KO aorta. Therefore, we reduced the CaCl2 incubation period to 10 minutes. Even in that protocol, 2 weeks after CaCl2 application, the aortas in both types of mice were significantly dilated when compared with those in the sham–operated on mice. Mice were anesthetized by IP injection with pentobarbital (100 mg/kg) and then underwent a laparotomy. An incision of ≈2 cm was made, and the small and large intestines were moved away to expose the infrarenal aorta. Small pieces of gauzes soaked with 0.6 mol/L CaCl2 were placed circumferentially on the infrarenal aorta for 10 minutes. Then, the gauze was removed and the aorta was rinsed with 0.95% NaCl and wiped with a cotton swab. NaCl (0.95%) was substituted for CaCl2 in sham–operated on mice. The incision was closed, and mice were placed on a warm plate until they had recovered from anesthesia. After the surgical procedure, the animals were allowed free access to food and water until the day of euthanasia.An abdominal aorta injury was induced in 8‐ to 14‐week‐old KO and WT male mice by periaortic application of CaClTwo weeks after the operation, mice were euthanized with an overdose of pentobarbital. The vascular system was flushed with cold PBS from the left ventricle. The abdominal aorta was carefully dissected under a microscope, cleaned of extraneous tissue, and then photographed for diameter measurement.Approximately 2 to 3 mm of infrarenal aorta was fixed with 4% paraformaldehyde overnight, embedded in paraffin, cut into 4‐μm cross sections. Then, cross sections were deparaffinized 3 times in xylene and dehydrated in 80%, 90%, and 100% ethanol for 5 minutes each. Elastin was visualized using the fluorescence properties of hematoxylin and eosin.2. Then, adventitia was rolled off the medial layer. Medial layers were minced and further incubated for 2 hours at 37°C with agitation. Resuspended cells were centrifuged for 5 minutes at 300g. The supernatant was aspirated, and the cell pellet was resuspended in an appropriate volume of DMEM supplemented with 10% fetal bovine serum (FBS). Finally, cell suspensions were cultured in collagenase type 1 coated culture dishes and maintained in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin in a 5% CO2/water‐saturated incubator at 37°C. Each kind of VSMCs was isolated from 1 mouse of KO, KA, KD, and littermate WT, and individual samples that used in vitro experiments were harvested from separately cultured dishes of cells.VSMCs from the aorta of littermate mice were isolated on the basis of a protocol described by Villa‐Bellosta and Hamczyk.KO VSMCs were transfected with 1 μg of IKKβ‐KD or IKKβ‐KA plasmids using transfection reagent Effectene , according to the manufacturer's instructions. Two days after transfection, VSMCs were harvested and then used in Western blotting. Plasmids encoding IKK‐2 K44M (IKKβ‐KD) and IKK‐2 S177E S181E (IKKβ‐KA) were from Anjana Rao Laboratory and procured through Addgene .VSMCs were harvested from culture dishes using cell scraper tools. Protein extraction was performed on ice and using a cell lysis buffer  with a protease inhibitor cocktail. After the total protein concentration was measured, the same amount of extracted protein was loaded for SDS‐PAGE immunoblot analysis and transferred to nitrocellulose membranes. Next, membranes were blocked with 5% nonfat milk or 5% BSA in Tris buffer solution containing 0.05% Tween‐20 for 1 hour at room temperature, with gentle agitation. The membrane was immunoblotted with primary antibody overnight at 4°C. The membrane was washed 3 times with Tris buffer solution containing 0.05% Tween‐20 for 10 minutes each. This was followed by a 1‐hour incubation with a horseradish peroxidase–conjugated secondary antibody. Blots were visualized using the ECL Western Blotting Detection kit . Each band was normalized by the corresponding value of β‐actin or GAPDH as an internal control. Densitometric analysis was performed by ImageJ Software. All experiments were performed at least in triplicate.Total RNA was extracted from cultured VSMCs using an RNAqueous total RNA isolation kit  according to the manufacturer's instructions. cDNA was generated from total RNA using The High Capacity RNA‐to‐cDNA Kit (4387406). Quantitative PCR was performed using the QuantiTect SYBR Green PCR Kit and real‐time PCR system . Relative mRNA level of ostrix, alkaline phosphatase, and osteocalcin was normalized to the Rn18s mRNA level in the same sample.Antibodies used include anti‐IKKβ , anti‐Transgelin , anti‐GAPDH, Runx2, anti‐mouse IgG horseradish peroxidase–linked antibody and anti‐rabbit IgG horseradish peroxidase–linked antibody (CST), active β‐catenin, total β‐catenin , anti‐p65 (Santa Cruz), monoubiquitinylated and polyubiquitinylated conjugate monoclonal antibody (ENZO), goat anti‐rabbit IgG, Alexa Fluor 488 goat anti‐mouse IgG, Alexa Fluor 594 donkey anti‐goat IgG, and Alexa Fluor 488 donkey anti‐goat IgG . The reagents used included interleukin‐1β  and NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Thermo).t test or 1‐ and 2‐way ANOVA, followed by the Tukey test. P<0.05 was considered statistically significant.Experimental results are expressed as the mean±SD. Groups were compared using the 2‐tailed Student To inactivate NF‐κB signaling in VSMCs, we produce mice with a deletion of IKKβ selectively in smooth muscle cells (KO mice), as previously described. Mice were born according to mendelian frequency, and there was no significant difference in growth between WT and KO mice Figure A. ImmunoNext, we examined the NF‐κB activity in IKKβ‐deficient VSMCs. IKKβ activates NF‐κB via the phosphorylation and degradation of IκB and eventually phosphorylates p65, a major subunit of NF‐κB.2‐induced aorta injury model. Unexpectedly, KO mice showed more significant calcification in aorta, and those aortas were more dilated compared with WT mice , as described previously,e Figure A, and no2 Figure B. Wester2 Figure C and 4D,2 Figure A. Furthe2 Figure B. These 2‐injured aortas of WT, KO, and KA mice. As shown in Figure 2 induces staining of active β‐catenin and Runx2 in nuclei of smooth muscle layers in KO aorta compared with that in sham group. In contrast, no apparent staining of active β‐catenin and Runx2 was observed in both KA and WT aorta, even after CaCl2‐induced aortic injury. These results are consistent with observations seen in cultured VSMCs.According to previous reports, β‐catenin is inactivated by ubiquitination via the ubiquitin‐proteasome pathway after it is phosphorylated.2 treatment. In contrast, terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed a remarkable increase of apoptotic cells after CaCl2‐induced aortic injury in KA mice, compared with WT and KO mice  mice, according to the previous report showing that point mutation of an amino acid inactivates kinase functionTo make it clear, we produced mice expressing kinase‐dead mutant (KD) of IKKβ in VSMCs on the background of IKKβf/f sm22CreNext, we examined β‐catenin and Runx2 signaling in KD VSMCs. Consistent with the previous results, active β‐catenin and Runx2 in KD VSMCs were significantly downregulated compared with KO VSMCs, clearly indicating that the kinase‐independent function of IKKβ is involved in the downregulation of β‐catenin Figure A. We alsRecent clinical studies have indicated that vascular calcification is different from atherosclerosis, but the mechanisms of vascular calcification have not been explored adequately. In this study, we show that the deletion of IKKβ, an essential kinase for NF‐κB activation, in VSMCs promotes vascular calcification. We also show that it is mediated by the upregulation of β‐catenin, Runx2, and transcription of osteoblast genes. Furthermore, we found that kinase‐independent function of IKKβ is involved in the regulation of such signaling. Considering that the accelerating roles of NF‐κB in atherosclerosis have been well analyzed, this finding would mechanistically explain the difference between atherosclerosis and calcification.The difference between atherosclerosis and vascular calcification has been only empirically suggested, but recent clinical studies have clearly demonstrated it.It was also an unexpected observation that more aortic dilatation was seen in KO mice. It has been reported that aortic calcification induces mechanical wall stress by decreasing biomechanical stability, which also associated with destruction and elongation of the elastic lamella in the medial layer.OSX), alkaline phosphatase (ALP), and osteocalcin (OCN), promote the transdifferentiation of VSMCs into osteoblast‐like cells.The molecular mechanisms of calcification in vessel walls and bone are reported to be similar, as indicated by the observation that the phenotypic transdifferentiation of VSMCs into the osteoblast‐like cells is seen in vascular calcification.Although the roles of β‐catenin in vascular calcification and bone formation have been well analyzed, as previously mentioned, those of IKKβ/NF‐κB signaling have not been adequately unveiled to date. In the bone research, it was reported that the deletion of IKKβ significantly enhanced bone formation by activating β‐catenin ubiquitination and degradation.Considering that kinase‐independent function of IKKβ regulates calcification, as shown in this study, the regulation of protein expression of IKKβ should be more carefully recognized. Interestingly, IKKβ is the target of miR148a, miR503, and miR199a,The findings in this study explain not only the difference between atherosclerosis and calcification but also the disappointing results reported in clinical studies that examined the effect of aspirin on calcification. Aspirin has a suppressive effect on inflammatory responses, including NF‐κB. Indeed, Yin et al reported that aspirin is a specific inhibitor of IKKβ.2 model on mice with modification of p65 or IκB in VSMCs would make it clear.There are strengths and limitations in this study. The strength is that kinase‐independent function of IKKβ in suppressing calcification is unveiled. The kinase‐dependent functions of IKKβ have been well analyzed; however, its kinase‐independent function has been rarely reported, except in our previous study.This work was supported by The Japan Society for the Promotion of Science KAKENHI grants 25461497 and 16H05297, a sponsored research program by Otsuka Pharmaceutical Co Ltd.None.