In this study, we unravel the atomic structure of a covalent resin near boundaries such as surfaces and composite constituents. For this, a molecular simulation analysis of epoxy resin hardening under various boundary conditions was performed. On the atomic level of detail, molecular dynamics simulations were employed to study crosslinking reactions and self-organization of the polymer network within nm scale slab models. The resulting structures were then coarsened into a graph theoretical description for connectivity analysis of the nodes and combined with characterization of the node-to-node vector orientation. On this basis, we show that the local bonding of epoxy resins near interfaces tends to avoid under-coordinated linker sites. For both epoxy–vacuum surface models and epoxy–silica/epoxy cellulose interfaces, we find almost fully cured polymer networks. These feature a local increase in network linking lateral to the surface/interface, rather than the dangling of unreacted epoxy groups. Consequently, interface tension is low (as compared to the work of separating bulk epoxy), and the reactivity of the resin surface appears negligible. Epoxy polymer-based composites are of increasing importance for many technological developments aiming at the combination of low syntheses costs and tailor-made material properties. While the thermosetting polymer offers excellent processing characteristics, the mechanical properties of pure epoxy resins are unsuited for most applications. This motivated concepts of polymer reinforcement, particularly by the embedding of fibers or (nano-) particles to increase toughness in a controlled manner. The range of such composite formulations is immense, and combinations of favorable effects are often employed. For example, toughening of epoxy resins by particle insertion may also provide better resilience to abrasion. In parallel, the embedding of fibers may account for improvements in the characteristics of wear and fracture ,2,3.To name just one example from the vast number of different devices made of toughened epoxy resins, wet clutches are typically based on epoxy composites including both particles  and fibers . The production process is akin to that of paper, entailing soaking of the fibers by water–particle mixtures, followed by pressing and drying of sheets or discs. Once dried, this “base paper” is then soaked by epoxy and hardener species, leading to the curing of the resin within a matrix of fibers and particles ,5,6. TheFor the understanding of such materials at the atomic/molecular scale, however, we fall short of experimental evidence. In turn, molecular simulation techniques offer in-depth insights into the manifold of atomic interactions accounting for the nature of the resin and its interplay with the composite constituents. This, however, requires careful preparation of the underlying simulation models, since basically no a priori knowledge of the polymer network structure is available apart from the almost 100%) degree of curing observed for industrial epoxies. We argue that the most reliable approach to model polymer network structures is to simulate the actual formation process. For this purpose, we combined a reactive molecular mechanics model with molecular dynamics simulation protocols, thus following the evolution of chemical bonding and structural relaxation during the curing process ,8. On th0% degreeIn the present work, we transfer these simulation protocols to heterogeneous systems, namely slab models mimicking epoxy surfaces and interfaces. In line with the composite applications described above, the investigated interfaces are focused on epoxy contacts to silica and cellulose. For all of these systems, we shall revisit the curing process such that epoxy network formation is elucidated in the presence of the corresponding boundary condition. On this basis, unprejudiced insights into the local nature of polymer network arrangement near surfaces and interfaces may be achieved from direct comparison to the bulk resin.The epoxy matrix for each model  was created from a 3D periodic simulation box featuring 1024 EPON and 512 DETDA molecules . Atomic 2. In turn, the silica–epoxy and cellulose-epoxy interfaces are described as sandwich models featuring 7.2 × 7.6 nm2 and 6.0 × 6.0 nm2 sized layer segments, respectively, in 3D periodic simulation boxes. In line with typical experiments, the curing is investigated at 460 K and 1 atm pressure [−1nm−2. After completion of the curing procedure, the repulsive walls are removed and the epoxy–vacuum slabs were allowed to relax at zero pressure within a 100 ns run at 300 K.Unlike our previous study on bulk epoxy , here, wpressure . This al2, which was stacked five times, resulting in slab of 1.8 nm thickness  by edges. In turn, both ends of the edges (the two –COC moieties of EPON) will then be connected to the linkers. While molecular simulations are used for the modeling of linking reactions and self-organization of the epoxy network, once the curing process of the thermosetting resin is accomplished, coarsening into a description inspired by graph theory offers a more general understanding of connectivity. For this, we define the chemical linker species, DETDA, as network nodes, whereas the EPON molecules are considered as network edges. In the fully cured epoxy resin, i.e., crosslinking degree of η = 100%, each node is 4-fold coordinated , and moving to global quantities such as degree distributions and degree–degree correlations . In our see also . Next, tThe statistical analysis of the angular occurrence profiles To investigate the local structure of epoxy surfaces and interfaces in an unprejudiced manner, each of the three sandwich-type simulation boxes was subjected to in-depth curing analyses from independent molecular simulation runs. Rather than simply attaching blocks of different constituents, our composite models describe resins that were cured in the presence of specific surface/interface boundaries. To ensure full comparability of the sandwich models to the bulk resin, the modeling and simulation protocols are implemented in full analogy to our earlier study of bulk epoxy resin curing.non-reacting (standard OPLS-AA models) DEDTA and EPON species [We hence start from stoichiometric mixtures of randomly arranged  species . In sepaUpon switching to our reactive forcefield (see Ref.  for deta3 (before curing) to 1.16 g/cm3 (fully cured), thus suggesting convergence of the curing process also in terms of resin density.Upon increasing the degree of crosslinking, the viscosity of thermosetting polymer increases drastically and full curing is thus beyond the scope of direct molecular dynamics simulations. To enable further crosslinking, we therefore employ the Monte Carlo procedure reported in Ref. . In fullOn this basis, in each of three sandwich models, the epoxy resin of was cured within ~100 ns scale simulation runs, achieving crosslinking degrees of η = 96–98% in the bulk of the epoxy slabs , Table 1Intuitively, one might expect a large number of dangling epoxy linker moieties at the boundaries of the resin at the end of the curing runs. However, slab − Qbulk indicates the disfavoring of epoxy–vapor boundary formation. For the sake of comparability, this difference in heat of polymerization ∆Q = −0.06 J/m2 is normalized per surface area (slab to the expected heat of polymerization of a bulk epoxy model (with the same number of molecules), but at a <η> = 96% degree of crosslinking , which leads to mechanical stress reduction that compensates about two-thirds of the loss in formation energy ΔQreaction stemming from the 3% lack of linking reactions.To demonstrate the interplay of crosslinking reaction energy and the mechanical stress in the thermosetting resin, we calculated the heat of polymerization, directly assessable from our reactive forcefield as the sum of all epoxy–epoxy interaction energy terms . Along tace area . To discslinking , namely To elucidate the change in the polymer network induced by the epoxy surface, we furthermore analyzed the orientation of the EPON entities with respect to the surface/interface normal vector. In To characterize the two interface models quantitatively, we first evaluated the heat of polymerization in analogy to the epoxy–vacuum system. In comparison to the surface model, the epoxy–silica and epoxy–cellulose interfaces show even stronger reductions ∆Q in the heat of polymerization per boundary area. However, the observed loss in epoxy–epoxy reaction energy was found to be over-compensated by epoxy–silica and epoxy–cellulose interactions, namely hydrogen bonding at the interface. This is reflected by the overall interface energy σ, which we define as the difference in total energy (per interface area) of the sandwich model as compared to the bulk epoxy resin and isolated silica/cellulose slabs. On this basis, we find negative (thus exothermic) interface formation energy for both silica and cellulose incorporation into the epoxy resin .2 for the bulk model of Ref. [While the energetics discussed above refer to the processes of resin and resin-based composite formation, the work of separation is of key importance for mechanical characterization. In our recent study on bulk epoxy deformation and fracture , the fra of Ref. . In contIn the past few years, molecular simulations achieved the in-depth analyses of the structure of polymer networks, including thermosetting resins ,20,21,22In the present study, we unravel the atomic structure of DEDTA/EPON thermosetting polymers next to three rather different boundaries to the resin. Inspection of the curing reactions, however, led to rather similar findings regarding the degree of linking and the arrangement of links between the nodes of the polymer networks. For both the epoxy surface and the interface models related to epoxy–cellulose and epoxy–silica composites, we found the polymer network to arrange parallel to the boundaries. The implication for the nature of epoxy-based composites is illustrated in Our findings underpin the importance of explicitly simulating the curing process in order to achieve realistic molecular simulation models of epoxy-based composites. Likewise, the proper account of nano-fibers and nano-scale layers of epoxy resins calls for the careful analysis of polymerization reactions in the presence of appropriate boundary conditions ,24,25,26While the present study was focused on flat boundary scenarios, we caution that studies of epoxy composites featuring particles of small dimensions  may call for explicitly considering curvature effects. This also applies to the consideration of chemical modification of particle surfaces, such as the introduction to amine moieties to achieve linker-type functionalization of silica particles that enable covalent incorporation into the epoxy network ,27,28,29


To better understand how MRAP2 modifies GHSR1a signaling, here we investigated the role of several phosphorylation sites within the C-terminal tail and third intracellular loop of GHSR1a, as well as the mechanism behind MRAP2-mediated inhibition of β-arrestin recruitment. We show that Ser252 and Thr261 in the third intracellular loop of GHSR1a contribute to β-arrestin recruitment, whereas the C-terminal region is not essential for β-arrestin interaction. Additionally, we found that MRAP2 inhibits GHSR1a phosphorylation by blocking the interaction of GRK2 and PKC with the receptor. Taken together, these data suggest that MRAP2 alters GHSR1a signaling by directly impacting the phosphorylation state of the receptor and that the C-terminal tail of GHSR1a prevents rather than contribute to β-arrestin recruitment.The melanocortin receptor accessory protein 2 (MRAP2) is essential for several physiological functions of the ghrelin receptor growth hormone secretagogue receptor 1a (GHSR1a), including increasing appetite and suppressing insulin secretion. In the absence of MRAP2, GHSR1a displays high constitutive activity and a weak G-protein–mediated response to ghrelin and readily recruits β-arrestin. In the presence of MRAP2, however, G-protein–mediated signaling  The “hunger hormone” ghrelin is secreted by X/A cells of the oxyntic mucosa of the stomach in response to a low energetic state, which leads to an increase in appetite , 2 and pq/11, thus stimulating the production of intracellular inositol triphosphate (IP) 3. Like other GPCRs, agonist stimulation results in phosphorylation of GHSR1a by kinases, including GPCR kinase 2 (GRK2) and PKC , MRAP2 is not present in every GHSR1a-expressing tissue. Consequently, it is possible that β-arrestin signaling plays an important role in the physiological function of ghrelin in tissues where MRAP2 is absent. Whereas, the inhibition of β-arrestin recruitment to GHSR1a by MRAP2 is well established and the domains of MRAP2 required for this function have been identified , the mol362, Ser363, and Thr366, have previously been shown to contribute to β-arrestin recruitment (A). β-arrestin recruitment was measured using the previously described NanoBiT recombination assay . Similar results were obtained after testing β-arrestin1 recruitment to GHSR1a and GHSR1aΔp-A , although MRAP2 only decreased β-arrestin1 recruitment by 40%. To test that the GHSR1aΔp-A mutant was functional, we measured ghrelin-stimulated IP1 accumulation in cells transfected with either GHSR1a or GHSR1aΔp-A alone or in the presence of MRAP2. Similar to WT GHSR1a .In Chinese hamster ovary (CHO) cells expressing WT receptor, ghrelin triggered a rapid and readily measurable recruitment of β-arrestin2; as previously shown, coexpression of MRAP2 blunted this effect Fig. 1B, ). In conB, . In cFig. 1D, ), GHSR1aF) with no significant difference in the total expression measured in permeabilized cells (G), suggesting that mutations in GHSR1aΔp-A result in impaired trafficking to the plasma membrane. This finding complicates the interpretation of the β-arrestin recruitment assay since the decrease in receptor surface density could explain the loss of β-arrestin recruitment. Therefore, to further assess the importance of the distal C-terminal tail of GHSR1a and its nine putative phosphorylation sites in modulation of β-arrestin recruitment, we generated a truncated form of GHSR1a by deleting the amino acids after phenylalanine 343 (H). Surprisingly, truncation of the distal C-terminal tail fragment did not impair either β-arrestin2 or β-arrestin1 recruitment (I and J). In fact, β-arrestin1 recruitment was increased by 50%. However, the inhibitory effect of MRAP2 on β-arrestin recruitment was also enhanced (I and J). In contrast to GHSR1aΔp-A, the surface density of GHSR1aΔc.t. was slightly higher than the WT receptor (K) with no difference in total expression (L).We then measured the surface density of 3HA-GHSR1a-LgBiT and 3HA-GHSR1aΔp-A-LgBiT cotransfected with SmBiT-β-arrestin using ELISA in nonpermeabilized cells. Whereas, GHSR1aΔp-A signaling was similar to the WT receptor, the surface density of GHSR1aΔp-A was greatly reduced F with noed cells G, suggesnine 343 H. Surpriruitment I and J. enhanced I and J. receptor K with nopression L.Together, these experiments show that, in accordance with previous reports , phospho252 and Thr261). To determine whether either residue is required to recruit β-arrestin, we generated two mutants of GHSr1a-LgBiT, in which either Ser252 or Thr261 were substituted for an alanine. LgBiT-fused receptors with either mutation were transfected in CHO cells with SmBiT-β-arrestin1 or SmBiT-β-arrestin2 in the presence or absence of MRAP2.Since the deletion of the distal C-terminal tail region of GHSR1a containing the putative phosphorylation sites did not prevent β-arrestin recruitment, we tested the importance of two putative phosphorylation sites contained within the ICL3 . In contrast, ghrelin-stimulated IP3 production was similar between WT and GHSR1a-S252A expressing cells; coexpression with MRAP2 reduced constitutive activity and enhanced the amplitude of the ghrelin-stimulated response similarly for the WT and mutated receptor (C). Surface expression of GHSR1a-S252A was about 40% lower than GHSR1a, suggesting the presence of spare receptors since signaling was not affected (D). Interestingly, the T261A mutation largely abrogated the ability of GHSR1a to recruit both β-arrestin2 and β-arrestin1 (E and F), and, like GHSR1a-S252A, signaling of GHSR1a-T261A was similar to the WT receptor while surface expression was about 40% lower (G and H). To determine whether the change in surface expression of the Ser252 and Thr261 mutants could explain the decrease in β-arrestin recruitment, we transfected cells with several concentrations of the 3HA-GHSR1a plasmid and measured both ghrelin-stimulated β-arrestin recruitment and receptor surface density. We found that β-arrestin recruitment is linearly proportional to the amount of receptor expressed at the cell surface (I and J); consequently, while the decrease in surface density of GHSR1a-S252A may be in part responsible for the decrease in β-arrestin2 recruitment observed for this mutant (A), the virtually complete abrogation of β-arrestin1 recruitment to GHSR1a-S252A (B) and the loss of recruitment of both β-arrestins to GHSR1a-T261A (E and F) are not due to the 30% decrease in receptor density. These results suggest an important role for the two phosphorylation sites within the ICL3 of GHSR1a for β-arrestin recruitment.Mutation of Serbrogated , A and Breceptor C. Surfacaffected D. Intererrestin1 , E and F0% lower , G and H surface I and J; s mutant A, the vi1a-S252A B and the1a-T261A , E and F32P) within GHSR1a. For this, we generated a bigenic plasmid containing the coding sequence for 3HA-GHSR1a and MRAP2 driven by the HSVTK promoter and a tetracycline-inducible promoter, respectively. To test the construct, we transfected CHO-T-REx cells with mock (GFP) plasmid or the bigenic construct in the presence or absence of tetracycline. CHO-T-REx cells were used because they display very low leakiness of the tetracycline-inducible promoter. This was verified by Western blot detection of MRAP2 in lysates from cells transfected with the bigenic plasmid. We found that, using this plasmid, MRAP2 was only detectable after treatment with tetracycline (A). The activity of MRAP2 on GHSR1a in this system was verified by measuring ghrelin-stimulated IP3 production in CHO-T-Rex cells transfected with the bigenic construct in the presence and absence of tetracycline. Here again, the signaling profile of cells without or with tetracycline matched previous results using cells transfected with GHSR1a alone or with MRAP2, respectively (B). The choice of using a bigenic construct for the phosphorylation studies was made because it allows measurement of GHSR1a 32P incorporation with or without MRAP2 expression in cells issued from the same transfection, thereby improving the reliability and accuracy of the assay.Since MRAP2 suppresses ghrelin-mediated β-arrestin recruitment to GHSR1a and phosphorylation of GPCRs is a critical step leading to β-arrestin recruitment, we next investigated whether expression of MRAP2 modified ghrelin-stimulated incorporation of phosphorus-32 ; however, in cells treated with tetracycline (thus expressing MRAP2), no phosphorylation of the receptor was detectable following ghrelin treatment (D). Phosphorylation changes in both conditions were quantified by densitometry analysis (E and F). These results establish that MRAP2 prevents ghrelin-stimulated GHSR1a phosphorylation, which provides a mechanism for the previously observed inhibition of β-arrestin recruitment by MRAP2.CHO-TREX cells were transfected with GFP or the bigenic plasmid in the presence or absence of tetracycline, incubated with [f GHSR1a C; howevereatment D. Phosphanalysis , E and F32P incorporation in GHSR1a in the presence or absence of the GRK2/3 inhibitor Compound 101. We found that Compound 101 significantly decreased ghrelin-stimulated GHSR1a phosphorylation (A–C), thus confirming that GHSR1a is a substrate of GRK2 and/or GRK3. Next, to determine if GRK2 inhibition reduces β-arrestin2 recruitment to GHSR1a, CHO cells transfected with GHSR1a-LgBiT and SmBiT-β-arrestin2 in the presence or absence of MRAP2 were treated with vehicle or Compound 101. Compound 101 treatment inhibited ghrelin-stimulated β-arrestin2 recruitment by about 50%, further confirming that GRK2-mediated phosphorylation of GHSR1a is involved in recruitment of β-arrestin (D). Again, MRAP2 inhibited β-arrestin recruitment by about 70% and the effect of MRAP2 and Compound 101 were not additive (D), suggesting that MRAP2-mediated reduction of β-arrestin recruitment to GHSR1a may involve the inhibition of multiple kinases as inhibition of GRK2 alone did not completely replicate the MRAP2 effect. We then performed the same experiment in cells transfected with GHSR1a-LgBiT and SmBiT-β-arrestin1. In contrast to β-arrestin2, pretreatment of cells with Compound 101 did not prevent β-arrestin1 recruitment to GHSR1a following ghrelin stimulation (E). This suggests that GRK2 and/or GRK3-mediated phosphorylation of GHSR1a contributes to β-arrestin2 but not β-arrestin1 recruitment.GRK2 and PKC appear to be the main kinases responsible for GHSR1a phosphorylation . Since Mrylation , A–C, tharrestin D. Again,additive D, suggesmulation E. This sF), thus demonstrating that MRAP2 interferes with the interaction of GRK2 with GHSR1a. This action of MRAP2 explains the decreased phosphorylation of the receptor and inhibition of β-arrestin2 recruitment observed.To determine if MRAP2 affects the interaction of GRK2 and GHSR1a, we generated NanoBiT constructs to kinetically and quantitatively measure the recruitment of GRK2 to GHSR1a. Our original approach involved fusing SmBiT to the N terminus of unmodified GRK2 and cotransfect the tagged kinase with GHSR1a-LgBiT; however, this assay did not display sufficient sensitivity likely due to the transient nature of the interaction between the kinase and the receptor. Therefore, to improve the assay and obtain measurable signal, we fused SmBiT to the N terminus of the kinase dead dominant negative mutant GRK2-K220R with which the interaction with receptors is more sustained. As such, luminescence recorded from CHO cells transfected with SmBiT-GRK2DN and GHSR1a-LgBiT was readily detectable. Upon addition of ghrelin, we observed a rapid increase in GRK2 recruitment, which was significantly inhibited by coexpression of MRAP2 F, thus dA, B and D). In contrast, either deletion of amino acids 24 to 33 (MRAP2Δ24–33), replacement of the transmembrane domain of MRAP2 with the transmembrane domain of RAMP3 (MRAP2-RTM), or substituting the C-terminal region of MRAP2 with the C-terminal region of MRAP1 (MRAP2-N2T2C1) resulted in a significant reduction in MRAP2-mediated inhibition of GRK2 recruitment . This experiment demonstrates that the regions of MRAP2 implicated in the inhibition of β-arrestin2 recruitment to GHSR1a are also important for the inhibition of GRK2 recruitment, which further implies that the inhibition of β-arrestin2 recruitment by MRAP2 is, at least in part, due to the inhibition of GRK2-mediated GHSR1a phosphorylation.We have previously shown that amino acids 24 to 33, the transmembrane domain, and the C-terminal region of MRAP2 are essential for the inhibitory function of MRAP2 on ghrelin-stimulated β-arrestin2 recruitment to GHRS1a . Therefo32P incorporation in GHSR1a, which was drastically reduced in the presence of Go6983 (A–C). Compound 101 and the combination of Go6983 and Compound 101 also blocked GHSR1a phosphorylation (A–C). To test the importance of PKC for ghrelin-stimulated β-arrestin recruitment, CHO cells expressing GHSR1a-LgBiT and either SmBiT-β-arrestin2 or SmBiT-β-arrestin1 with or without MRAP2 were treated with vehicle or the PKC inhibitor Go6983 before measuring ghrelin-stimulated β-arrestin recruitment. Pharmacological PKC inhibition resulted in a 50% reduction in both ghrelin-stimulated β-arrestin2 (D) and β-arrestin1 (E) recruitment, suggesting that, unlike for GRK2, GHSR1a phosphorylation by PKC is involved in the recruitment of both β-arrestin isoforms. Here again, the inhibition of β-arrestin recruitment by MRAP2 was greater than the inhibition mediated by Go6983, suggesting that inhibition of PKC alone is not sufficient to fully replicate the effect of MRAP2. Interestingly, inhibition of both GRK2 and PKC by combining Compound 101 and Go6983 resulted in further inhibition of β-arrestin2 recruitment (F), thereby mimicking the effect of MRAP2, consistent with the hypothesis that MRAP2 prevents the interaction of both GRK2 and PKC with GHSR1a. To verify that MRAP2 also blocks PKCα recruitment to GHSR1a, we generated a SmBiT-PKCα construct. Ghrelin-stimulated PKCα interaction with GHSR1a was measured in real time by transfecting CHO cells with GHSR1a-LgBiT and SmBiT-PKCα. In contrast to the assay, we developed for GRK2; recruitment of SmBiT-PKCα to GHSR1a-LgBiT was readily measurable without requiring the use of a kinase dead form of the enzyme. Whereas, ghrelin caused a rapid yet transient recruitment of PKCα to GHSR1a, expression of MRAP2 almost completely abrogated this effect (G). These results demonstrate that the inhibition of GHSR1a phosphorylation by MRAP2 is achieved by preventing GRK2 and PKCα interaction with GHSR1a.While the inhibition of GRK2 decreased β-arrestin2 recruitment to GHSR1a, it did not achieve the same inhibition as MRAP2. Additionally, the inhibitory actions of MRAP2 and Compound 101 were not additive suggesting that MRAP2 may prevent the recruitment of additional kinases to the receptor. For this reason, we tested whether PKC phosphorylates GHSR1a and participates in the initiation of β-arrestin recruitment. To determine if PKC phosphorylates GHSR1a, we transfected CHO cells with 3HA-GHSR1a, loaded the cells with 32P, and stimulated GHSR1a with ghrelin with or without the PKC inhibitor Go6983 and/or the GRK2 inhibitor Compound 101. Ghrelin caused a significant increase in f Go6983 , A–C. Corylation , A–C. Torrestin2 D and β-arrestin1 E recruitruitment F, therebs effect G. These GPCRs regulate numerous physiological functions by transmitting extracellular signals to intracellular domains utilizing G-protein–dependent and β-arrestin-dependent pathways. Biased ligands that preferentially activate either the G-protein or β-arrestin signaling pathway are valuable tools to study the functional consequence of each pathway separately or to promote the development of drugs with improved clinical outcomes , 21, 22.In this study, we first investigated the importance of different phosphorylation sites within GHSR1a for its interaction with β-arrestin. While the canonical interaction of β-arrestin with GPCRs is thought to be initiated by the engagement of β-arrestin with the phosphorylated C-terminal tail of the receptor and then, in some cases, progresses with the interaction of β-arrestin with one or more intracellular loops of the receptor, several other interaction modes have been described , 31. We 362, Ser363, and Thr366) were shown to be significant for β-arrestin recruitment , ghrelin preferentially stimulates Gαq/11-dependent signaling. However, it is also likely that in cells that express GHSR1a but not MRAP2 (like cardiomyocytes), ghrelin preferentially activates a signaling cascade downstream of β-arrestin. Ultimately, these results have potentially important implications when it comes to identifying drugs targeting GHSR1a. For example, it is possible that a β-arrestin-biased agonist of GHSR1a could provide cardioprotection without increasing appetite through activation of AGRP neurons. Finally, it is also possible that molecules that interfere with the interaction of GHSR1a with MRAP2 could reduce ghrelin action centrally to treat obesity without having deleterious cardiovascular effects.As MRAP2 enhances the ghrelin-stimulated activation of Gα2. Cells were transfected with LipoD293 DNA Transfection Reagent from SignaGen laboratory .CHO-K1 and CHO-T-REx cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 (ThermoFisher) supplemented with 5% v/v of a 1/1 mix of calf bovine serum and fetal bovine serum and 1% penicillin–streptomycin. Cells were incubated at 37 °C in a humidified atmosphere containing 5% COThe 3HA-GHSR1a plasmid was obtained from the Missouri S&T cDNA Resource Center. Plasmids coding for mutants of MRAP2 and β-arrestin recruitment were previously described . GRK2-K2CHO cells were plated in 12-well plates, and triplicate wells were transfected with indicated plasmids. The next day, cells were rinsed with PBS and fixed for 10 min in 4% paraformaldehyde. Paraformaldehyde was washed three times with PBS, and cells were blocked with 5% nonfat dried milk in either PBS (nonpermeabilized) or radioimmunoprecipitation assay buffer (permeabilized) for 1 h at room temperature (RT) on a shaker. Cells were incubated with primary antibody ant-HA (mHA.11) diluted at 1/5000 in blocking buffer for 2 h at RT on a shaker. Cells were washed three times for 5 min with PBS at RT on a shaker and incubated with secondary antibody  in blocking buffer for 1 h at RT. Cells were washed three times for 5 min with PBS before adding 200 μl of 3,3′,5,5′tetramethylbenzidine (Sigma–Aldrich). The reaction was stopped with 200 μl of 10% sulfuric acid. Three hundred liters of each sample was transferred to a 96-well plate, and absorbance was measured at 450 nm using a SpectraMax i3 plate reader.CHO cells were plated in 6-well plates and transfected with the indicated plasmids. Using TrypLE Express (Thermo Fisher Scientific), cells were then lifted and resuspended in 500 μl of DMEM/F12. Cell suspension (7 μl/well) was transferred in a white opaque 384-well plate. IP1 accumulation assay was performed using the IP-One kit (Cisbio) following manufacturer’s manual. Seven microliters of agonist diluted in stimulation buffer was added to each well and incubated for 1 h at 37 °C. Cells were then lysed and incubated with the detection reagents. Homogenous time-resolved fluorescence reading was performed on a SpectraMax i3 plate reader (Molecular Devices). Each condition was run in triplicate, and experiments were repeated independently at least three times.CHO-K1 cells seeded in 6-well plates were transfected with HA-GHSR1a-LgBiT and either SmlBiT-β-arrestin2, SmlBiT-β-arrestin1, SmlBiT-GRK2DN, or SmlBiT-PKCa with either empty vector or MRAP2. Cells were plated in a white 384-well plate (clear bottom), Nanobit substrate was added, and the plate was read with a FLIPR Tetra automated kinetic plate reader (Molecular Devices). A separate plate with agonist was prepared and placed in the FLIPR Tetra. Luminescence was measured in every well at a sampling rate of 3 to 5 s. After 5 min of recording without agonist to allow measurement of the basal luminescence signal, agonist was injected and luminescence was measured for an additional 15 min. Baseline signal was subtracted for each well. Ghrelin was injected at a final concentration of 1 μM and when indicated, cells were preincubated with Compound 101 and/or Go6983 for 30 min before the experiment at 30 μM and 1 μM, respectively. Each condition was run in triplicate and experiments were repeated independently at least three times.To test the effect of MRAP2 on GHSR1a phosphorylation, CHO-T-Rex cells were seeded in 6-well plates and transfected with the bigenic GHSR1a-TetO2-MRAP2 plasmid sin the presence or absence of 1 μg/ml tetracycline. To test the effect of Compound 101 and Go6983, we used CHO cells transfected with 3HA-GHSR1a. Twenty-four hours after transfection, cells were serum starved for 4 h, washed three times with phosphate-free Krebs/Hepes buffer, and incubated with 140 μCi/well of phosphorus-32 in phosphate-free Krebs/Hepes buffer for 1 h at 37 °C. Cells were then stimulated with 1 μM ghrelin for the indicated time, placed on ice, and lysed. Lysates were centrifuged at 13,000 rpm for 10 min at 4 °C, and supernatants were incubated with anti-HA antibody 1/5000  overnight at 4 °C before purifying immunocomplexes by incubating with Dynabeads protein G for 1 h 4 °C. Beads were resuspended in lithium dodecyl sulfate sample buffer, heated at 60 °C for 30 min, and separated by SDS-PAGE.For Western blot, proteins were transferred on polyvinylidene difluoride membrane. Membrane was blocked with 5% nonfat dry milk in PBS with Tween 20 (PBST), incubated with primary antibody  at 1/5000, and rabbit polyclonal anti-MRAP2 antibody 1/1000) in blocking buffer overnight at 4 °C. Membranes were washed three times 5 min with PBST, incubated with secondary antibody  1/5000 in 5% milk PBST for 1 h at RT, and washed time 5 min with PBST before imaging using SuperSignal West Pico PLUS Chemiluminescent Substrate  and an iBright imager.32P incorporation, gels were dried and phosphorimaging was performed using a Typhoon FLA 9500 .For measurement of All data described are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


Decidualization is a prerequisite for successful embryo implantation, in which elongated fibroblast-like endometrial stromal cells differentiate into more rounded decidual cells. Accumulating evidence has stressed the important role of the defective eutopic endometrium in infertility in endometriosis patients. However, the role of arginine methylation in the process of physiological decidualization and pathological decidualization defects is not clear. Here, we observed that the expression level of PRMT5, the main type II PRMT, was decreased in the endometrium of endometriosis patients, predominantly in stromal cells. Compared with the undecidualized state, PRMT5 was increased in the stromal cells of normal secretory endometrium in humans and in the decidua of normal pregnant mice or mice with artificially induced decidualization. The inhibition of PRMT5 resulted in a significant decrease in uterine weight and decidualization-related regulator expression, including FOXO1, HOXA10 and WNT4, in mice and IGFBP1 and prolactin levels in human endometrial stromal cells. Transcriptome analysis showed that decreased PRMT5 activity led to NF-κB signaling activation by inducing p65 translocation to the nucleus, which was also observed in endometriosis patients. Finally, overexpression of PRMT5 rescued the defective expression of IGFBP1 and prolactin in primary endometrial stromal cells from endometriosis patients. Our results indicate that promotion of PRMT5 may provide novel therapeutic strategies for the treatment of decidualization defects in infertile women, such as those with endometriosis. Endometriosis is a reproductive disorder in which endometrial tissue is aberrantly located outside the uterus. Almost 10% of reproductive-age women suffer from endometriosis related infertility and pelvic pain , 3. AccuDecidualization occurs during the secretory phase of the menstrual cycle controlled by progesterone and other ovarian hormones, which is a prerequisite for successful embryo implantation . DeciduaProtein arginine methyltransferases (PRMTs) are responsible for arginine methylation by transferring methyl groups from S-adenosylmethionine to a guanidine nitrogen of arginine in proteins, to generate three types of methylated arginine residues: monomethylarginine (MMA), asymmetric dimethylarginine (aDMA), and symmetric dimethylarginine (sDMA). PRMTs have been proven to be implicated in the regulation of many biological processes, including development and cancer . DependiPRMT5 mRNA from several published gene expression profiles of endometriosis [PRMT5 were significantly decreased in the ectopic endometrium of endometriosis patients compared to the endometrium of healthy controls  but expanded to uterine luminal and glandular epithelial cells and stromal cells at 3.5 dpc when the uteri entered receptive status . With thTo clarify the role of PRMT5 in endometrial decidualization, we applied GSK591, a specific inhibitor of PRMT5 , to an aWe next assessed the significance of PRMT5 on the decidualization of human endometrial stromal cells (hEnSCs). Immunofluorescence staining showed that PRMT5 was predominantly localized in the epithelial cells of the proliferative endometrium, while its localization was expanded to epithelial and stromal cells in the secretory endometrium Fig. . The expTP53 and FOXO1, while increased mRNA level of CREB1 in the mid-secretory phase eutopic endometrium compared with the fertile controls  and interleukin 1-β (IL1B) were increased significantly in GSK591-treated decidualized hEnSCs  analysis and Gene Set Enrichment Analysis (GSEA) further showed an enrichment of the NF-κB signaling pathway in GSK591-treated decidualized hEnSCs compared with decidualized hEnSCs Fig. . ImmunofSCs Fig. , which wSCs Fig. . All theIGFBP1 mRNA expression and secreted PRL levels. However, PRMT5 supplementation obviously promoted IGFBP1 mRNA expression  and secreted PRL levels   Fig. , indicatDecidualization defects are responsible for impaired endometrial receptivity and embryo implantation failure, contributing to reproductive disorders, such as endometriosis, recurrent implantation failure and recurrent pregnancy loss –28. ArgiPRMT mRNAs from several published gene expression profiles of endometriosis , we observed that PRMT5, a major type II enzyme, was decreased in the ectopic endometrium of endometriosis patients compared to the eutopic endometrium of healthy controls. In humans, circulating progesterone levels are increased following ovulation and maintained at elevated levels during the secretory phase, which is responsible for the decidualization of hEnSCs in nonpregnant cycles [Recently, some studies have reported increased expression of PRMT1 and PRMT3, two type I enzymes, in the endometrium of LPS-induced endometritis rats and the decidua of recurrent miscarriage patients, respectively , 30. By t cycles . Our datt cycles . PRMT5 wt cycles . We founNF-κB is a family of transcription factors composed of homodimers or heterodimers of related Rel proteins, including RelA p65), p105/p50, p100/p52, c-Rel, and RelB . The ups5, p105/pIn addition to being regulated by IKK, various posttranslational modifications have been found to be involved in the regulation of NF-κB, including ubiquitination, phosphorylation, acetylation, sumoylation, nitrosylation and methylation –46. PrevIn summary, we identified PRMT5 as a critical regulator in decidualization both in mice and humans, partly due to its effect on the NF-κB signaling pathway. Since several PRMT5 inhibitors are in ongoing preclinical and clinical studies for the treatment of cancer , it is w3, digested with 0.1% collagenase, centrifuged and filtered through a screen to obtain the primary human endometrial stromal cells (hEnSCs) for in vitro decidualization experiments as described previously [Endometrial biopsy samples were taken from women undergoing treatment at the Reproductive Medicine Center of Nanjing Drum Tower Hospital (2013-408 081-01). Informed consent was signed by the patients before sample collection. The women in the study were aged between 20 and 40, and none of them had received hormone therapy for at least 3 months prior to tissue collection. The endometriosis group was comprised of women with endometriosis ovarian cysts diagnosed by ultrasound, elevated serum CA125, or endometriosis ovarian cysts confirmed by laparoscopic surgery. The normal group was comprised of women receiving assisted reproductive treatment because of male infertility factors. Mid-secretory endometria timed 6–8 days after ovulation in the natural cycle, as determined by ultrasonography, were obtained from 26 normal women and 26 women with endometriosis for qRT–PCR, western blotting and immunohistochemical staining. Proliferative endometria were obtained from three normal women for immunofluorescence staining. Freshly collected endometrial tissues from 11 normal women and three endometriosis patients were cut into fragments of ~1 mmeviously . Detailen = 32) were purchased from the Lab Animal Center of Yangzhou University . Fourteen mice were subjected to a normal pregnancy assay. To induce in vivo decidualization, 6–8-wk-old female mice were ovariectomized (n = 25) with appropriate analgesics. After 14 days, the mice were injected with 100 ng E2  subcutaneously for three consecutive days. After 2 days of rest, the mice were injected with 1 mg P4  and 10 ng E2 subcutaneously for three consecutive days. After the last hormone injection, artificial decidualization was induced in one uterine horn by injection of 20 μL of sesame oil into the lumen. The other uterine horn was left unstimulated as a control. Daily hormone treatments with 1 mg P4 and 10 ng E2 were continued for another 5 days. During this process, the mice were treated with GSK591 (n = 3) , Ad-shPrmt5 (n = 6) and vehicle (n = 9) at times outlined in the procedure in Fig. ICR (Institute of Cancer Research) mice n = 2 were pu2 banding, followed by dialysis against 20 mmol/l Tris-buffered saline with 10% glycerol.An adenovirus harboring PRMT5 (Ad-PRMT5) was generated using AdMax  as previously described . The priThe primary endometrial stromal cells were isolated and cultured as described previously . Before 2O2 in deionized water for 15 min to block endogenous peroxidase activity, and nonspecific binding sites were blocked with 10% normal goat serum for 30 min at room temperature. The sections were then incubated with primary antibodies  at 95 °C for 10 min. The sections were then incubated with 3% HhEnSCs were fixed with 4% paraformaldehyde for 20 min at RT and then permeabilized with 0.1% TritonX-100 in PBS for 5 min at RT. Nonspecific sites were blocked with 1% BSA in PBS for 1 h at 37 °C. Endogenous proteins were stained with primary antibodies  in collected supernatants according to the manufacturer’s instructions. Samples were assayed in duplicate, and the concentrations were expressed as ng/mL cell supernatant.Protein was extracted from the tissues or cells using RIPA buffer with a phosphatase inhibitor  and protease inhibitors . The supernatant was extracted after high-speed centrifugation at 4 °C. The protein concentration was determined using the BCA Protein Assay kit . Equal amounts of protein (20 μg) were separated by SDS–PAGE and transferred to polyvinylidene difluoride membranes . The membranes were blocked with 5% skimmed milk for 1 h and then incubated overnight at 4 °C with primary antibodies  following the manufacturer’s instructions. RNA purity was assessed by measuring the OD at 260 nm and 280 nm, and RNA integrity was assessed by agarose gel electrophoresis. The first strand of DNA (cDNA) was synthesized from total RNA (1 μg) using the Takara PrimeScriptTM RT reagent kit . The expression levels of genes were analyzed by real-time PCR with SYBR Premix Ex Taq kits  using appropriate primers . ClusterProfiler49 was used to perform GO, KEGG and GSEA analyses. The transcription factor regulatory network was analyzed by KnockTF (http://www.licpathway.net/KnockTF/index.php).Primary endometrial stromal cells were cultured in 60 mm dishes and treated with GSK591  or solvent for 24 h and then decidualized for another 48 h. Total RNA was extracted from cells using TRIzol . RNA-seq and data analysis were performed by OE Biotech Co., Ltd. . Clean reads were mapped to the human reference genome using HISAT2. The DESeqR software package was used to identify DEGs (the threshold was set as Renilla luciferase activity.Preconfluent (60%) hEnSCs in 24-well plates were transfected with the indicated plasmids using Lipofectamine 3000 Reagent . NF-κB-Luc was purchased from Beyotime Biotech (#D2206). Dual-Luciferase Assay System  was used to analyze the luciferase activities with a luminescence counter (Berthold Technologies) according to the manufacturer’s instructions. Firefly luciferase activity was normalized to the corresponding t-test was used to compare data between two groups, and one-way ANOVA with Tukey’s multiple comparisons was used to compare data from more than two groups. Two-way ANOVA with Tukey’s correction for multiple comparisons was applied for data from two groups with three time points. Data quantification was expressed as the mean ± standard error (X ± SEM), and P < 0.05 indicated a significant difference in all cases. Ns = not significant.GraphPad Prism 9.0 was used for data statistics and analysis. No statistical methods were used to predetermine the sample size. Mice were randomly allocated to experimental groups. No blinding method was used for animal studies. There was no animal exclusion criteria. The variance was similar between the groups that were being statistically compared. A Student’s Supplementary Figures and Figure legendsSupplementary Table 1. Clinical characteristics of women recruited in the studySupplementary Table 2. Primers used for Ad-shPrmt5 (5'-3')Supplementary Table 3. Primers used for qRT-PCR (5'-3')Supplemental Table 4. Details of antibodies usedOriginal Data File


A family of signal transduction pathways known as wingless type (Wnt) signaling pathways is essential to developmental processes like cell division and proliferation. Mutation in Wnt signaling results in a variety of diseases, including cancers of the breast, colon, and skin, metabolic disease, and neurodegenerative disease; thus, the Wnt signaling pathways have been attractive targets for disease treatment. However, the complicatedness and large involveness of the pathway often hampers pinpointing the specific targets of the metabolic process. In our current study, we investigated the differential metabolic regulation by the overexpression of the Wnt signaling pathway in a timely-resolved manner by applying high-throughput and un-targeted metabolite profiling. We have detected and annotated 321 metabolite peaks from a total of 36 human embryonic kidney (HEK) 293 cells using GC-TOF MS and LC-Orbitrap MS. The un-targeted metabolomic analysis identified the radical reprogramming of a range of central carbon/nitrogen metabolism pathways, including glycolysis, TCA cycle, and glutaminolysis, and fatty acid pathways. The investigation, combined with targeted mRNA profiles, elucidated an explicit understanding of activated fatty acid metabolism (β-oxidation and biosynthesis). The findings proposed detailed mechanistic biochemical dynamics in response to Wnt-driven metabolic changes, which may help design precise therapeutic targets for Wnt-related diseases. One Clinical importance has been proposed for Wnt/β-catenin signaling pathway in both normal homeostasis and abnormal status of organs . The dys8HEK293 cells  were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1%penicillin/streptomycin (Pen/Strep). Wnt3a-secreting L cells (ATCC) were utilized for Wnt3a-conditioned medium (Wnt3a-CM). The development of HEK293-firefly luciferase (FL) reporter cells was conducted by transfection of β-catenin/TCF-dependent FL reporter (TOPFlash) as previously described . The DuaFreeze-dried cell samples (1 mg) were dissolved in 100 μl of lysis buffer. Lysis buffer consisted of protein inhibitor and radioimmunoprecipitation assay (RIPA) buffer. To determine the quantity of protein, Bradford assay was employed. Equal volumes of lysates were separated by SDS-PAGE  and transported onto nitrocellulose membranes . The blocking for western blot analysis was performed by soaking in SuperBlock Blocking Buffer  for one hour. Anti-β-catenin  was utilized for western blot detection of β-catenin. β-actin was chosen as a loading control using anti-actin . Blotting was visualized and quantified using the ECL system  and the ImageJ software .Total RNA was isolated using RNeasy mini kit  and the techniques in the associated protocol. cDNA was synthesized with the RT2 First Strand kit . cDNA was mixed with RT2 qPCR master mix for performance of SYBR Green-based real-time PCR . The CFXg at 4°C).Freeze-dried cell samples were disrupted using a Mixer Mill MM400  with a single steel bead . A 750 μl of extraction solvent  was used to extract the metabolites of the powder samples. The samples were homogenized in Mixer Mill MM400 at frequency of 25 Hz for 10 sec, followed by sonication (5 min) and centrifugation  derivatization was performed in two steps , 15. Fir library .m/z tolerance, 0.005 Da; Product ion m/z tolerance, 0.05 Da, MS/MS similarity score: 70%)Details of the metabolic profiling methods can be found in our previous publication . In summhttps://github.com/CHKim5/LMSstat). Principle component analysis (PCA), partial least squares-discriminant analysis (PLS-DA) were performed using SIMCA 17 software . Significance analysis of microarrays (SAM) and hierarchical clustering heat map were performed using MetaboAnalyst 4.0 . The metabolite-protein interaction network was constructed using STITCH (http://stitch.embl.de) . Th. Thq-valerase 1C  29]..q-value p-value < 0.05) to the 41 genes  based on in-silico chemical-protein association. The association included experimental evidence of direct chemical-protein interaction and enzyme-substrate relatedness from metabolic pathway, which is all deposited and extractable from STITCH . The baited proteins and metabolites without interactions were excluded from network. The resultant network identified palmitic acid as a primary hub, which has 16 interactions including 4 metabolites and 8 genes (ACSL1 (Acyl-CoA synthetase long-chain family member 1) showed the highest connectivity in the network. ASCL1 is one of the members associated with fatty acid ligase activity and long-chain fatty acid-CoA ligase activity. ACSL1, long-chain acyl-CoA synthases, synthesizes fatty acyl-CoAs from long-chain fatty acid and accelerates the early stage of fatty acid metabolism . CP. CPp-valxidation .In our current study, given the activation of Wnt/β-catenin signaling by Wnt3a-CM, we identified the treatment-specific alteration of the metabolome with high temporal resolution. Of note, hexose, including glucose and fructose, showed down-regulation in cell with activated Wnt/β-catenin signaling, which may be associated with promotion of cell proliferation by β-catenin. Cells in proliferation state require the primary carbon source for building blocks and energy production. Our results supported the metabolic evidence from coordinately regulated central carbon/nitrogen metabolism, including glycolysis, TCA cycle, and glutaminolysis. Of note, the activation of fatty acid metabolism was determined at both metabolite and gene levels. The hyper-activity may meet the need for increased energy production via β-oxidation in CRT-positive cells . At the http://jmb.or.kr.Supplementary data for this paper are available on-line only at


Yet, satire differentially affected patriotic attitudes. This effect was moderated by country and age: younger U.S. citizens became less patriotic after exposure to the one-sided satire that targeted their country and decreased their positive emotions; older U.S. citizens, in contrast, became more patriotic after exposure to this one-sided satire that particularly increased their negative emotions. The Dutch audience’s level of patriotism remained stable irrespective of satire sidedness. Altogether, this study demonstrates how humor type, country-of-reception, and age matter for satire effects.The differential satire effects across domestic and foreign audiences are largely unknown; yet, this is of growing relevance as political satire increasingly reaches international audiences. A two-country experiment was conducted in which satirical stimuli from the Netherlands with either a one-sided (only targeting the United States) or two-sided humorous message (targeting both the U.S. and the Netherlands) was presented to a domestic (in-group) or foreign (out-group) audience. Specifically, this study examines political satire’s differential emotional and attitudinal impact on audiences located in the country-of-production (Netherlands) or abroad (U.S.). Results show that satire sidedness  While more research regarding satire has emerged in other continents e.g., , these sThis prompts questions regarding what effects take place when satire crosses national borders and whether domestic versus foreign audiences are influenced differently. The current study addressed these questions, using an experiment to compare effects of Dutch satire on viewers from the U.S. (foreign audience) and the Netherlands (domestic audience). Furthermore, we explicitly differentiated between a one-sided satire that only targets the other country  versus a two-sided mode of humor that simultaneously included self-mockery towards the in-group (the Dutch in this case) and jokes targeting the foreign out-group (U.S.).highly educated among student samples  is often more Juvenalian , more feExposure to one-sided satire (a) reduces positive emotion and (b) increases negative emotion compared to two-sided satire.1.1.2others’ misfortune or disparaging an unaffiliated target, one might experience amusement via enhanced self-esteem  or with the source of the message . After all, superiority theory explains that humor may cause positive feelings through a downward comparison . By makif-esteem . This elf-esteem , which pf-esteem , one couLinking this back to the Benign-Violation Hypothesis, people are more likely to perceive a violation as benign when they identify less with the satire target . Althougless likely for two-sided satire in which multiple groups are simultaneously targeted, including the in-group of the satire producer. Two-sided joking is by itself perceived as less aggressive, more sympathetic  reduces positive emotion and (b) evokes negative emotion compared to two-sided satire, and this effect is stronger among the out-group whose identity is targeted by the one-sided satire than among the in-group.1.1.3Previous research found that younger audiences process satirical texts more easily than older audiences , and thaBecause younger people are more engaged with satirical content than older people , they wiExposure to one-sided satire (a) reduces positive emotion and (b) evokes negative emotion compared to two-sided satire, and this effect is stronger among younger than among older people.1.21.2.1The other-deprecating message in one-sided satire likely causes a stronger identity threat among the targeted out-group people compared to the in-group people who are not targeted by this one-sided satire . This diConcretely, satire targeting one’s own country could pose a perceived threat to one’s national identity , which mpatriotism as dependent variable.However, the opposite is also possible. Identity threat caused by one-sided satire could instigate a process of motivated reasoning and counterarguing among the out-group, which eventually could reinforce their identity. To examine this, the current study operationalizes this potential process by studying From the literature, it is also not obvious whether one-sided satire or two-sided satire results in the highest level of patriotism among the in-group audience. When a political actor engages in self-directed humor, citizens generally develop a more positive attitude towards the politician . By engaHowever, it is also true that this in-group is only confronted with criticism of their own identity within the two-sided satire. When combined with the persuasive power of satire , this coHow does exposure to one-sided satire influence patriotic attitudes compared to exposure to two-sided satire, and (b) how does this effect differ between the in-group and the out-group?(a) 1.2.2The well-established Impressionable Years Hypothesis  states tPrevious research found that younger people are more narratively engaged in satirical content than older viewers . NarratiAccordingly, it is rather unlikely that younger foreign viewers (in our case from the U.S.) engage in counter-arguing to defend their social identity when exposed to one-sided satire from another country that targets their nation hence, their level of patriotism likely decreases. In contrast, older audiences will be relatively less engaged in the satirical narrative and therefore will likely experience more reactance, feeling more disparaged by the one-sided satire. In such case, we would expect the older audiences to counterargue the message, thereby potentially strengthening their patriotism to protect their self-identity.Put simply, we expect that narrative engagement among younger audiences reduces their propensity to engage in identity protection in response to the harsher critique in one-sided satire, while more reactance  among the relatively less engaged older audiences stimulates their need for identity protection. Thus, a cleaved age moderation effect would ensue. Previous research has also found that older citizens learn less from political comedy exposure than younger people  as they Exposure to one-sided satire that targets the out-group alone compared to two-sided satire that simultaneously targets both the in-group and the out-group will cause a stronger decrease of patriotism among younger out-group citizens than among older out-group citizens.Similarly, age was expected to moderate the effect of satire sidedness on patriotism among in-group citizens (in our case: Dutch viewers). Also for the in-group, a less critical processing of the satire would be expected among the younger audiences that will be more narratively engaged and therefore more easily persuaded . YoungerExposure to two-sided satire that simultaneously targets the in-group and the out-group compared to one-sided satire that targets the out-group alone will cause a stronger decrease of patriotism among younger in-group citizens than among older in-group citizens.2SSI/ResearchNow. Quotas were set on age , gender , and political ideology to ensure a diverse sample on at least these characteristics. To ensure data quality, participants who did not recall the topic of the video (n = 37) or who were outliers on response time (following n = 51) were excluded and dropped from the dataset prior to data analysis.1Initially, the cut-off point of 60 minutes or more was used as rule-of-thumb, leading to substantially the same results. However, Tukey’s method is a stronger standard in the literature and provides the necessary robustness against outliers at the tails . Average response time was 21 min (SD = 6.89).A randomized experiment was simultaneously conducted in the Netherlands and the United States using samples from 2.12F = 0.01, p = 0.942, gender χ2(1) = 1.26, p = 0.262, education, F = 1.88, p = 0.171, nor political ideology, F = 0.32, p = 0.570.No differences between conditions were found for age,  Both videos were produced and broadcasted by the Dutch satire show Zondag Met Lubach. Participants were explicitly informed about the Dutch origin of the clip on the survey-page before stimulus exposure. The original videos were already narrated in English, which made them understandable for a foreign English-speaking audience (in this case: U.S. audience) and explains why the videos could go viral outside of the domestic Dutch context. Both videos satirized a conservative/right-wing political subject directly related to the United States.Participants were randomly exposed to one of two satire clips that lasted between 3 and 4 min: randomization was successful; hence, no control variables were needed in the statistical analyses.2.1.13https://www.youtube.com/watch?v=a-o9pwWUzz0.See:  It criticized the weapon policy of the United States and describes a “terrible epidemic: nonsensical rifle addiction, NRA.” The video continues by claiming that NRA is a “constitutional disorder that is caused by a dysfunction of the prefrontal second amendment in the nonsensical cortex causing patients to shoot. It starts with an innocent Colt, but soon patients will show signs of shotguns, sniper rifles, and M-16s even. Often, patients use silencers to hide their condition.” After describing the danger for the environment of people with NRA, it stated that “NRA is highly contagious. Parents often pass it on to their children: this happens automatically or semi-automatically.” By the end of the clip, it showed a list of bullet points—because these bullets do not kill people – stating that the Red Cross can help the situation with “water, blankets, facts, insights, statistics, and truth bombs.” Importantly, the one-sided satire clip did not contain any reference to the Netherlands; it was fully other-directed.The one-sided condition satirized the United States through the National Rifle Association of America (NRA)—a gun rights advocacy group.2.1.24https://www.youtube.com/watch?v=ELD2AwFN9Nc.See:  It did so with two-sided jokes that simultaneously satirized the U.S. and its new President (and thereby the U.S. electorate) but also mocked the home country of the satirist (the Netherlands). The video ironically presented topics for which the Dutch could be similarly ‘proud’ as U.S. citizens. Examples included a major dam  and amusement park particularly known for its pony rides: “The best pony park in the world. It’s true. They are the best ponies. You can ride them, you can date them, you can grab ‘em by the pony. It’s fantastic.” The satire continues: “In December, we have this scandalous tradition of Black Pete. It’s the most offensive, the most racist thing you have ever seen. You’ll love it, it’s great.” And later on: “We also have a disabled politician for you to make fun of (….), she is from the Ministery of Silly Walks.” By the end of the clip, the narrator asked Trump for support: “We totally understand it is going to be America first. But can we just say, the Netherlands second?” Thus, this two-sided satire targeted both the other  and the self .The second video introduced the Netherlands to Donald Trump upon his inauguration speech in which he emphasized “America First”.2.1.32-test confirmed that the two clips functioned in the intended way regarding satire sidedness, χ2(4) = 251.78, p < 0.001: the one-sided satire clip was perceived by 82.8% of participants to solely be “criticizing (making fun of or joking about)” one country  only. The dominant perception in the two-sided satire condition was instead that the clip criticized the two nations simultaneously (81.5%).A χ2.22.2.1Hedonic enjoyment. Enjoyment was measured with two separate scales to represent that people may experience both positive and negative emotions independent from each other : surprised, happy, proud. A scale of negative emotion was composed of the following emotions : angry, concerned, frustrated, afraid, offended. Positive and negative emotion only correlated weakly , which demonstrated that these are two separate constructs requiring separate analysis.From this list of emotions, a scale of Patriotism. Four items from the scale of α = 0.82, M = 2.10, SD = 0.96): (a) I support my country’s political leaders even if I disagree with their actions; (b) people who do not wholeheartedly support America/Netherlands should live elsewhere; (c) I believe that U.S./Dutch policies are almost always good for our country; and (d) America/Netherlands is a better country than most other countries.2.2.2Age category. Age of participants ranged from 19 to 67. The PROCESS 3.3-tool , they represented the theoretical out-group in this study. 237 people were recruited from the Netherlands (50.2%), who represented the theoretical in-group coming from the same country as where the satire had been produced.33.13.1.1M = 3.52, SD = 2.55) had a strong suppressing effect on the positive emotions that were experienced while viewing the clip compared to exposure to the two-sided satire condition , t(470) = −5.60, p < 0.001, Cohen’s d = 0.52. This provides evidence in line with Hypothesis 1a.The sidedness of the satire stimuli affected audience emotions in the hypothesized direction. One-sided satire (t(470) = 3.66, p < 0.001, Cohen’s d = 0.34. One-sided satire  evoked more negative emotion than exposure to two-sided satire . This provided evidence in line with Hypothesis 1b. As can be inferred from the Cohen’s d-values, the effect of satire sidedness on positive emotions was stronger than the effect on negative emotions.Negative emotion was also strongly affected in the expected direction, 3.1.2F = 0.78, p = 0.377; and for negative emotion, F = 0.142, p = 0.706. reduced positive emotion (graph above) for both the in-group of Dutch viewers and the out-group of U.S. participants compared to the two-sided satire. Moreover, the one-sided satire also uniformly increased negative emotion (graph below) among both the in-group and the out-group compared to the two-sided satire. Thus, no support was found for Hypothesis 2.The out-group viewers from the targeted country in the one-sided satire (United States) did not experience stronger decreases of positive emotion nor stronger increases of negative emotion compared to the in-group of Dutch viewers. The interaction effects between satire sidedness and the country from which participants originated were insignificant for both indicators of hedonic enjoyment: for positive emotion, 3.1.3b = −0.08, SE = 0.03, p = 0.017. This implies that the effect of satire sidedness on hedonic enjoyment was indeed conditional upon age, but that the strength of the moderation effect depended on the specific country.We hypothesized that the emotions of younger viewers should be more strongly affected by the sidedness of satire compared to older viewers. Running a full factorial model  compared to the older viewers . The difference in effect strength was significant and in the expected direction: relatively less positive emotion was elicited by the one-sided satire versus the two-sided satire among the younger people.Looking at the U.S sample  than among the younger participants . Hypothesis 3a was thus only partially supported: one-sided satire indeed reduced positive emotion most strongly among younger viewers, but only in the satirized out-group of U.S. viewers.An opposite pattern was found in the Netherlands (p = 0.741). Examining this interaction effect while leaving out the country factor, though, we found that the positive effect of one-sided satire on negative emotions strengthens with higher age, b = −0.05, SE = 0.02, p = 0.004. The Johnson-Neyman significance region begins at age 39: b = −0.18, p = 0.580), whereas negative emotions significantly increased after exposure to the one-sided versus the two-sided satire for the older viewers . So, Hypothesis 3b was rejected: one-sided satire elicited negative emotions particularly among the older audience – and this was not moderated by country.With respect to negative emotion, the three-way interaction between satire sidedness, age, and country was insignificant  = −0.26, p = 0.799.No across-the-board effect of satire sidedness on patriotism was yielded in response to RQ3.2.2F = 0.62, p = 0.686. The answer to RQ1b was, thus, that exposure to one-sided satire versus two-sided satire did not influence patriotism differently between the in-group and out-group, because neither were influenced across-the-board.No moderated effect between country and satire sidedness on patriotism was found either, 3.2.3b = −0.02, SE = 0.01, p = 0.049. Zooming in on this finding, the interaction effect between satire sidedness and age on patriotism was only significant in the U.S. (p = 0.008), but not in the Netherlands (p = 0.816). Hypothesis 4b is thus rejected, because that sub-hypothesis expected an interaction effect among the in-group of Dutch viewers. However, no effects of satire sidedness on patriotism were found among the in-group participants from the satire’s country of production .A full factorial OLS regression model was run using satire sidedness (one-sided vs. two-sided satire), age, and country of residence together with all their possible interaction effects as independent variables and patriotism as dependent variable. A three-way interaction effect on patriotism was yielded, F = 7.04, p = 0.009. As predicted, a small positive effect on patriotism was found in exposure to the one-sided satire for the older U.S. citizens; thus, indicating a potential process of reactance among the older out-group. This corresponds with the stronger negative emotion they experienced in response to the one-sided satire.Regarding the U.S. sample, an interesting pattern was found. Results showed that the effect of exposure to one-sided versus two-sided satire on patriotism indeed varied for younger versus older participants. The Johnson-Neyman significance region was found at value below 33.2 years old (for negative effect on patriotism) and above the value of 65.3 years (for a positive effect on patriotism); these age boundaries closely replicate the moderated effect in earlier work . Figure a, which predicted that one-sided satire targeting the U.S. compared to two-sided satire would result in a higher level of patriotism among older U.S. citizens compared to younger U.S. citizens. The latter (young U.S. citizens) instead became less patriotic due to the one-sided humoristic attacks on their country.For younger U.S. citizens, in contrast, a negative effect of exposure to the one-sided satire on patriotism was found. Thus, indicating a process of message acceptance, which corresponds to their lower level of positive emotions after the one-sided satire. Altogether, this finding confirms Hypothesis 446https://twitter.com/samuelljackson/status/966060490219597824.E.g.,  interacted with on social media  alone. This suggests that it is not just the (downward) social comparison in one-sided satire that restricts the process toward enjoyment e.g., . Insteadlower levels of patriotism after exposure to the one-sided satire compared to the two-sided satire. Older U.S. audiences, in contrast, maintained a relatively higher level of patriotism after exposure to the one-sided satire that criticized their country. Two theoretical explanations help to understand this conditional response in the non-domestic out-group. First, younger audiences probably were more narratively engaged with the story line of the satire  when exposed to the one-sided satire. In contrast, negative emotions  were particularly elicited among older U.S. citizens by the one-sided satire, which suggests that a motivated reasoning process was activated amongst them. Analyses of indirect effects, subsequently, showed that both the negative and positive emotions functioned as significant mediators to shape patriotic attitudes. Whereas patriotism decreased among young U.S. audiences due to message-acceptance of the one-sided satire that targeted their home country , the opposite occurred for the older U.S. viewers who ostensibly engaged in motivated reasoning (via elicited negative emotion) to defend their national pride when confronted with the one-sided satire critiquing their nation. Exploratory analyses showed that the interactions with age have not been caused by differences in political ideology .Additional analyses of emotional response as a mediating mechanism between satire sidedness and patriotism confirmed that both outcome variables could have influenced each other see .7For reaThe current study thus shows that context matters when predicting the effects of satire. Moreover, we have further explored the boundary conditions of the genre’s impact: when satire reaches a foreign audience with comedy that targets another nation, it is especially likely the younger foreign audience’s attitudes will be influenced in line with the satirical message, whereas the older foreign audience is more likely to experience a boomerang effect. Meanwhile, the domestic audience’s patriotic attitudes remained unaffected. This finding is especially relevant during a time when questions of foreign influence on domestic elections are rising throughout the world. Does exported satire have the power and influence to affect democratic processes in foreign nations? While likely constrained by reach and viewership, the potential for such influence merits further investigation.Just as many studies on satire before, the current investigation tried to strike a balance between internal and external validity of the study design. This is always complicated for satire research, because externally valid stimuli (a) require high-quality audiovisual materials that (b) are still sufficiently humorous. This is difficult to craft oneself, making that most experimental studies (including our own) relied on existing clips. Consequently, one must compromise on internal validity: often studies compared clips of different shows with different hosts, which could be confounding factors. In our case, video clips have been used of the same show and of a similar length, but the targets of satire were different . Although these targets were consciously chosen for their relative ideological similarity, the target is still a potentially confounding factor, and thus a limitation, of our experiment. To elevate political satire research to a next level, future research should consider producing stimuli in cooperation with professional satire makers to create funny though comparable clips of different humor styles.


Tumor killing and wound healing are two complementary and influential processes during the treatment of melanoma. Herein, a two-layered microneedle platform was developed with bifunctional effect of chemo-photothermal synergistic melanoma therapy and skin regeneration. The bifunctional platform composed of embeddable curcumin nanodrugs/new Indocyanine Green/hyaluronic acid (Cur NDs/IR820/HA) microneedles and sodium alginate/gelatin/hyaluronic acid (SA/Ge/HA) supporting backing layer was prepared through a two-step casting process. With uniform incorporation of curcumin nanodrugs and IR820, the microneedles exhibited excellent photothermal performance under external near-infrared (NIR) light stimulation and tumor co-therapy ability. Once the embeddable microneedles were inserted into skin, they would rapidly dissolve and activate drug release successfully for tumor treatment. Moreover, the SA/Ge/HA supporting backing layer was left behind to cover the wound and promote the proliferation of endothelial and fibroblasts cells for enhanced skin regeneration. The two-layered microneedles platform can simultaneously eliminate the tumor and accelerate wounding healing, which may be potentially employed as a competitive strategy for the treatment of melanoma. As the largest organ of human body, skin tissue is a crucial defense that protects the human body from outside environmental disturbance , 2. NowaTo address this issue, we designed a two-layered microneedle platform with a dissolvable microneedle part and a biodegradable backing cover. As a kind of transdermal drug delivery system, microneedle (MN) is famous for its use in biomacromolecules delivery –14, due Surgical resection is the most common modality for treating melanoma in clinic. The large skin defect from surgery is difficult to heal and could cause chronic wounds. Unfortunately, few studies addressed the issues of tumor-induced skin repair . AlthougBecause melanoma is a superficial skin cancer, which could rapidly spread into the surrounding skin, local therapy by MNs along with PTT is a more suitable method to melanoma than the systemic therapy. The local therapy strategy can enhance delivery efficiency to the target tumor site, avoid excessive circulation and side effects to normal tissues , 40. In Herein, a two-layered MN system was developed via a two-step casting process. As displayed in Fig. Cur NDs were synthesized via a reprecipitation approach. Due to strong intermolecular interaction, the Cur molecules self-aggregate and precipitate to form Cur NDs , 33. As The SEM image of lyophilized SA/Ge/HA scaffold of the backing layer presented a porous and interconnected structure, which had uniform size and distributed homogenously Fig. F. The avFigure −4 N/needle) to 500 g (~ 1.225 × 10−2 N/needle) weights on MNs [To clarify whether the HA MNs are mechanically strong enough to penetrate through the stratum corneum, the morphology changes of MNs tips against static force were observed. As displayed in Fig. s on MNs . The micTo verify whether the HA MNs could successfully penetrate into the skin, the MNs were applied to nude mice skin. The brightfield photograph of mice skin displayed the uniform microchannels created due to the insertion of HA MNs Fig. C. The ro2) were captured by the near-infrared thermal camera and the temperature change curves  in G2, G5 and G6 were recorded, which demonstrated the changing temperature of the tumor site : Control group, HA MNs group, and Cur NDs/IR820/HA MNs group. At day 7, all the three groups showed a decrease of the wound area, while better wound closure was observed in HA MNs group and Cur NDs/IR820/HA MNs group compared to control group  the microneedle-based system is relatively simple, convenient, and noninvasive, (2) photothermal/chemo co-therapy could efficiently control the progression of tumor, (3) the curcumin nanodrugs could not only inhibit the melanoma cells growth, but also reduce inflammation and promote skin regeneration process, (4) the SA/Ge/HA backing layer is biocompatible and could promote wound healing. This bifunctional two-layered microneedle system is a desirable candidate for photothermal/chemo skin tumor therapy and simultaneously treating wound defect.3 kDa) was obtained from Freda Biochem Co., Ltd . Curcumin was purchased from Dalian Meilun Biological Co., Ltd. Sodium alginate, IR820, and gelatin (from porcine skin) were obtained from Sigma-Aldrich (USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and all other cell culture media and reagents were bought from Gibco™ . Live/dead cell imaging kit was acquired from Thermo Fisher Scientific Inc . CCK-8 kit was purchased from Houston, Texas, USA. The polydimethylsiloxane (PDMS) microneedle templates were acquired from Guangzhou SLLK Co. Ltd .Sodium hyaluronic acid  were purchased from Chengdu Dashuo Animal Experiment Co. Ltd . C57BL/6 mice  and nude BALB/c mice  were obtained from Beijing HFK Bioscience Co. Ltd. . The animal researches were admitted and supervised by the animal committee of West China Hospital of Stomatology, Sichuan University.The Cur NDs/IR820/HA MN patches were prepared through a two-step casting process. Firstly, the Cur NDs were synthesized via a reprecipitation approach . In brieThe bright-field microscopic photographs of MNs were captured by a stereomicroscope . The morphology of the microneedle structures, the Cur NDs and the lyophilized SA/Ge/HA backing layer were characterized by scanning electron microscopy . The hydrodynamic size of the Cur NDs was determined by a DLS instrument . The average pore size of lyophilized SA/Ge/HA backing layer was estimated with the Smile-View software. Briefly, 100 pores were chosen randomly from the SEM images of the lyophilized SA/Ge/HA backing layer scaffold to calculate the average pore size using the “Length Measurement” tool.For in vitro dissolution analysis of MNs, the Cur NDs/HA MNs were applied onto porcine skin and fixed with a gum tape. The MNs were taken off from the skin at indicated time intervals  and imaged by brightfield stereomicroscopy to visualize the dissolution of microneedles.To evaluate the mechanical strength of HA MNs, the MNs were tested with a serial of weights. Briefly, the MN patches were positioned vertically (microneedle tips facing up), and weights of 10, 50, 100, 250, and 500 g were put on the tips of MN patches. Afterwards, the deformation of the microneedles was visualized with stereomicroscopy.The in vitro skin insertion ability of HA MNs was assessed by inserting the MNs into the separated back skin of nude BALB/c mice with a force of 10 N and remained for 5 min. After removal, the treated skin was observed using stereomicroscopy and trypan blue staining method. In brief, the inserted mice skin was stained with trypan blue dye for 5 min, and the residual dye was washed off with phosphate-buffered saline (PBS). The skin insertion capacity was observed by stereomicroscopy. In addition, the collected skin samples were evaluated by H&E staining.2 for 5 min. The real-time thermal images and temperature changes of the MNs were recorded by a Fluke Ti32 Infrared thermal camera  every 30 s.To evaluate the photothermal performance of MNs, the MNs were exposed to 808 nm NIR laser irradiation  at an output power of 0.75 W/cm2, 37 ℃) for 24 h, respectively. The cell culture medium was then replaced by HA MNs solution  with different concentrations. After another 24 h of incubation, the CCK-8 kit was utilized to detect the cell viability according to the manufacturer’s protocol. In brief, 100 µl CCK-8 working solution (10%) was added into each well. After incubation in the dark (37 ℃) for 1–4 h, the absorbance values were measured at 450 nm via Beckman DU7400 spectrophotometer . To investigate the cell compatibility of the backing layer material (SA/Ge/HA), the NIH3T3 cells were employed and the experimental procedure was carried out as described above. Moreover, the CCK-8 assay was also used to quantitatively analyze the NIH3T3 cell proliferation with the leaching solution from two-layered HA microneedle material (HA microneedle tip + SA/Ge/HA backing layer) for 1, 2, and 3 days after cell seeding.To study the cell compatibility of microneedle materials, two kinds of cells (B16F10 and NIH3T3 cells) were used. Firstly, the cytotoxicity of microneedle tip material (HA) was verified by B16F10 and NIH3T3 cells. B16F10 or NIH3T3 cells (5000/well) were seeded into the 96-well plates and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in an incubator (5% CO5 cells/well), and different groups of microneedles were placed on the chambers of Transwell. Then the microneedles were irradiated with an NIR laser for 5 min . After 2 h, the cells were stained with live-dead cell imaging kit and observed by fluorescence microscope .The in vitro anticancer activity of MNs was evaluated using live/dead staining. B16F10 cells were seeded into a 6-well plate , and then cultured for 48 h. The cell survival rate of B16F10 was tested via the CCK-8 kit.In addition, CCK-8 assay was also employed to determine the cell viability after MNs treatment. The dissolved solutions of HA MNs, Cur NDs/HA MNs, IR820/HA MNs and Cur NDs/IR820/HA MNs were added into the culture wells, respectively. Subsequently, the cells were irradiated with an NIR laser light . When the tumor size grew to approximately 50–70 mm3, the mice were randomized into 6 groups (n = 5) as follows: Control (G1); HA MNs (G2); Cur NDs/HA MNs (G3); Cur NDs/IR820/HA MNs (G4); IR820 MNs + laser (G5); Cur NDs/IR820/HA MNs + laser (G6). After anesthetization via isoflurane, the mice were treated with microneedles according to the grouping. The G5 and G6 were exposed to the NIR laser . The tumor surface temperature and thermal images were captured timely via the NIR thermal imaging camera. The body weight of mice and size of tumor were measured every 2 days. The tumor volume was calculated as follows: tumor volume (V) = (tumor length) × (tumor width)2/2. At day 14 after treatment, the mice were sacrificed and the tumor tissues was harvested, weighed and photographed. After fixation with 4% paraformaldehyde, the tumor samples were dehydrated with graded ethanol series, embedded into paraffin, sectioned into serially 5 μm thick slices, and then stained with Ki67. Additionally, the key organs of mice including heart, liver, spleen, lung, and kidney were all collected for histological analysis.The subcutaneous melanoma tumor model was established by injecting 100 µl of 1 × 10Skin repair experiment was carried out on SD rats . After anesthetization, the dorsal hair of SD rats was shaved and a 1.5 cm × 1.5 cm skin defect was constructed on the back. The rats were randomized into 3 groups (n = 8): Control group, HA MNs group, and Cur NDs/IR820/HA MNs group. The microneedles were inserted into the defect area, after which the defects of all groups were protected with Tegaderm . At day 0, 7 and 14, the wound healing was photographed and the wound areas were measured by Image J software. Subsequently, four rats were sacrificed in each group at day 7 and day 14, respectively, and the whole wound sites  were excised and stained with H&E and Masson staining. At day 7 and 14, the number of inflammatory cells in H&E stained micrographs was calculated through and Image J software.All quantitative data were presented as means ± standard deviation (SD). SPSS 11.0 software  was utilized for statistical analysis. Statistical differences of groups were analyzed by student’s t-test or one-way ANOVA, and the P value < 0.05 was considered to be statistically significant.


Casein kinase 1δ/ϵ (CK1δ/ϵ) are well-established positive modulators of the Wnt/β-catenin signaling pathway. However, the molecular mechanisms involved in the regulation of β-catenin transcriptional activity by CK1δ/ϵ remain unclear. In this study, we found that CK1δ/ϵ could enhance β-catenin-mediated transcription through regulating β-catenin acetylation. CK1δ/ϵ interacted with Tip60 and facilitated the recruitment of Tip60 to β-catenin complex, resulting in increasing β-catenin acetylation at K49. Importantly, Tip60 significantly enhanced the SuperTopFlash reporter activity induced by CK1δ/ϵ or/and β-catenin. Furthermore, a CK1δ/CK1ϵ/β-catenin/Tip60 complex was detected in colon cancer cells. Simultaneous knockdown of CK1δ and CK1ϵ significantly attenuated the interaction between β-catenin and Tip60. Notably, inhibition of CK1δ/ϵ or Tip60, with shRNA or small molecular inhibitors downregulated the level of β-catenin acetylation at K49 in colon cancer cells. Finally, combined treatment with CK1 inhibitor SR3029 and Tip60 inhibitor MG149 had more potent inhibitory effect on β-catenin acetylation, the transcription of Wnt target genes and the viability and proliferation in colon cancer cells. Taken together, our results revealed that the transcriptional activity of β-catenin could be modulated by the CK1δ/ϵ-β-catenin-Tip60 axis, which may be a potential therapeutic target for colon cancer. The Wnt/β-catenin signaling pathway plays crucial roles in embryonic development, tissue homeostasis, stem cell renewal and tumorigenesis , 2. In tTAT-interactive protein 60 kDa (Tip60), a member of the Moz, Ybf2/Sas3, Sas2 and Tip60 (MYST) family of histone acetyltransferases (HATs), is a crucial regulator of the DNA damage response and transcriptional co-activator –11. ThisCK1 belongs to a family of serine/threonine protein kinases, composed of seven family members α, β, γ1, γ2, γ3, δ and ϵ in human. Among them, CK1δ and CK1ϵ share the highest homology, with 98% amino acid sequence identity in their kinase domain , 26. CK1In this study, we explored the effect of CK1 on β-catenin-mediated Wnt signaling. We found that CK1δ/ϵ could increase β-catenin-mediated transcription through regulating β-catenin acetylation. We further investigated the molecular mechanism by which CK1δ/ϵ modulated the acetylation of β-catenin. Our results revealed that a novel CK1δ/ϵ-Tip60-β-catenin axis is involved in regulation of Wnt/β-catenin signaling.2. Cells in the logarithmic phase of growth were used for the subsequent experiments.The human embryonic kidney 293T (HEK293T) cells, and colon cancer SW480 and HCT116 cells were obtained from American Type Culture Collection . These cells were maintained in DMEM  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  in a humidified incubator at 37°C with 5% COSR3029, D4476, MG149, NU9056, MG132 and cycloheximide (CHX) were purchased from MedChemExpress . The following primary antibodies were used: anti-CK1δ, anti-β-catenin, anti-p300, anti-acetyl-lysine, IgG , anti-CBP, anti-acetyl-β-catenin (K49), anti-phospho-β-catenin (S45), anti-Flag, anti-V5, anti-CK1ϵ, anti-GFP, anti-mouse IgG , anti-Tip60 , and anti-GAPDH antibody . The Tip60-Flag, PCAF-Flag, p300-Flag and pEGFP-N1 plasmids were purchased from Vigenebio . The SuperTopFlash reporter plasmid was provided by Karl Willert . The expression plasmids encoding β-catenin, pCMXβgal , CK1α-V5, CK1δ-V5, CK1ϵ-V5, CK1α-Flag, CK1δ-Flag, CK1ϵ-Flag, CK1γ-Flag have been described previously , 36. ForThe CK1 shRNA oligos were cloned into pLKO.1-GFP vector, and the Tip60 shRNA oligos were inserted into pLKO.1-TRC vector. The resulting constructs were validated by sequencing. The shRNA oligos used have been previously described , 38. ForHEK293T cells were transfected with the SuperTopFlash reporter, control vector, and the indicated expression plasmids in 24-well plates using Lipofectamine 2000 . After 48 h, the cells were harvested and lysates were used to examine the expression of luciferase by a luciferase assay kit , according to the manufacturer’s instructions. The luciferase values were normalized using the β-gal internal control to determine the variation in transfection efficiency. The results are presented as means ± SD of at least three independent experiments. The values for luciferase activity were presented as fold induction over control.The cells were lysed in RIPA buffer containing 50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, protease inhibitors , and phosphatase inhibitors . After quantifying the concentration of protein using the BCA protein assay kit , 30 µg of protein was loaded and separated by SDS-PAGE, followed by transferring to poly-vinylidenefluoride (PVDF) membranes . Immunoblotting was performed with the indicated primary antibodies at 4°C overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody  at room temperature for 2 h. After incubating the membranes with ECL Plus Western Blotting Substrate , immunolabeled proteins were detected using X-ray film or Chemiluminescent Imaging System .For immunoprecipitation assay, cell lysates were harvested in 500 µL RIPA buffer supplemented with protease inhibitor, phosphatase inhibitor cocktails and 1 mM PMSF, followed by centrifugation at 15000 rpm for 15 min at 4°C. Protein concentration in the supernatant was quantified by the BCA Protein Assay Kit. The supernatant was incubated with specific primary antibody or control IgG at 4°C overnight, then further incubated with protein A/G magnetic beads  for 6 h. The immunoprecipitated pellet was washed with RIPA buffer at least three times, and proteins were eluted by boiling in SDS loading buffer. The eluted proteins (30 µg) were analyzed by SDS-PAGE and immunoblotting.-ΔΔCt, where ΔΔCt = ΔCttreated -ΔCtcontrol. Ct is the cycle number at which fluorescence first exceeds the threshold. The ΔCt values from each target gene were obtained by subtracting the values for GAPDH Ct from the sample Ct. The primer pairs used for quantitative PCR amplification were as follows: Survivin sense, 5’-AGGACCACCGCATCTCTACAT-3’ and antisense, 5’-AAGTCTGGCTCGTTCTCAGTG-3’; Cyclin D1 sense, 5’-AATGACCCCGCACGATTTC-3’ and antisense, 5’-TCAGGTTCAGGCCTTGCAC-3’; PROM1 sense, 5’-AGGCTACTTTGAACATTATCTGC-3’ and antisense, 5’-GGCTTGTCATAACAGGATTGT-3’; GAPDH sense, 5’-CCAGAACATCATCCCTGCCTCTACT-3’ and antisense, 5’-GGTTTTTCTAGACGGCAGGTCAGGT.Total RNA was extracted using RNAiso Plus  and then reverse-transcribed into cDNA using the PrimeScript RT reagent kit  according to the manufacturer’s instructions. Prepared cDNA was then subjected to quantitative PCR analysis using 2×SYBR Green qPCR Master Mix . Real-time PCR assays were performed to quantify mRNA levels of human Survivin, Cyclin D1 and Prominin 1 (PROM1) genes. The comparative Ct method was used to analyze relative expression of genes. The data was presented as the fold change. The fold change was calculated as 2SW480 cells were treated with 240 nM SR3029 for 24 h, and ChIP assay was performed with ChIP-IT Express Enzymatic Chromatin Immunoprecipitation Kit  according to the manufacturer’s protocols. The eluted DNA was amplified by quantitative PCR analysis using an ABI Prism 7500 Real-Time PCR System . Primer sequences were shown as follows: Survivin sense, 5’-GCGTTCTTTGAAAGCAGT-3’ and antisense, 5’-ATCTGGCGGTTAATGGCG-3’; Axin2 sense, 5’-TCTGGTAGCATTATGGCCATCGCA-3’ and antisense, 5’-AAAGTCCTCCAAGCCCAAATTCCC-3’; The antibodies used were anti-β-catenin and anti-mouse IgG.SW480 and HCT116 cells were seeded in 96-well plates at a density of 5000 cells per well and cultured for 24 h. The cells were treated with SR3029 and MG149, alone or combined, at indicated concentrations for 48 h. Subsequently, 10 μL of MTT solution (5 mg/mL) was added into each well. The plate was further incubated for 4 h at 37°C and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the insoluble formazan. The absorbance of the formazan solution was measured at 570 nm. Each treatment was performed in three replicates.SW480 and HCT116 cells were plated on 96-well plates at a density of 5000 cells per well and cultured for 24 h. The cells were treated with SR3029 and MG149, alone or combined, at indicated concentrations for 48 h. The BrdU incorporation assay was performed using the Cell Proliferation ELISA BrdU Colorimetric Kit  according to the manufacturer’s instructions. BrdU incorporation was detected by measuring the absorbance at 450 nm. Each treatment was performed in three replicates.t-test was applied to determine the significance of difference between the two groups. One-way analysis of variance (ANOVA) with Dunn’s multiple comparisons test were utilized to compare the means of several groups. P<0.05 was defined as statistically significant.Statistical analyses were conducted using GraphPad Prism 7 software . The experiments were repeated three times, and data were exhibited as mean ± standard deviation (SD). The Student’s . Since β-catenin can be acetylated at lysine 49 (K49), which is required for the activation of Wnt/β-catenin signaling  markedly decreased the K49 acetylation of β-catenin in SW480 and HCT116 cells, while depletion of CK1δ/ϵ or treatment with small molecular CK1 inhibitors had little effect on the expression of Tip60 and β-catenin . In contrast, proteasome inhibitor MG132 increased the expression of acetylated β-catenin and total β-catenin in SW480 and HCT116 cells  and MG149 (5 and 10 μM), alone or combined. Our results showed that combined treatment markedly downregulated the level of β-catenin acetylation at K49  and the  and the via modulating the acetylation of β-catenin at K49. The interaction of CK1δ/ϵ with Tip60 recruited Tip60 to β-catenin complex, leading to β-catenin acetylation and the activation of Wnt signaling. Our results revealed a novel molecular mechanism by which CK1δ/ϵ enhances β-catenin-mediated transcription in colon cancer.Aberrant activation of canonical Wnt/β-catenin signaling plays a crucial role in proliferation, cellular stemness and chemoresistance in colon cancer . Mutatio. However, CK1α had little effect on Tip60 phosphorylation and the interaction between β-catenin with Tip60. These results suggest that CK1δ/ϵ-mediated phosphorylation of β-catenin at S45 may not be related to the positive regulatory function of CK1δ/ϵ in the Wnt pathway.Previous studies have reported that different CK1 family members could exert a coordinated action in the activation of Wnt signaling . CK1α haIncreasing evidence shows that the acetylation of β-catenin has been implicated in regulating its stability, subcellular location, specific interactions, and transactivational activity , 8, 49. In conclusion, our study demonstrated that CK1δ/ϵ could increase the transcriptional activity of β-catenin through regulating β-catenin acetylation at K49. CK1δ/ϵ potentiated the association of β-catenin with Tip60, and facilitated the recruitment of Tip60 to β-catenin complex, thereby leading to the acetylation of β-catenin at K49. Blockade of CK1δ/ϵ or/and Tip60 downregulated β-catenin acetylation and the transcription of Wnt target genes, resulting in growth inhibition of colon cancer cells. Our study define a novel CK1δ/ϵ-β-catenin-Tip60 axis which is implicated in modulation of β-catenin-mediated transcription.The original contributions presented in the study are included in the article/JN and DL developed the concept and designed this work. JN, QS, ZS, LT, YT, SS, and HL performed the experiments, carried out the data acquisition. JN, QS, ZS, VX, SL, XC, and DL performed data analysis. JN, QS, and DL edited and revised the manuscript. DL supervised this study. All authors read and approved this manuscript.This work was supported by the National Natural Science Foundation of China (31970739 and 81802662), the Natural Science Foundation of Guangdong Province (2020A1515010340 and 2020A1515010543), the Shenzhen Key Basic Research Program (JCYJ20200109105001821), the Shenzhen Natural Science Fund (the Stable Support Plan Program) (20200826134656001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.