Research suggests that to restore equity, third parties prefer compensation of a victim over the punishment of a perpetrator. It remains unclear, however, whether this preference for compensation is stable or specific to certain situations. In six experimental studies, we find that adjustments in the characteristics of the situation or in the available behavioral options hardly modify the preference of compensation over punishment. This preference for compensation was found even in cases where punishment might refrain a perpetrator from acting unfairly again in the future, and even when punishment has a greater impact in restoring equity than compensation does. Thus, the preference of compensation over punishment appears to be quite robust. Implications and ideas for future research are discussed. Injustice and unfairness are among the most agitating and frustrating experiences. We can experience injustice done to ourselves, but we can also observe injustice being done to someone else. Research shows that in cases where people receive an unjust treatment, they are motivated to punish the transgressor to restore justice , whereas compensation allows for relationship building with the victim , and when punishment has a greater impact than compensation (Experiment 4). In Experiment 5, we tested a methodological constraint, namely whether people increase punishment when compensation is not possible. In Experiment 6, we investigated whether people would shift to a preference for punishment when they do not have to do the punishment themselves. All studies also include the option to keep one’s endowment, making both punishment and compensation costly. This option has been included in the previous research, as well  were randomly assigned to the single game condition or multiple games condition. Participants in the single (multiple) game condition read the following scenario:Three hundred and eight U.S. Amazon Mechanical Turk (“MTurk”) workersYou observe a game (a series of games) played by Mark and Rick. In this (every) game, $100 needs to be divided. (In every game) Mark gets to decide how to divide this money between himself and Rick. Rick has no influence on the division of the money. (In the first game) Mark decides to give Rick $20 and to keep $80.Next, we assessed our primary dependent variable, the choice for compensation, punishment, or keeping the money. This was done as follows in all studies (unless indicated differently). Participants read that they themselves owned $50, and that they could choose one of the three options to use the dollars they owned: They could compensate Rick; every dollar that they used for compensation would increase Rick’s amount with $3. They could punish Mark; every dollar that they used for punishment would decrease Mark’s amount with $3. Or they could keep the money themselves. When they choose to punish or to compensate, they also indicated how many dollars they would spend. We also included the option for participants to keep the money, to investigate whether the potential absence of punitiveness is driven by people’s  motivation to compensate or by people’s motivation not to act at all  = 1.33, p  = 0.515. There were also no differences in the amount of money spent on compensation between the single game condition  and the multiple game condition , t(102) = − 0.49, p = 0.627. Participants in the single game condition, on average, spent $27.20 (SD = 19.25) on punishment; participants in the multiple games condition $20.11 (SD = 9.94). We could not compare the amount of money spent on punishment between conditions, and the amount of money spent on punishment and compensation within each condition, because of the low number of participants choosing punishment  = 23.20, p < 0.001, and the multiple game condition, χ2  = 35.84, p < 0.001. Finally, participants more often chose for keeping the money themselves than for compensation, in both the single game condition, χ2  = 14.90, p < 0.001, and the multiple game condition, χ2  = 6.16, p  = 0.013.Participants more often chose for compensation than punishment, in both the single game condition, It thus appears that people are reluctant to punish when they have an opportunity to compensate a victim, even if a perpetrator might act unfairly again in the future. In the next study, adjusted versions of the scenarios used in Experiment 1 were used. It might not have been clear for participants how many games which Mark would play in the multiple games condition and that Mark could be repeatedly unfair. We also made it clear that it was a purely random choice to let Mark decide how to distribute the money.Mage = 19.44, SD = 2.39) were randomly assigned to the single game condition or multiple game condition and received course credit for their participation. Participants in the single (multiple) game condition read the following scenario and next indicated what to do with the $50 :One hundred and thirty-seven university studentsYou observe a single game (a series of 20 games) played by Mark and Rick in which (and in every game) €100 needs to be divided. By the toss of a coin, Mark is selected to decide how to divide this money between himself and Rick (in every game). Rick has no influence on the division of the money. (In the first game) Mark decides to give Rick €20 and to keep €80.χ2  = 1.64, p  = 0.441. There were also no differences in the amount of money spent on compensation between the single game condition  and the multiple game condition , t(72) = 0.29, p = 0.770. Participants in the single game condition on average spent $16.25 (SD = 7.50) on punishment; participants in the multiple games condition $20.25 (SD = 7.30). We could not compare the amount of money spent on compensation with the amount of money spent on punishment, because of the low number of participants choosing punishment.The choices are shown in Table χ2  = 25.33, p < 0.001, and the multiple game condition, χ2  = 15.51, p < 0.001. Finally, participants marginally significantly more often chose for compensation than for keeping the money themselves in the single game condition, χ2  = 3.16, p  = 0.075. The same pattern is found for participants in the multiple game condition, χ2  = 3.27, p  = 0.071.Participants more often choose for compensation than for punishment, in both the single game condition, From these results, we can conclude that when a perpetrator could be repeatedly unfair, people are still reluctant to punish and prefer compensation over punishment. However, from these results, we cannot determine whether participants choose to compensate the victim, because Mark could be unfair to this victim multiple times, making compensation for this particular victim the more ‘necessary’, or whether participants would also choose to compensate this victim if Mark had been unfair to multiple other victims. Hence, in the next experiment, we wanted to investigate whether people would still prefer compensation when we vary to whom the unfairness was done. We again adjusted the scenario, so that participants are confronted with Mark acting unfairly multiple times to multiple victims.Mage = 33.65, SD = 11.01) were randomly assigned to the repeated unfairness condition or repeated fairness condition (acting as a control condition). Participants in the repeated unfairness condition read the following scenario:Two hundred and one U.S. MTurk-workers  condition read the same scenario, but here Mark divided the money between himself and the other players equally. Next, participants read that:Shortly, Mark will play a fourth game with Daniel. You own $50 in this fourth game. How would you spend the $50 as an observer of this game? You have three options to spend each dollar: You can compensate Daniel; every dollar that you use for compensation will increase Daniel’s amount with $3. You can punish Mark; every dollar that you use for punishment will decrease Mark’s amount with $3. Or you can keep the money.In the repeated unfairness condition, participants subsequently read: “In the fourth game, Mark decides to give Daniel $20 and to keep $80”. In the repeated fairness condition, participants read: “In the fourth game, Mark decides to give Daniel $50 and to keep $50”. Finally, participants were asked which option  they would choose.χ2  = 29.32, p < 0.001, whereas participants in the latter condition more often chose to keep the money. There were again no differences in the amount of money spent on compensation between the repeated unfairness condition  and the repeated fairness condition , t(59) = 1.37, p = 0.177t(55) = − 1.28, p = 0.205.The choices are shown in Table χ2  = 6.12, p  = 0.013. Participants in the repeated fairness condition more often chose for compensation over punishment, as well, χ2  = 17.64, p < 0.001. Furthermore, within the repeated unfairness condition, the choice for compensation or keeping the money did not differ, χ2  = 0.50, p  = 0.823. Participants in the repeated fairness condition more often chose for keeping the money themselves, χ2  = 28.37, p < 0.001.Although the amount of money spent on compensation and punishment did not differ within the repeated unfairness condition, participants more often chose for compensation than for punishment within this condition, The first series of experiments leads us to the preliminary conclusion that irrespective of whether a perpetrator can act unfairly again in the future or has been repeatedly unfair, people seem to prefer compensation over punishment. This finding might be surprising, as the compensation in the current experiment is given specifically to one victim, whereas the punishment could be viewed as given to all transgressions. Furthermore, punishment might be seen as a more viable option to deter the perpetrator from future offenses.The preference for compensation might stem from the impression that the perpetrator is someone that will not change his behavior, because of the repeated unfairness already described in the scenario. Compensation has the sure benefit of restoring the harm done to the victim, as opposed to punishment in which the potential benefit of a perpetrator (or others in society) not committing any subsequent transgressions is more insecure. This could create a threshold in which people only consider punishment when its expected impact on the restoration of fairness is large enough compared to disutility of not compensating the victim. Hence, to make people choose for punishment, the benefit of this option should probably be greater than that of compensation. In Experiment 4, we, therefore, varied the impact of punishment and compensation in restoring injustice. Participants will read that the impact of compensation and punishment is equal or unequal. In the unequal condition, the impact of punishment is greater than compensation, which might motivate people to choose punishment over compensation.Mage = 31.28, SD = 9.33) were randomly assigned to the equal impact condition or unequal impact condition. Participants read the scenario from the single game condition of Experiment 1. In the equal impact condition, the dependent variable was the same as in Experiment 1. In the unequal impact condition, the impact of punishment was greater than that of compensation: Every dollar used for punishment would decrease Mark’s amount with $3, while $1 compensation would increase Rick’s amount with $1.Two hundred and two U.S. MTurk-workers  = 4.34, p  = 0.114. When looking specifically at the choice to compensate or not, we do see that participants in the equal impact condition more often chose for compensation than participants in the unequal impact condition, χ2  = 4.29, p  = 0.038, but participants in the unequal impact condition  spent more on compensation than participants in the equal impact condition , t(67) = − 2.83, p = 0.006. Participants in the equal impact condition on average spent $24.50 (SD = 10.85) on punishment; participants in the unequal impact condition $22.91 (SD = 10.44). We could not compare the amount of money spent on compensation with the amount of money spent on punishment, because of the low number of participants choosing punishment.The choices are shown in Table χ2  = 23.12, p < 0.001, and the unequal impact condition, χ2  = 7.41, p  = 0.006. In the unequal impact condition, participants more often chose for keeping the money themselves than for compensation, χ2  = 12.84, p < 0.001. These findings indicate that when compensation has a low(er) impact, it is less preferred. In the equal impact condition, this difference was not significant, χ2  = 0.87, p  = 0.351.Participants more often chose for compensation than for punishment in both the equal impact condition, In line with the previous experiments, people seem to prefer compensation over punishment, despite the fact that the impact of punishment was greater than that of compensation. We do find that when punishment has a greater impact, people less often choose for compensation. However, instead of seeing a larger total of people choosing for punishment, we see a larger total of people choosing to keep the money themselves. A potential explanation for this finding is that a confrontation with the perception that punishment has a greater impact than compensation might make people think that compensation is a ‘waste’ of their money. However, as the alternative of punishment is a response people generally do not want to opt for in the first place, they decide to keep the money themselves.This latter finding made us wonder whether people would also opt for keeping the money if the option to compensate is not present at all, as in the case of the classic third-party punishment studies. Zhang and Ortmann  have shoMage = 31.74, SD = 9.21) were randomly assigned to the two options (punishment/keeping the money) condition or three options (punishment/compensation/keeping the money) condition. Participants read the scenario from the single game condition of Experiment 1, and responded to the same dependent variable. The only difference was that, in the two-option condition, they could only choose between punishment and keeping the money.Two hundred and four U.S. MTurk-workers  = 49.83, p < 0.001. Participants in the two-option condition more often chose to keep the money than to punish. Participants in the three-option condition more often chose to compensate than to punish, χ2  = 27.52, p < 0.001, and more often chose for keeping the money themselves than for compensation, χ2  = 4.94, p  = 0.026. This experiment thus again documents a preference for compensation over punishment. Participants in the two-option condition on average spent $13.31 (SD = 6.62) on punishment; participants in the three-option condition $15.50 (SD = 7.14). Because of the low number of people in the three-option condition choosing punishment, we could not test for statistical differences between these amounts.The choices are shown in Table who is distributing the punishment and compensation. That is, in the final experiment, we investigate whether people are less reluctant to punish if the act is performed by someone else. Therefore, in what follows either participants themselves were responsible for deciding how to spend the $50 or someone else was responsible for deciding how to spend the money.As variations in the unfair situation and the spectrum of behavioral options did not seem to alter the preference for compensation, we sought to investigate whether it makes a difference Mage = 31.98, SD = 10.71) were randomly assigned to the third-party decision condition, fourth-party own decision or fourth-party other decision condition. Participants read the scenario from the single game condition of Experiment 1, with the small adjustment that participants in the third-party decision condition read that they observed this game between Mark and Rick and participants in the two-fourth-party conditions read that they and another person observed the game between Mark and Rick (and together owned $50). The dependent variable was again the choice for compensation, punishment, or keeping the money. In the fourth-party own decision condition, participants read that they were the person responsible for choosing. In the fourth-party other decision condition, participants read that the other person was responsible for choosing and they were asked how they would like to other person to spend the $50. Hence, the difference between conditions lies in (1) whether there is a fourth party, and (2) whether the participant (as a third party) or the fourth party is responsible for the decision.Three hundred and three U.S. MTurk-workers  = 1.16, p  = 0.885. There were also no differences in the amount of money spent on compensation, F = 2.56, p = 0.082, ηp2 = 0.044. Participants in the third-party decision condition  spent an equal amount of money on compensation as those in the fourth-party own decision condition , p = 0.358, and the fourth-party other decision condition , p = 0.068. Participants in the third-party decision condition on average spent $16.88 (SD = 5.30) on punishment; participants in the fourth-party own decision condition $12.78 (SD = 5.65); and participants in the fourth-party other decision condition $19.40 (SD = 12.83). We could not compare the amount of money spent on compensation with the amount of money spent on punishment because of the low number of participants choosing punishment.The choices are shown in Table χ2  = 19.57, p < 0.001, in the fourth-party own decision condition, χ2  = 14.70, p < 0.001, and in the fourth-party other decision condition, χ2  = 16.49, p < 0.001. Finally, the choice for compensation or keeping the money did not differ within the third-party decision condition, χ2  = 2.18, p  = 0.140, and within the fourth-party other decision condition, χ2  = 1.11, p  = 0.292. However, within the fourth-party own decision condition participants more often chose for keeping the money, χ2  = 5.15, p  = 0.023. From this experiment, it can be concluded that irrespective of whether people are responsible for punishment or compensation themselves, people are not more inclined to punish.Participants chose more often for compensation than for punishment in the third-party decision condition, The previous research seemed to indicate that third parties prefer compensation of a victim over punishment of a perpetrator in cases of unfair distributions of money  not committing any subsequent transgressions is more insecure. Related to that, people might be more reluctant to punish than compensate, because, in economic games, punishment both costs the punisher and destroys value of the punished. Compensation on the other hand could be considered more of a transfer of money in which the victim gains money.Furthermore, although it has not been explicitly mentioned that the participant would engage in future contact with the victim or perpetrator, it might be the case that compensation is preferred because of (implicit) relational concerns , they might be more inclined to choose punishment over compensation.Our studies also included the option not to act (keeping the money). When comparing the prosocial act with not acting at all results do not indicate a clear preference. Deciding to keep the money might be viewed as the self-interested option. However, in addition to that, it might be the case that people chose to not act, because they judge their act as interfering between other people’s business. This might especially be the case in situations that involve people that the participants were not acquainted with. In the case of friends or family, people might be more inclined to act  than not to act. For example, research has illustrated that people show more prosocial behavior when a victim is a  close other or when feelings of distance between victims and potential benefactors are reduced  punishment might never match up to the impact that compensation establishes in observers’ eyes.Taken together, the results show that third parties have a strong preference for compensation over punishment to restore equity. Although, in some situations, the percentage of people that want to punish the perpetrator does increase, the preference for compensation of victims seems to be a strong one. Even when punishment might refrain a perpetrator from acting unfairly again in the future, and even when punishment has a greater impact in restoring equity than compensation does, the preference for compensation remains. These findings imply that third parties prefer a prosocial option over an antagonistic option which has implications for theories on punishment, justice, and law.


CTNNB1 gene encoding β-catenin, or inactivating APC and AXIN gene mutations. However, these mutations are uncommon in breast and pancreatic cancers despite nuclear β-catenin localization, indicative of pathway activation. Notably, the low-density lipoprotein receptor-related protein 6 (LRP6), an indispensable co-receptor for WNT, is frequently overexpressed in colorectal, liver, breast and pancreatic adenocarcinomas in association with increased WNT/β -catenin signaling. Moreover, LRP6 is hyperphosphorylated in KRAS-mutated cells and in patient-derived colorectal tumours. Polymorphisms in the LRP6 gene are also associated with different susceptibility to developing specific types of lung, bladder and colorectal cancers. Additionally, recent observations suggest that LRP6 dysfunction may be involved in carcinogenesis. Indeed, reducing LRP6 expression and/or activity inhibits cancer cell proliferation and delays tumour growth in vivo. This review summarizes current knowledge regarding the biological function and regulation of LRP6 in the development of epithelial cancers—especially colorectal, liver, breast and pancreatic cancers.The WNT/β-catenin signaling pathway controls stem and progenitor cell proliferation, survival and differentiation in epithelial tissues. Aberrant stimulation of this pathway is therefore frequently observed in cancers from epithelial origin. For instance, colorectal and hepatic cancers display activating mutations in the  Cell proliferation and differentiation are finely regulated during epithelial tissue development and renewal. One of the most important signaling pathways involved in this regulation is the ubiquitous WNT/β-catenin pathway due to its crucial role in adult stem cell regulation . AccordiCTNNB1, acts as the main signal transducer. In the absence of WNT factors, β-catenin cytoplasmic concentrations are kept low because of active degradation through the proteasome . Indeed, palmitoylation-deficient LRP6 mutants are retained in the ER due to Lys-1403 mono-ubiquitination, resulting in defective WNT/β-catenin pathway activation . ConversLike many membrane-anchored proteins, LRP6 folds inefficiently. LRP6’s folding and membrane targeting require the assistance of chaperones and enzymes. For example, the mesoderm development LRP chaperone (MESD) is a specialized chaperone preventing intermolecular disulfide bond formation in the extracellular domain which induces LRP6 aggregation in the ER . MoreoveOn the cell surface, the presence of large LRP5/6-containing complexes suggests that the monomeric mode of an FZD-LRP6 coreceptor complex is not privileged. In fact, FZD receptors (FZDs) and LRP5/6 undergo multiple polymerizations on the cell surface upon WNT ligand binding, to form oligomers. WNT ligands selectively bind different states of LRP5/6-FZD receptor oligomerization . For insSeveral groups have reported a crucial role for the five proline-rich PPP(S/T)P motifs, containing serine or threonine residues  in LRP6, in the activation of downstream signaling A [6]. PhEndocytosis is required for sustained WNT/β-catenin signal activation. Indeed, endocytosis of the LRP5/6-FZD receptor complex occurs quickly after WNT binding . InteresCaveolin-dependent internalization occurs mostly at lipid rafts, a platform for receptor-mediated signaling, which facilitates the sequestration of receptors and signaling molecules within caveolae . In factLrp6 have been developed in order to determine the role of LRP6 during development. Global Lrp6 deletion in mice induces a severe phenotype with developmental defects in eye, limb and neural tube . Be. Be47]. RP6 gene . Aside fRP6 gene ,65,66.RUNX2 and VEGFa, which in turn decrease CRC metastasis .,54.27,54tastasis . Additiotastasis .APC mutations—inhibits constitutive WNT pathway activation, indicating that LRP6 at least partially mediates the positive effect of APC inactivation on the WNT pathway . . DKK1 prvitamin D receptor gene has been recently identified as a determinant of survival in patients with pancreatic cancer [Interestingly, the c cancer . In thisc cancer ,119. Notc cancer ,121. In c cancer .Helicobacter pylori or Mycoplasma hyorhinis induced rapid phosphorylation of LRP6 and downstream activation of Wnt/β -catenin signaling [Some evidence also suggests a role for LRP6 in the development of other epithelial cancers, including prostate, gastric, bladder, non-small-cell lung (NSCL) and papillary thyroid cancers. For instance, androgen-dependent LRP6 expression is necessary for prostate cancer cell growth . As obseignaling ,126. Addignaling ,128. In ignaling ,130. Regignaling . Interesignaling . Likewisignaling . FurtherApc-mutant intestinal tumoroids [Rnf43 mutant intestinal organoid growth and survival [Until now, the clinical trials that were conducted to inhibit the Wnt/β-catenin pathway mostly involved the use of porcupine inhibitors to block WNT secretion or the use of adenoviruses to overexpress DKK3, an antagonist of LRP5/6 . Inhibitumoroids . Nonethesurvival . Thus, tFrom these previous studies, it is clear that LRP6 is a key player in the activation of the WNT/β-catenin signaling pathway and, consequently, in the regulation of tissue homeostasis under physiological and pathological conditions. However, β-catenin-independent roles of WNT/LRP6 signaling have recently been reported . How exa


It has been shown to have an important role in formation of the neuromuscular junction. Our previous studies showed that in the absence of β-catenin, the resting membrane potential (RMP) is depolarized in muscle cells and expression of the α2 subunit of sodium/potassium adenosine triphosphatase (α2NKA) is reduced. To understand the underlying mechanisms, we investigated the electrophysiologic properties of a primary cell line derived from mouse myoblasts (C2C12 cells) that were transfected with small-interfering RNAs and over-expressed plasmids targeting β-catenin. We found that the RMP was depolarized in β-catenin knocked-down C2C12 cells and was unchanged in β-catenin over-expressed muscle cells. An action potential (AP) was not released by knockdown or over-expression of β-catenin. α2NKA expression was reduced by β-catenin knockdown, and increased by β-catenin over-expression. We showed that β-catenin could interact physically with α2NKA (but not with α1NKA) in muscle cells. NKA activity and α2NKA content in the cell membranes of skeletal muscle cells were modulated positively by β-catenin. These results suggested that β-catenin (at least in part) regulates the RMP and AP in muscle cells, and does so by regulating α2NKA. StudiesIn the peripheral nervous system, the neuromuscular junction (NMJ), which is the synapse between a motor neuron and a muscle fiber, plays an important part in muscle contraction. Appropriate interactions between motor neurons and muscle cells are required for information transmission at the NMJ .Agrin is released by motor neurons and binds to low-density lipoprotein receptor-related protein (LRP)-4 and activates muscle-specific kinase (MuSK). Agrin, LRP4, and MuSK are needed for NMJ formation . In contResearch has revealed that most mutant mice deficient in β-catenin  die a few hours after birth . PreviouIn the present study, we showed that β-catenin plays a part in the RMP and action potential (AP) of skeletal muscle cells at the NMJ. We recorded depolarization following knockdown of the β-catenin gene, but variance was not seen if β-catenin was over-expressed in skeletal muscle cells. The AP was inhibited by up-regulation and down-regulation of functional expression of β-catenin. Also, α2NKA expression was reduced by knocking down β-catenin and was increased by over-expressing β-catenin. We found that α2NKA and β-catenin displayed a “physical” interaction in muscle cells. Finally, NKA activity and α2NKA content in the cell membrane were found to be modulated positively by β-catenin.We were able to suggest a mechanism underlying the electrophysiologic changes caused by β-catenin expression in muscle cells. We also provided additional evidence that supports a key role for β-catenin at the NMJ. Our data could be used to advance research on the intricate role of β-catenin in neurologic disorders.+-voltage-gated channel subfamily C member 4  and glyceraldehyde-3-phosphate dehydrogenase  were purchased from Affinity . Antibodies against α1NKA  were obtained from GeneTex . Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)G , HRP-conjugated goat anti-rabbit IgG , Cy3-conjugaed goat anti-rabbit IgG , and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG  were purchased from Wuhan Boster . A Na+/K+-ATP Enzyme Test kit (BC0060) was obtained from Solarbio Life Sciences . A Cell Surface Protein Isolation kit (89881) was purchased from Pierce .Antibodies targeted against β-catenin and α2NKA were obtained from Abcam . Antibodies against KChemically modified small interfering (si)RNA oligonucleotides were obtained from Shanghai GenePharma . The 5′-termini of all siRNAs were labeled with a fluorescent conjugated dye (carboxyfluorescein). The sequences of β-catenin siRNAs  were: 5′-CACCU CCCAA GUCCU UUAUT T-3′ and 5′-AUAAA GGACU UGGGA GGUGT T-3′ for siRNA-459; 5′-CCAGG UGGUA GUUAA UAAAT T-3′ and 5′-UUUAU UAACU ACCAC CUGGT T-3′ for siRNA-777; 5′-GGGUU CCGAU GAUAU AAAUT T-3′ and 5′-AUUUA UAUCA UCGGA ACCCT T-3′ for siRNA-1512; 5′-UUCUC CGAAC GUGUC ACGUT T-3′ and 5′-ACGUG ACACG UUCGG AGAAT T-3′ for the negative control (carboxyfluorescein).The cDNA of β-catenin was obtained by reverse transcription-polymerase chain reaction (RT-PCR) using the primer sequences  5′-GGACT CAGAT CTCGA GATGG CTACT CAAGC TGACC T-3′ and 5′-GTCGA CTGCA GAATT CTTAC AGGTC AGTAT CAAAC CAG-3′. This was followed by sub-cloning into the pIRES2-ZsGreen1 plasmid . The oveC2C12 myoblasts were obtained from American Type Culture Collection . They were cultured in high-glucose Dulbecco’s modified Eagle’s medium  supplemented with 100 U/mL penicillin–streptomycin, 10% fetal bovine serum , and 1 mM GlutaMAX  in 60-mm dishes. Fusion of myoblasts into myotubes was induced by culture in differentiation medium (high-glucose DMEM supplemented with 2% horse serum). Myoblasts at 70–80% confluence were transfected with Lipofectamine 2000  according to manufacturer instructions, and then switched to differentiation medium 24-h later.2, 1 EGTA, 10 HEPES, 1 MgCl2, and 2 Mg-ATP . Cells were perfused throughout recordings with an external solution containing (in mM): 140 NaCl, 3.5 KCl, 1 MgCl2, 2 CaCl2, 10 D-glucose, 10 HEPES, and 1.25 NaH2PO4 (pH 7.4 with NaOH and 300 mOsm). Only cells with elongated (rather than flat) morphology were selected. Slightly positive pressure was applied while the pipette was advanced into the bath with the microelectrode manipulator. Once the glass pipette made contact with the cell surface, positive pressure was removed. Then, negative pressure was used to form a pipette-cell seal at >1 GΩ, and gentle suction was applied to rupture the patch membrane. The slow capacitance was compensated, and the membrane capacitance and series resistance were recorded. Seal formation and whole-cell recording were carried out in “current clamp” mode. The RMP was recorded at zero current immediately after breaking the seal between the membrane and the pipette. Cells with a series resistance <20 MΩ and changes <20% throughout recording were used for analyses. Current signals were Bessel-filtered at 5 kHz and sampled at 10 kHz.Fully differentiated myotubes transfected with siRNAs and pIRES2-ZsGreen1-β-catenin were visualized with an inverted microscope . They were recorded using whole-cell recording techniques employing an amplifier (EPC10), a Patchmaster v2 × 73 analog-to-digital converter, and IGOR 6.0.1.0 . Recording pipettes were pulled to a tip resistance of 3–5 MΩ when filled with an internal solution containing (in mM): 140 potassium gluconate, 5 NaCl, 0.1 CaClIndependent APs were evoked by intracellular injection of depolarizing current in a series of 600–1100 pA (at increments of 10–100 pA) rectangular pulses with 10 mS at 20 Hz . All expTM imaging system .C2C12 cells were collected and lysed with lysate buffer supplemented with a “protease inhibitor cocktail”  . The concentration of protein lysates was measured using the Bradford method . Subsequently, 10 μg of cell lysate with 5 μg of sample buffer  was added to a 10% polyacrylamide gel. Samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . After sealing with 4% milk containing 0.1% Tween, the PVDF membranes were incubated overnight with primary antibodies against β-catenin, α2NKA, KV3.4, α1NKA, and GAPDH, respectively, at 4°C. They were then washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween. Next, 4% skimmed milk containing 0.1% Tween and HRP-conjugated antibody were added and incubated for 2 h at room temperature. After an electrochemiluminescence developer  had been added to PVDF membranes, photographs were obtained using the Gel Doc® Premix Ex TaqTM II kit  in a real-time PCR instrument  with pre-denaturation at 50°C for 10 min, denaturation at 95°C for 30 s, and annealing at 60°C for 30 s for a total of 40 cycles. The primer sequences  were: 5′-CTTAC GGCTA CAGAG AGGGG-3′ and 5′-GCAGA GGGAA GCCGT AGTAT-3′ for α1NKA; 5′-ATCAA TGCAG AGGAG GTGGT-3′ and 5′-TGAAC TCAGG AGAAC GGGTC-3′ for α2NKA; 5′-AGACA GCTCG TTGTA CTGCT-3′ and 5′-GTGTC GTGAT GGCGT AGAAC-3′ for β-catenin; 5′-ATGGG TGTGA ACCAC GAGA-3′ and 5′-CAGGG ATGAT GTTCT GGGCA-3′ for GAPDH.mRNA samples from C2C12 cells were prepared using an RNeasy mini-kit  and digested using an RNase-Free DNase kit . mRNA samples were reverse-transcribed into cDNA using a high-capacity cDNA Reverse Transcription kit (4368813) according to manufacturer  instructions. qRT-PCR was done with a SYBRC2C12 cells were fixed by immersion in 4% paraformaldehyde in PBS for 30 min at 4°C. After addition of Triton X-100 to a final concentration of 0.1%, sections were mounted on gelatin-coated glass slides. Non-specific binding sites were blocked by preincubation for 1 h at room temperature in bovine serum albumin in PBS (PBS/BSA), followed by incubation with the primary antibody in PBS/BSA overnight at 4°C. Bound antibody was visualized by labeling with a FITC-conjugated anti-rabbit antibody or Cy3-conjugated anti-mouse antibody. Secondary antibodies were used at 1:2000 dilution. Fluorescence images were captured using a confocal laser scanning microscope and software .g for 10 min at 4°C. The supernatant was precleared with 30 μL of protein A + G agarose beads for 2 h at 4°C to eliminate non-specific binding and reduce the background reading. Samples were incubated with 3 μg of β-catenin antibody at 4°C overnight. Immune complexes were harvested with another 30 μL of protein A + G agarose beads for 3–6 h at a 4°C. The beads were resuspended in 30 μL of 2× SDS–PAGE buffer and washed three times with PBS. α2NKA and α1NKA were immunoprecipitated under identical conditions using monoclonal antibodies. After heating for 5 min at 100°C in 2× sample buffer, immunoprecipitated proteins were subjected to SDS–PAGE and immunoblotting as described above.C2C12 cells were resuspended in immunoprecipitation buffer supplemented with protease inhibitors and phosphatase inhibitors for 10–15 min on ice, and centrifugated at 10,000 × g. Then, the homogenate was centrifuged at 1000 × g for 10 min at 4°C to obtain the precipitate, which was then resuspended in 1 mL of homogenizing buffer  for 10 min at 37°C. The homogenized sample was centrifuged for 10 min at 4000 × g and 4°C. The supernatant obtained interfered with subsequent phosphate analyses, so it was mixed with a phosphorus reagent for 10 min in a 40°C water bath, and then cooled to room temperature. Absorbance was determined at 660 nM using an automatic microplate spectrophotometer .Cell suspensions of myoblasts at 80–90% confluence were thawed on ice and homogenized for 10 min at1000 × +/K+-ATPase activity were determined as described by Assays for NaATPase activity (U/mg protein) = C standard × (A estimated – A control)/(A standard – A blank) × V total/(protein concentration of the sample × V sample)/T = 7.5 × (A estimated – A control)/(A standard – A blank)/protein concentration of the sample.A, enzymatic activity (the amount of inorganic phosphorus produced by enzymatic decomposition of ATP per milligram of tissue protein and per hour as a unit of enzymatic activity); C, concentration, V, volume, T, the reaction time.TM Sulfo-NHS-SS-Biotin  on ice for 30 min.  The liquid was suctioned. Cells were incubated with 4 mL of 100 mmol/L glycine at 4°C for 5 min. The step described above was repeated. Cells were washed and harvested by scraping in pre-cooled cell lysate buffer containing 1% Triton-X100 and protease inhibitors . The supernatant was harvested and boiled in 4× buffer solution for 10 min. For pull down of biotinylated proteins, 500 μL of the protein extract was incubated with 30 μL of high-capacity streptavidin agarose beads  overnight at 4°C. Beads were washed, eluted by incubation in Laemmli buffer containing dithiothreitol at 100°C for 10 min, and analyzed by western immunoblotting. The lysate input and elutes were subjected to SDS–PAGE and analyzed by western immunoblotting.Cells were starved with serum-free DMEM overnight, washed with ice-cold PBS, and incubated with PBS containing 0.5 mg/mL non-permeable EZ-linkP < 0.05.Data are the mean ± SEM. Data were calculated using one-way analysis of variance (ANOVA) with Tukey’s honestly significantly different test. Changes were considered significant if alpha values satisfied the limit of –/– pups died hours after birth having presented with global cyanosis, consistent with previous reports . β-Catenin siRNA459 and a number 1 bacterial solution of pIRES2-ZsGreen-β-catenin were selected for subsequent studies. To ascertain if the RMP of β-catenin siRNA- and pIRES2-ZsGreen1-β-catenin plasmid-transfected cells had changed, we undertook whole-cell patch-clamp recording. We found that, in control cells, the RMP was −76 ± 2.83 mV (P = 0.00067) and −80.75 ± 1.50 mV, respectively  was given (increased in increments of 10–100 pA). The AP of muscle cells was evoked in control cells, and the morphology and platform stage were normal . HoweverOur data showed that β-catenin knockdown depolarized skeletal muscle cells. Furthermore, the AP was not evoked in β-catenin knocked-down or in over-expressed C2C12 cells. Thus, we further explored the possible mechanisms underlying this phenomenon.Sodium/potassium adenosine triphosphatase and Kv3.4 channels  are crucMoreover, β-catenin regulates the expression and transcription of target genes in the Wnt canonical pathway . Thus, wP < 0.0001 for β-catenin siRNA) (P = 0.0002 for pIRES2-ZsGreen1-β-catenin) . By contrast, enhanced expression of β-catenin increased α2NKA transcription markedly (We undertook immunoblotting and qRT-PCR in β-catenin knocked-down and over-expressed cells. We measured the protein expression of α1NKA and α2NKA in transfected C2C12 cells. Transfection with β-catenin siRNA caused a significant reduction of α2NKA expression (n siRNA) , and tracatenin) . α2NKA ecatenin) . No signcatenin) . The decmarkedly .These data suggested that β-catenin regulates RMP maintenance and partially triggers the AP by altering the transcription and expression of α2NKA in skeletal muscle cells.We found that β-catenin transfection regulated α2NKA (but not α1NKA) expression positively, so we investigated if there was an interaction between α2NKA and β-catenin. Co-immunoprecipitation studies showed that β-catenin could pull down α2NKA protein but not α1NKA protein in C2C12 cells . FurtherConfocal microscopy revealed that β-catenin protein co-localized with the proteins of α2NKA and α1NKA in C2C12 cells . In addiThe results mentioned above showed that β-catenin regulates the electrophysiologic properties of muscle cells and α2NKA expression. In addition, NKA activity has an important role in maintenance of the RMP in skeletal muscles. We determined whether deletion and increases in β-catenin expression affected NKA activity.P = 0.0046). Compared with the PIRES2-ZsGreen1 group, the percentage activity of NKA was increased significantly in the β-catenin over-expressed group (P = 0.0002).The percentage activity of NKA was not significantly different among the siRNA control group, PIRES2-ZsGreen1 group, or control group. When compared with siRNA control cells, the percentage activity of NKA was reduced significantly in the β-catenin knockdown group . However, the content of total α2NKA, α2NKA in the cell membrane, and α2NKA/total α2NKA protein in the cell membrane was not significantly different between the two groups (P = 0.0032). The content of total α2NKA, α2NKA in the cell membrane, and α2NKA/total α2NKA protein in the cell membrane was not significantly different when comparing the two groups /+ mice  are viable at birth, whereas HSA-β-cat–/– mice  die soon after birth  it acts as a co-transcriptional activator of T-cell factor/lymphocyte enhancer factor-1 and (ii) it regulates expression of target genes . β-CatenFurthermore, our results suggested that β-catenin and α2NKA exerted a physical interaction with C2C12 cells. We also revealed that NKA activity and α2NKA content in the cell membrane were modulated positively by β-catenin.We provided further evidence that β-catenin is crucial for maintaining the RMP and evoking the AP in skeletal muscle cells. Skeletal muscle cells lacking β-catenin expression and with high expression of β-catenin showed decreased expression and increased expression of α2NKA, respectively. Hence, β-catenin appears to regulate the electrical properties and affects the normal functioning of skeletal muscle cells.We showed that β-catenin could interact physically with α2NKA (but not with α1NKA) in muscle cells. β-Catenin could regulate the enzymatic activity and content of α2NKA in cell membranes. These results suggested that β-catenin (at least in part) regulates the RMP and AP in muscle cells, and does so by regulating α2NKA.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.FG, CZ, KJ, YY, and JS conceptualized and designed the study. CZ, YY, YZ, JS, LJ, GS, LX, and WZ acquired and analyzed the data. CZ, SM, and PJ drafted the text and prepared the figures. All authors approved the final version of the manuscript to be published.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.


Additionally the IMD-Si@GO sheets could catalyze the curing reaction of epoxy resin-anhydride system significantly. Moreover, the improved thermal conductivities and thermal stabilities of epoxy composites filled with IMD-Si@GO were also demonstrated.Surface modification of graphene oxide (GO) is one of the most important issues to produce high performance GO/epoxy composites. In this paper, the imidazole ionic liquid (IMD-Si) was introduced onto the surface of GO sheets by a cheap and simple method, to prepare a reinforcing filler, as well as a catalyst in epoxy resin. The interlayer spacing of GO sheets was obviously increased by the intercalation of IMD-Si, which strongly facilitated the dispersibility of graphene oxide in organic solvents and epoxy matrix. The addition of 0.4 wt % imidazolium ionic liquid modified graphene oxide (IMD-Si@GO), yielded a 12% increase in flexural strength (141.3 MPa), a 26% increase in flexural modulus (4.69 GPa), and a 52% increase in impact strength (18.7 kJ/m Nevertheless, inherent brittleness strongly hinders the applications of epoxy resins in many fields. A large number of researchers have focused on the reinforcement of epoxy matrices with nano-fillers, such as SiOes, etc. ,2,3,4. Aes, etc. . Howeveres, etc. .Graphene oxide (GO), as an oxide form of graphene, contains a large number of oxygenated groups on the basal planes and along the edges. As a result, GO has high organic compatibility with matrices and can be a suitable nano-filler to reinforce epoxy resins . HoweverN,N-dimethylformamide (DMF), and dimethyl sulphoxide (DMSO). They thought the good dispersibility was attributed to the improved solubility and electrostatic repulsion of modified graphene nanosheets because of the ionic liquid incorporation. Due to the good dispersibility provided by ionic liquids, we think that the ionic liquid modified graphene or graphene oxide is of great value for reinforcing polymers. However, most reports about ionic liquid modified graphene or graphene oxide just focus on the electrochemical applications, such as electrodes, sensors, and supercapacitors [Ionic liquids have attracted a great deal of attention for their unique properties, such as excellent chemical and thermal stabilities, good electrical conductivity, and high ionic mobility ,15. Yangpacitors ,18. We tMeanwhile, it is worth mentioning that a majority of ionic liquids contain imidazole groups. As is well known, the imidazole rings can obviously catalyze the curing reactions of epoxy resins . Pour etHerein, we fabricated the imidazolium ionic liquid (IMD-Si) modified graphene oxide (IMD-Si@GO) by a cheap and simple method, based on the silanization reaction. The IMD-Si@GO nanosheets were incorporated into an epoxy resin-anhydride system to prepare the novel epoxy composites with high performances. The dispersibility of IMD-Si@GO, and the reinforcing and catalytic effects of IMD-Si@GO in the epoxy resin-anhydride system, have been investigated. Additionally, the thermal conductivities and thermal stabilities of IMD-Si@GO/epoxy (IMD-Si@GO/EP) composites were studied.Diglycidyl ether of bisphenol A (DGEBA) epoxy with an epoxy value of 0.48–0.54 mol/100 g was purchased from Wuxi Resin Factory of Bluestar New Chemical Materials Co., Ltd., Wuxi, China. Methyltetrahydrophthalic anhydride  was used as the curing agent. GO sheets were prepared from natural graphite flakes  by a modified Hummers method . Imidazo2 atmosphere [2 at 50 °C. After refluxing for 4 h, the mixture was filtered, washed with anhydrous alcohol, and dried in a vacuum oven at 60 °C.A schematic representation of the synthesis of IMD-Si@GO is shown in mosphere . The ionThe IMD-Si@GO/EP were prepared by casting method with different contents of fillers . The IMD-Si@GO sheets were first dispersed in acetone by sonication and then mixed with DGEBA epoxy. Afterwards, the mixture was put into a vacuum oven at 70 °C to remove solvent. Then, a stoichiometric amount of the curing agent MTHPA was added to the above mixture, followed by stirring for 10 min. Finally, the mixture was poured into a mold, cured following the schedule of 140 °C/2 h + 160 °C/4 h + 180 °C/3 h. As a reference, the pure epoxy resin was also prepared in similar procedures to perform contrast experiments.1H Nuclear magnetic resonance  spectra were recorded in deuterated DMSO solvent using a Bruker Avance spectrometer . Atomic force microscope (AFM) images were taken in a tapping mode using Hitachi Nanonavi E-sweep  with the size of 6 μm × 6 μm. X-ray photoelectron spectra (XPS) were performed on a PHI Quantum 2000 Scanning ESCA Microprobe system . All XPS spectra were corrected using the C 1s line at 284.8 eV. Fourier transform infrared (FTIR) spectra were characterized by a PerkinElmer-283B FTIR  spectrometer ranging from 4000 to 400 cm−1. Elemental analysis was carried out with a VarioEL III Analyser . X-ray diffraction (XRD) patterns were recorded with a Bruker D8 ADVANCE X-ray diffractometer  . The XRD data were collected from 5° to 85° with the scanning rate of 0.02°/s. Thermogravimetric analysis (TGA) was performed on a TGA Q50  at a heating rate of 10 °C/min, in an argon atmosphere.3 and (80 ± 0.2) × (10 ± 0.2) × (4 ± 0.2) mm3, respectively. The fractured surface of epoxy composites obtained from the impact test was observed using a scanning electronic microscope  and the samples were sputter-coated with gold. Differential scanning calorimetry (DSC) was conducted on a TA Instruments DSC 2920  using N2 as a purge gas from 30 to 300 °C with a heating rate of 10 °C/min. Thermal conductivities of the samples were measured at room temperature by a Hot Disk instrument .The dispersion of IMD-Si@GO in epoxy matrix was verified by using transmission optical microscopy . Bending tests and impact tests were carried out in a universal testing machine  following the Chinese standard GB/T2567-2008, and at least five tests were carried out for each sample. The samples’ dimensions for bending tests and impact tests were (80 ± 0.2) × (15 ± 0.2) × (4 ± 0.2) mm1H NMR , respectively. Additionally it shows a maximum increase of 26% in flexural modulus (4.69 GPa) for epoxy composites containing 0.4 wt % IMD-Si@GO. The enhanced strength and modulus of epoxy by adding a small amount of IMD-Si@GO is considered to be due to the strong interfacial bonding between fillers and epoxy matrix. The imidazole rings and residual oxygenated groups on the IMD-Si@GO surface can participate in the curing reaction. Consequently, the IMD-Si@GO is not only a nano-filler, but also a co-curing agent in epoxy matrix, which can strongly improve the interfacial interactions [The mechanical properties of epoxy resin with different contents of IMD-Si@GO fillers were experimentally studied. The flexural strength, flexural modulus, and impact strength of IMD-Si@GO/EP composites are shown in ractions . Meanwhiractions . By incrractions ,33.To further study the reinforcing mechanism of IMD-Si@GO in epoxy matrix, the fracture surfaces of epoxy and its composites were investigated by SEM. In Imidazole and its derivatives are widely used as a type of accelerators in epoxy resin-anhydride systems, which can initiate alternating copolymerization between anhydride and epoxy groups, as a result, initiate the esterification reaction and improve the curing speed dramatically. Thence, the catalytic effect of IMD-Si@GO in epoxy resin-anhydride system was investigated by DSC . For theThe main catalytic mechanism of IMD-Si@GO in epoxy resin-anhydride system is anionic ring-opening alternating copolymerization, which is shown in Thermal conductivity is also effectively affected by carbon fillers within the epoxy matrices . Figure Td (the temperatures at 5 wt % weight loss) and Tmax (the temperatures at maximum weight loss) of 225.2 and 399.0 °C, respectively. By adding 0.8 wt % IMD-Si@GO, the Td and Tmax (267.3 and 408.1 °C) are both higher than these of neat epoxy, suggesting that IMD-Si@GO can efficiently inhibit the mass loss during thermal degradation process. As for the char yield at 800 °C, the char residue of 0.8 wt % IMD-Si@GO/EP dramatically increases to 6.41 wt %, 2.2 times compared to that of unfilled epoxy (2.91 wt %). The enhancement of thermal stabilities can be explained by the barrier effect of modified GO sheets, uniform dispersion of IMD-Si@GO in matrix, and also strong interfacial bonding between IMD-Si@GO sheets and epoxy matrix [Thermal stabilities of the neat epoxy and IMD-Si@GO/EP composites loading 0.8 wt % of nano-fillers were investigated by TGA analysis. TGA and derivative thermogravimetry (DTG) curves are shown in y matrix .In this study, imidazolium ionic liquid modified graphene oxide was prepared by a cheap and simple method. The results from AFM, XPS, FTIR, XRD, and TGA indicate the successful preparation of IMD-Si@GO, and the grafting ratio is calculated to be 22.7% from the elemental analysis. The interlayer spacing of GO sheets is increased by intercalation of IMD-Si, which strongly facilitates the dispersibility of graphene oxide in organic solvents and epoxy matrix. The flexural strength, flexural modulus and impact strength of the IMD-Si@GO/EP composites are optimal with 0.4 wt % fillers, increased by 12%, 26%, and 52%, respectively, compared to the neat epoxy. Additionally, DSC reveals that the IMD-Si@GO sheets can catalyze the curing reaction of epoxy resin-anhydride system at very low IMD-Si@GO loadings. Moreover, a little inclusion of IMD-Si@GO into epoxy matrix can remarkably improve the thermal conductivities and thermal stabilities. These enhancements can be attributed to the uniform dispersion of IMD-Si@GO and strong interfacial interactions with the epoxy matrix. Consequently, the modification of GO sheets by covalent grafting of ionic liquid could be a promising method in the application of reinforced polymers by nanomaterials.


The Wnt/β-catenin pathway is involved in the osteogenic differentiation of human adipose-derived stem cells (hASCs) under cyclic strain. Very little is known about the role of microRNAs in these events.Cells were obtained using enzyme digestion methods, and proliferation was detected using Cell Counting Kit 8. Cell cycles and immunophenotypes were detected by flow cytometry. The multilineage potential of hASCs was induced by induction media. Cyclic strain was applied to hASCs  to induce osteogenic differentiation and miRNA changes. Bioinformatic and dual-luciferase analyses confirmed lymphoid enhancer factor 1 (LEF1) as a potential target of let-7i-3p. The effect of let-7i-3p on LEF1 in hASCs transfected with a let-7i-3p mimic and inhibitor was analyzed by immunofluorescence. hASCs were transfected with a let-7i-3p mimic, inhibitor, or small interfering RNA (siRNA) against LEF1 and β-catenin. Quantitative real-time PCR (qPCR) and western blotting were performed to examine the osteogenic markers and Wnt/β-catenin pathway at the mRNA and protein levels, respectively. Immunofluorescence and western blotting were performed to confirm the activation of the Wnt/β-catenin pathway.Flow cytometry showed that 82.12% ± 5.83% of the cells were in G1 phase and 17.88% ± 2.59% of the cells were in S/G2 phase; hASCs were positive for CD29, CD90, and CD105. hASCs could have the potential for osteogenic, chondrogenic, and adipogenic differentiation. MicroRNA screening via microarray showed that let-7i-3p expression was decreased under cyclic strain. Bioinformatic and dual-luciferase analyses confirmed that LEF1 in the Wnt/β-catenin pathway was the target of let-7i-3p. Under cyclic strain, the osteogenic differentiation of hASCs was promoted by overexpression of LEF1and β-catenin and inhibited by overexpression of let-7i-3p. hASCs were transfected with let-7i-3p mimics and inhibitor. Gain- or loss-of-function analyses of let-7i-3p showed that the osteogenic differentiation of hASCs was promoted by decreased let-7i-3p expression and inhibited by increased let-7i-3p expression. Furthermore, high LEF1 expression inactivated the Wnt/β-catenin pathway in let-7i-3p-enhanced hASCs. In contrast, let-7i-3p inhibition activated the Wnt/β-catenin pathway.Let-7i-3p, acting as a negative regulator of the Wnt/β-catenin pathway by targeting LEF1, inhibits the osteogenic differentiation of hASCs under cyclic strain in vitro. Bone tissue engineering can be used to repair bone defects induced by various factors by construction of functional bone tissue in vitro . Seed ceBone marrow mesenchymal stem cells (BMSCs) are currently the most widely used seed cells in the field of bone tissue engineering . HoweverHow to promote the osteogenic differentiation of ASCs in vitro has become an important research area for bone tissue engineering. Mechanical loading plays an essential role in cellular biological responses, such as the regulation of protein synthesis, cell proliferation, and differentiation . Some stThe Wnt signaling pathway is involved in the regulation of many critical biological processes, including embryogenesis and development, stem cell maintenance and differentiation, energy metabolism balance, and tissue stability maintenance. The Wnt/β-catenin pathway plays a complicated role in bone biology. Some syndromes show low bone mass and, consequently, frequent bone fractures caused by loss of functional mutations of the coreceptor LRP5 , while gMicroRNAs (miRNAs) are highly conserved single-stranded noncoding RNA molecules consisting of 18–22 nucleotides. MiRNAs bind to the 3′UTRs of their target mRNAs, inhibiting their translation or promoting their degradation . miR-138In this study, a gene microarray was used to screen the altered expression levels of miRNAs in hASCs during osteogenic differentiation under cyclic strain. We analyzed miRNAs associated with Wnt/β-catenin by bioinformatics methods and found that the target gene regulated by hsa-let-7i-3p is most likely lymphoid enhancer factor 1 (LEF1) in the Wnt/β-catenin pathway. We hypothesize that cyclic strain activates the Wnt/β-catenin pathway by downregulating hsa-let-7i-3p, thereby promoting the osteogenic differentiation of hASCs. This study aimed to validate this hypothesis using cell and molecular biology methods.g for 5 min, after which the supernatant was discarded, and the pellet was resuspended twice. Resuspend cells were cultured in alpha-modified Eagle’s medium  containing 10% fetal bovine serum  at 37 °C in a 5% CO2 incubator. In this study, fourth-passage cells were used in the following experiments.Fat tissue was obtained from 6 donors who underwent flap reconstruction in the Oral and Maxillofacial Surgery Department at the Affiliated Stomatological Hospital of Nanjing Medical University, and all patients signed informed consent. The procedures were approved by the ethics committee of Nanjing Medical University. Adipose tissue was isolated from flaps and then washed three times with phosphate-buffered saline (PBS) to remove red blood cells. The adipose tissue was digested with 0.1% collagenase type I  at 37 °C for 1 h. After tissue digestion, the tissue was centrifuged at 3002 incubator. Cells in 6 wells were measured by adding 10 μl of Cell Counting Kit-8 solution to each well, followed by incubation at 37 °C for 2 h. Absorbance was measured by spectrophotometry  at 450 nm. Cell counting was performed for 8 consecutive days. The growth curve of the cells was drawn according to the above results.Cell proliferation was detected following the manufacturer’s manual by Cell Counting Kit-8 assays  according to the manufacturer’s instructions. Cells were seeded into 96-well plates at 3000 cells per well and then cultured in α-MEM supplemented with 10% FBS at 37 °C in a 5% COCells were collected by centrifugation and fixed with precooled 70% ethanol overnight. Cells were added to 50 μL of RNase stock solution (100 μg/mL), stained with 500 μL of PBS containing 50 μg/mL ethidium bromide (PI), and incubated at 4 °C for 30 min in the dark. Finally, the fluorescence intensity of DNA-PI was detected by a FACSVerse flow cytometer  for analysis of cell cycle changes.Immunophenotyping of the hASCs was performed by staining for 30 min on ice in the dark with the fluorochrome-labeled monoclonal antibodies CD29-APC , CD34-PE , CD45-PE , CD90-FITC , and CD105-PerCP cy5.5 . Untreated cells served as the negative control to adjust for the compensation of fluorochrome overlap. The fluorescence intensity was examined by a FACSVerse flow cytometer , and flow cytometry analysis was performed with FlowJo software 7.6.1 .hASCs were induced toward adipogenic, osteogenic, and chondrogenic differentiation to confirm their multilineage potential. Adipogenic medium  was used to induce adipogenic differentiation of hASCs; after 14 days, hASCs were stained with Oil Red O to determine the formation of lipid droplets. Osteogenic medium  was used to induce osteogenic differentiation of hASCs; 21 days later, hASCs were stained with Alizarin Red to confirm the existence of mineralized nodules. Chondrogenic medium  was used to induce chondrogenic differentiation of hASCs; 14 days later, hASCs were stained with Alcian Blue to verify the existence of proteoglycans.5 cells/ml and incubated in α-MEM containing 10% FBS at 37 °C in a 5% CO2 incubator. Twenty-four hours later, stable adherent cells in the BioFlex® Culture Plates were exposed to uniaxial cyclic strain  by a Flexcell® FX-5000™ Tension System . The control groups were cultured in α-MEM supplemented with 10% FBS at 37 °C in a 5% CO2 incubator for 6 days without cyclic strain loading. On the sixth day, hASCs were collected for real-time quantitative PCR (qPCR) and western blot analyses.hASCs were seeded on silicone rubber BioFlex® Culture Plates  at a density of 1.0 × 10mirVana™ miRNA Isolation Kit  according to the manufacturer’s instructions. Fluorescent labeling using the T4 RNA ligase labeling method was followed by precipitation with absolute ethanol, and the samples were then blown dry. The isolated RNA was dissolved in hybridization solution and incubated overnight at 42 °C. Then, the samples were washed with 0.2% sodium dodecyl sulfate (SDS) and 2 × SSC. After drying, the slides were scanned by the LuxScan 10K-A Dual Laser Scanner .Total RNA was isolated from hASCs by TRIzol Reagent  after cyclic strain for 6 days. miRNAs were isolated from total RNA using an Ambion® http://www.targetscan.org), miRWalk , and miRBase (http://www.mirbase.org).The conservative characteristics of let-7i-3p, the 3′UTR binding site for LEF1, and the targets of let-7i-3p were analyzed by the following three databases: TargetScan (The following genetic research tools were used in this study: LEF1 expression plasmid (EX-LEF1), LEF1 siRNA (siLEF1), negative control expression plasmid (EX-Ctrl), negative control siRNA (siR-Ctrl), let-7i-3p mimic, let-7i-3p inhibitor, negative control of the let-7i-3p mimic (miR-Ctrl mimic) and negative control of the let-7i-3p inhibitor (miR-Ctrl inhibitor), LEF1 3′UTR-wild type (wt-LEF1), and LEF1 3′UTR-mutant (mu-LEF1). These vectors were transfected into fourth-passage hASCs using Lipofectamine™ 2000 Transfection Reagent  according to the manufacturer’s instructions. The let-7i-3p mimic and inhibitor, siLEF1, miR-Ctrl mimic, miR-Ctrl inhibitor, and siR-Ctrl were synthesized by GenePharma Corporation . EX-LEF1 (pEZ-M02 vector), EX-Ctrl (pReceiver-M02 vector), wt-LEF1, and mu-LEF1 were supplied by GeneCopoeia Corporation . ORF sequence information for EX-LEF1 and EX-Ctrl is listed in the supplemental materials. Other sequences transfected in this process are listed in Table To change the expression of β-catenin, we transduced lentiviral particles containing an shRNA targeting β-catenin or an shRNA control  into SKOV3 cells. Lentiviral-transduced SKOV3 cells were selected with puromycin (1.5 μM/mL). Transient transfection was performed using a pool of 4 short interfering RNAs (siRNAs) targeting β-catenin  or individual siRNA sequences  and DreamFECT transfection reagent . The scrambled siRNA pool (Dharmacon) was used as a control.Renilla luciferase reporter gene. The sequences mentioned above are listed in Additional file Renilla luciferase activities 48 h after transfection. The firefly values were normalized to Renilla luciferase.Sequence fragments, such as LEF1 3′UTR-WT and LEF1 3′UTR-Mu, were inserted between the NotI and XhoI cleavage sites of the psiCHECK-2 vector  downstream of the The fourth-passage hASCs transfected with the let-7i-3p mimic and inhibitor were used to detect the effect of let-7i-3p on LEF1 by immunofluorescence. Two days after transfection, hASCs were seeded on coverslips in 9-well plates. After 48 h, hASCs were fixed with 4% paraformaldehyde for 15 min and washed three times with PBS. Triton X-100  was added to each well for 20 min at room temperature. After rinsing three times with PBS, hASCs were blocked with goat serum for 2 h. Primary antibodies specific for LEF1  were added to the cells and then incubated overnight at 4 °C. Fluorescent Cy3 secondary antibodies  were added and incubated for 1 h at 37 °C in the dark after rinsing three times with PBS Tween-20. The nucleus was then restained with 4′,6-diamidino-2-phenylindole . Cells were subsequently viewed by fluorescence microscopy .Total RNA was isolated from hASCs by TRIzol Reagent and detected by a NanoVue™ Plus spectrophotometer  to determine purity at 260/280 nm. cDNA was synthesized from RNA using PrimeScript RT Master Mix . PCR was performed on the ABI 7300 Real-Time PCR System . The following markers associated with osteogenesis and the Wnt/β-catenin pathway alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), secreted protein acidic and cysteine-rich (SPARC), LEF1, β-catenin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All primer sequences used in this study are shown in Table g, 20 min). The protein concentrations were determined using a BCA Protein Assay Kit . The protein was boiled at 100 °C for 5 min after the addition of 5 × SDS loading buffer . Protein samples were loaded onto SDS polyacrylamide gel electrophoresis gels and then electrophoresed at 100 V for 1.5 h. Then, the protein blots were transferred onto membranes , which were blocked with TBS containing 5% nonfat milk overnight at 4 °C. Then, the membranes were incubated with primary antibodies specific for ALP , RUNX2 , SPARC , LEF1 , β-catenin , and GAPDH  overnight at 4 °C. Afterward, the membranes were washed three times with TBS-0.05% Tween 20 and incubated with the corresponding secondary antibodies for 1 h at room temperature. Next, ECL solution  was added to the membranes and incubated for 1 min. The protein blots were visualized by exposure to enhanced chemiluminescence reagents . Grayscales on the blots were analyzed by Quantity One software .Adherent hASCs were lysed with cell lysis buffer for western blot and immunoprecipitation (IP) analyses  and Nuclear and Cytoplasmic Protein Extraction Kit  respectively. Then, samples centrifuged at 4 °C  One-way analysis of variance (ANOVA) was used for multiple group comparisons. Two-way ANOVA was used for two-group comparisons. All of the results are presented as the mean ± sem, and statistical significance was set at As shown by a light microscope , the fourth-passage hASCs had a typical fibroblast-like morphology , CD90 (98.43% ± 1.77%), and CD105 (92.21% ± 6.48%) and negative for CD34 (2.39% ± 0.72%) and CD45 (0.97% ± 0.41%) , 2.57 ± 0.09-fold (p = 0.004), and 2.13 ± 0.60-fold (p = 0.018), respectively; LEF1 and β-catenin, which are associated with the Wnt/β-catenin pathway, were increased 1.89 ± 0.45-fold (p = 0.012) and 2.22 ± 0.51-fold (p = 0.017), respectively , 1.39 ± 0.09-fold (p = 0.010), and 1.79 ± 0.21-fold (p = 0.005), respectively; LEF1 and β-catenin levels were increased 1.55 ± 0.39-fold (p = 0.030) and 1.63 ± 0.20-fold (p = 0.004), respectively . Among them, 150 miRNAs exhibited decreased in expression, and 87 miRNAs exhibited increased expression. Some miRNAs associated with Wnt/β-catenin and their fold changes are shown in Fig. The predicted binding sequences of both the 3′UTR in LEF1 and let-7i-3p are highly conserved across species Fig. a, b. Thep = 0.007) compared with that in the control group, which was a significant difference. In the reporter gene system in which the LEF1 mRNA 3′UTR binding sites were mutated, overexpression of let-7i-3p did not affect the reporter gene luciferase activity. Inhibition of let-7i-3p expression induced the luciferase activity of the reporter gene. Compared with that in the control group, the luciferase activity in the let-7i-3p inhibition group was increased 1.58 ± 0.37-fold (p = 0.011), and the difference was significant. Then, combination with the LEF1 mRNA 3′UTR was assessed. In the reporter gene system with a site mutation, inhibition of let-7i-3p expression did not affect the reporter gene luciferase activity. The results are shown in Fig. Overexpression of let-7i-3p significantly inhibited the luciferase activity of pMIR/LEF1-WT reporter genes, and the luciferase activity was decreased by 2.19 ± 0.49-fold (p = 0.013), as shown by qPCR, compared to that in hASCs transfected with the miR-Ctrl mimic. LEF1 mRNA expression in hASCs transfected with the let-7i-3p inhibitor was increased 1.66 ± 0.07-fold (p = 0.002), as shown by qPCR, compared to that in hASCs transfected with the miR-Ctrl inhibitor , as shown by western blot, compared to that in hASCs transfected with the miR-Ctrl mimic. LEF1 protein expression in hASCs transfected with the let-7i-3p inhibitor was increased 1.43 ± 0.10-fold (p = 0.013), as shown by western blot, compared to that in hASCs transfected with the miR-Ctrl inhibitor    Fig. a. Compar01) Fig. a.Fig. 6p < 0.001), 3.38 ± 0.64-fold (p = 0.002), 2.07 ± 0.35-fold (p = 0.008), 4.67 ± 0.47-fold (p = 0.008), and 2.33 ± 0.39-fold (p = 0.023), respectively , 1.65 ± 0.68-fold (p = 0.044), 3.04 ± 1.38-fold (p = 0.039), 3.40 ± 1.18-fold (p = 0.001), and 2.72 ± 0.44-fold (p = 0.003), respectively   , 3.76 ± 2.04-fold (p = 0.021), 1.85 ± 0.49-fold (p = 0.004), 2.64 ± 0.40-fold (p = 0.006), and 4.30 ± 1.12-fold (p = 0.001), respectively , 3.46 ± 0.79-fold (p = 0.009), 2.16 ± 0.02-fold (p = 0.010), 2.52 ± 0.61-fold (p = 0.001), and 4.15 ± 1.72-fold (p = 0.004), respectively   , 1.80 ± 0.15-fold (p = 0.012), 3.07 ± 0.86-fold (p < 0.001), 2.08 ± 0.73-fold (p = 0.018), and 3.60 ± 0.57-fold (p = 0.002), respectively  and 1.74 ± 0.24-fold (p = 0.019), respectively , 2.53 ± 0.49-fold (p = 0.008), 1.83 ± 0.28-fold (p = 0.012), 2.31 ± 1.03-fold (p = 0.046), and 2.86 ± 1.68-fold (p = 0.010), respectively  and 2.97 ± 0.19-fold (p = 0.001), respectively , 2.80 ± 1.62-fold (p = 0.005), 2.06 ± 0.30-fold (p = 0.006), 3.06 ± 0.52-fold (p = 0.029), and 2.75 ± 0.68-fold (p = 0.010), respectively, in hASCs cotransfected with the let-7i-3p mimic and EX-LEF1 , 2.03 ± 0.07-fold (p = 0.002), 2.54 ± 0.60-fold (p = 0.004), 2.91 ± 0.61-fold (p = 0.004), and 1.49 ± 0.17-fold (p = 0.022), respectively  and 1.40 ± 0.15-fold (p = 0.017), respectively, in hASCs cotransfected with the let-7i-3p mimic and EX-LEF1 , 3.12 ± 1.55-fold (p = 0.002), 2.55 ± 0.25-fold (p = 0.038), 3.92 ± 0.72-fold (p = 0.018), and 2.25 ± 1.24-fold (p = 0.030), respectively, in hASCs cotransfected with the let-7i-3p inhibitor and siLEF1 , 1.54 ± 0.17-fold (p = 0.013), 1.71 ± 0.19-fold (p = 0.003), 2.24 ± 0.28-fold (p = 0.001), and 1.55 ± 0.06-fold (p = 0.012), respectively  and 2.13 ± 0.35-fold (p = 0.001), respectively, in hASCs cotransfected with the let-7i-3p inhibitor and siLEF1 , 4.27 ± 1.78-fold (p = 0.002), 3.06 ± 0.41-fold (p = 0.001), and 3.17 ± 1.09-fold (p = 0.008), the mRNA expression levels of LEF1 was decreased 1.47 ± 0.21-fold (p = 0.016), respectively, in hASCs cotransfected with the let-7i-3p mimic and EX-β-catenin , 2.10 ± 0.03-fold (p = 0.001), 3.16 ± 1.01-fold (p = 0.002), and 1.50 ± 0.04-fold (p = 0.006); the protein expression levels of LEF1 were decreased 1.40 ± 0.13-fold (p = 0.023), respectively  and 2.23 ± 0.27-fold (p = 0.008), respectively, in hASCs cotransfected with the let-7i-3p mimic and EX-β-catenin , 2.63 ± 0.69-fold (p = 0.004), 2.36 ± 0.46-fold (p = 0.006), and 3.75 ± 0.21-fold (p = 0.001); the mRNA expression levels of LEF1 was increased 1.89 ± 0.74-fold (p = 0.009), respectively, in hASCs cotransfected with the let-7i-3p inhibitor and siβ-catenin , 2.12 ± 0.14-fold (p = 0.006), 1.40 ± 0.01-fold (p = 0.020), and 1.51 ± 0.17-fold (p = 0.011); the protein expression levels of LEF1 were increased 1.53 ± 0.19-fold (p = 0.006), respectively  and 1.83 ± 0.04-fold (p = 0.004), respectively, in hASCs cotransfected with the let-7i-3p inhibitor and siβ-catenin , is involved in many different types of diseases. Falzone et al. reported that abnormal let-7i-3p expression has the potential to predict recurrence in oral cancer patients . IndersiStudies have been conducted to directly regulate the expression of LEF1 by miRNAs, mainly focusing on tumor diseases. miR-34a increased chemosensitivity in BIU87/ADR cells by targeted inhibition of the TCF1/LEF1 axis . miR-22,Currently, some studies have confirmed that some miRNAs can affect the expression of LEF1 and the Wnt/β-catenin pathway in the process of osteogenesis. Overexpression of miR-539 increased the expression of β-catenin, LEF1, c-myc, cyclin D1, RUNX2, BGP, and BMP-2 in rat osteoblasts . HoweverTo determine the role of let-7i-3p in the process of hASC osteogenic differentiation under cyclic strain, we designed serial experiments to verify its function. Inhibition of let-7i-3p and overexpression of LEF1 promoted the osteogenic differentiation of hASCs in vitro. Furthermore, gain- and loss-of-function experiments on let-7i-3p showed that let-7i-3p negatively regulates the Wnt/β-catenin pathway via LEF1, which inhibits the osteogenic differentiation of hASCs under cyclic strain in vitro Fig. .Fig. 10Additional file 1. The sequences mentioned in the studyAdditional file 2: Figure S1. Microarray for miRNA changes under cyclic strain. There were 150 miRNAs decreased and 87 miRNAs increased significantly (>2-fold).


Biomimetics is the interdisciplinary scientific field focused on the study and imitation of biological systems, with the aim of solving complex technological problems. In this paper, we present a new bio-inspired design for microneedles (MNs) and MN arrays, intended for rapidly coating the MNs with drug/vaccine. The biomimetic approach consists in ornamenting the lateral sides of pyramidal MNs with structures inspired by the external scent efferent systems of some European true bugs, which facilitate a directional liquid transport. To realize these MNs, two-photon polymerization (TPP) technique was used. Liquid coating capabilities of structured and non-structured MNs were compared. Moreover, both in-vivo and ex-vivo skin tests were performed to prove that MNs pierce the skin. We show that the arrays of MNs can be accurately replicated using a micro-moulding technique. We believe this design will be beneficial for the process of drug/vaccine loading onto the needles’ surfaces, by making it more efficient and by reducing the drug/vaccine wastage during MN coating process.The online version of this article (10.1007/s10544-019-0456-z) contains supplementary material, which is available to authorized users. Microneedles (MNs) are sharp, needle-like projections, generally less than 1 mm tall and arranged in arrays covering surfaces ranging from a few square millimetres up to several square centimetres ; and finally - due to gravitational force - the liquid drug formulation tends to accumulate at the base of the MNs, resulting in drug loss  were proven to be efficient in the adhesion to, and interlocking with the soft tissues. These MN patches are much more flexible than conventional polymer-based MNs, therefore they are easily removed from the tissues, without damaging the tissue or the MNs  insertion into skin. This knowledge was implemented in MN design and production  lithography was used – an intrinsically three-dimensional (3D) micro-fabrication technique, which enables production of micro-scale devices with feature sizes below the resolution limit  were realized using SketchUp Make 2017 – a freeware for three-dimensional (3D) modelling.2, into which the MNs were fabricated. The microstructures building up the ‘arms’ of the vertical channel are oriented towards the MNs. The vertical channel measures 900 μm in length, the horizontal channel measures 1500 μm in length, and both channels have a width of 300 μm.The channel, consisting of periodic bug-inspired microstructures Fig. , is inteThe MNs were designed as 210 μm high pyramids with square bases, 160 μm long sides and 10 μm thick walls Fig. . The str2, and thickness of 170 ± 30 μm (Menzel-Gläser coverslips) at a speed of 20 μm s−1, with a laser excitation power of 12 mW.To write the guiding channel, a custom-made setup , wavelength = 800 nm, pulse length = 150 fs, repetition rate = 80 MHz) was used. The setup is described in more detail in  in a sandwich sample configuration . The photoresist used for MNs consisted of OrmoComp® and 1 wt% of Ciba® IRGACURE® 2959 (Sigma-Aldrich). In both cases, the unpolymerized photoresist was dissolved in OrmoDev developer (micro resist technology GmbH).Wetting tests were performed to prove the wettability of the lateral sides of the structured MNs. The liquid used for wetting tests and fluorescence microscopy imaging was a mixture of 1 to 10 of an ethanol solution of Alexa Fluor® 555 fluorophore  and distilled water. Furthermore, less than 1% of soap (DAWN liquid dish soap) was added, in order to adjust the liquid contact angle in a range of 40° to 50°, which is a prerequisite for the directional liquid movement , with a Texas Red filter cube, with excitation window at 540–580 nm, and emission window at 600–660 nm (Nikon Instruments Inc.) was used. Before performing the wetting test, the samples were imaged in the red channel, to assess their auto-fluorescence. Afterwards, a small volume of water-fluorophore solution was deposited in the close proximity of the microstructured channel  printed on a glass slide were replicated as described in . After the glue had dried, a mixture of 10:1 of the two components of polydimethylsiloxane SYLGARD 184 (Neyco) was poured inside the Petri dish, covering the MNs array. The Petri dish was then left overnight on a levelled shelf, and cured in the morning at 80 °C for 1 h.In a similar way, the two components of EPO-TEK® 353ND  were mixed together in a ratio of 10:1, degassed, and used to fill the SYLGARD® 184 mould. A flat disk of PDMS was used as rear mould and the ensemble was put on top of a vacuum table covered by a 500 g weight for 1 h. The EPO-TEK® 353ND was cured at 80 °C for 1 h and left at room temperature for 14 h to cool down. Finally, the two moulds peeled away from the MNs patch.The master template , as well as the epoxy replicas, were SEM imaged to assess the fidelity of the replication process. From the SEM images, the MNs’ base lengths, tip widths, as well as the heights, widths and lengths of individual microstructures were measured using the ImageJ software.2 was used to apply the epoxy MNs array with a surface of 4 × 4 mm2 to the skin in a repeatable and reliable manner. After the impact, the applicator maintained a force of 0.5 N on the MNs array, and retracted after 2 min. The test was repeated three times to confirm the reproducibility of the results. After each test, the impact areas were stained with methylene blue (Sigma-Aldrich) for 10 min, prior to PBS rinsing and tape stripping to remove the excess dye present on the surface of the skin , human skin was excised following plastic surgical operations and stored at −80 °C until needed. The skin was thawed and incubated in phosphate-buffered saline (PBS) solution (Sigma-Aldrich) for 30 min at 32 °C. Then, the skin samples were trimmed of the excess fat, cut into squares of approximately 3 cm × 3 cm and mounted on am artificial tissue substrate , to mimic the mechanical properties of underlying tissues. A custom-built, spring-loaded applicator, with a spring constant of 274 N/m, and impact energy of 0.4 ± 0.01 J/cmFor a second set of skin tests, an optical coherence tomography (OCT) system was used to observe the penetration depth of the MNs. The MNs array was pressed against the forearm skin of a healthy male volunteer and fixed with tape prior to recording the images. The commercially available TELESTO™ Spectral Domain OCT imaging system  using a super-luminescent diode, with a central wavelength at 1325 nm, with a 150 nm bandwidth was employed. The device offers a lateral resolution of 15 μm and an axial resolution of less than 7.5 μm. After performing several skin tests, the MNs were imaged using a scanning electron microscope.Figures The CAD designs of the structured and non-structured MNs are shown in Fig. An image sequence of the liquid movement in the guiding channel is presented in Fig. Figure Figure MNs with the same bug-inspired ornamentation, but pointing downwards, were also tested  are summarised in Table t-test revealed no significant difference between the three sets of data, as the p-values were always greater than 0.1. These results show a successful and exact replication of the MNs array.Measurements of the MNs’ base length yielded an average value of 155.01 ± 1.69 μm for the original one, and a value of 150.51 ± 1.38 μm for the replicated MN. The difference is due to post curing shrinkage. The tip diameters for both of MNs were about 6 μm. Microstructures’ lengths, base and tip widths mean values and standard deviations , which were subjected to several skin tests, showed no damage caused by the tests. This confirms the mechanical stability of the MNs: the MNs did not break and presented no risk of remaining in the skin.The wetting tests were performed using a water-based liquid on OrmoComp® MNs. To obtain a directional flow, it is essential that the dimensional parameters (structures heights and curvatures) are set in the correct range, and that the liquid contact angle is adjusted under 45°. As discussed in detail in  is the simplest of all coating techniques, but it leads to significant wastage of the active ingredient, since coatings are produced on the entire microneedle patch and only a small percentage is actually delivered , as a nAs microneedles are becoming a more auspicious tool for transdermal drug delivery, a large amount of research is being carried out to optimize MN aspect ratios, fabrication materials and techniques, as well as the methods used for coating their surfaces with drug and vaccine formulations. In this study we describe a novel biomimetic approach towards drug delivery and show proof of principle. We introduced a new MN design - the lateral faces of the MNs were ornamented with bug-inspired microstructures. When compared to the non-structured negative control MN, the structured MN proved to be easily loaded with liquid. We showed that the MN arrays can be rapidly replicated, with high accuracy, in medical-grade epoxy materials. Skin tests proved that the MNs are able to break the stratum corneum barrier and reach the epidermis, with no damage to the MNs, and no pain sensation for the volunteer. However further research is required to better characterise and demonstrate pharmacological loading capabilities of the MN and drug delivery efficiency both in vitro and in vivo models.In the future, we envisage an array of hundreds of MNs, interconnected by a net of guiding channels, consisting of bug-inspired structures. The arrays would be easily loaded with necessary drugs/vaccines, without recourse to complicated deposition procedures, and with minimal formulation wastage. To our knowledge, this is the first MN design that makes use of bio-inspired surface structures for passive liquid transportation on its lateral surfaces.Supplement 1Video showing a z-stack of confocal microscope images of the non-structured MN after the wetting test. First, one sees the structures in the guiding channel. Later on, one sees the non-structured MN’s lateral faces and corners from the bottom (z = 0 μm) till z = 60 μm. (MP4 1447 kb)Supplement 2Video showing a z-stack of confocal microscope images of the structured MN after the wetting test. First, one sees the structures in the channels. Later on, one sees the structured MN’s lateral faces and corners from the bottom (z = 0 μm) till z = 90 μm. Note that the height range here is considerably larger than in Supplement Supplement 3a) The CAD design of the test sample. (b) Optical microscope image of the T-shaped channel and the two MNs before the wetting test. (c) Fluorescent microscope image taken in the red channel after the wetting test performed with a fluorophore-soap-water solution. Scanning electron microscope (SEM) images of the (d) MN with surface structures pointing downwards, and of the (e) MN with the surface structures pointing upwards, taken at a 45° view angle . (f) Intensity profile along the white line in Supplement 3E . For this certain wetting test a fluorophore concentration of 1 to 100 was used, therefore the signal intensity in image (c) is lower than in the Fig. A T-shaped guiding channel containing two different structured MNs . (High Resolution Image (TIF 32858 kb)


AXIN1) gene expression, thereby attenuating Wnt/β-catenin signaling. C/EBP-β interacted with cis-regulatory element for C/EBP-β in the 5′-upstream sequences of the AXIN1 gene and increased AXIN1 promoter activity. Functional analysis using Drosophila and zebrafish models established that C/EBP-β negatively regulates the Wnt/β-catenin pathway. Small-molecule-based up-regulation of C/EBP-β induces AXIN1 gene expression and down-regulates the intracellular β-catenin level, thereby inhibiting hepatoma cell growth. Thus, our findings provide a unique mechanistic insight into the regulation of Axin homeostasis and present a novel strategy for the development of anticancer therapeutics targeting Wnt/β-catenin signaling.Axin1, a concentration-limiting component of the β-catenin destruction complex, negatively regulates the Wnt/β-catenin pathway. Axin1 concentration is reported to be regulated by proteasomal degradation; however, its transcriptional regulation has not yet been reported. Here, we demonstrated that CCAAT/enhancer-binding protein-β (C/EBP-β) activates axis inhibition protein 1 ( AXIN1) regulates the Wnt/β-catenin pathway and subsequently plays important roles in embryogenesis and tumorigenesis1. In this pathway, the scaffold protein Axin1 interacts with several proteins including casein kinase 1, glycogen synthase kinase-3β (GSK-3β), adenomatous polyposis coli (APC), and β-catenin through separate domains (which constitute the β-catenin destruction complex) to coordinate the sequential phosphorylation of β-catenin at residues Ser45, Thr41, Ser37, and Ser333. The occurrence of phosphorylation serves as a target for degradation by the ubiquitin-dependent proteasome pathway4. Axin1 is recruited to the phosphorylated low-density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors after the binding of Wnt ligands  to the Frizzled receptor and LRP5/65. This leads to negative regulation of GSK-3β in the β-catenin destruction complex and in turn stabilization of β-catenin6. Axin1 is present at lower concentrations in the β-catenin destruction complex compared with the other components, and therefore acts as a concentration-limiting factor for assembly of this complex7. Thus, the levels of Axin1 are the key regulators in the Wnt/β-catenin pathway.Alteration of the axis inhibition protein 1 , LAP2 , and LIP 9. These transcription factors regulate several classes of genes, such as proliferation/differentiation-related markers, metabolic enzymes, and cytokines10. In this study, we identify C/EBP-β as a bona fide transcriptional activator of the AXIN1 gene expression. C/EBP-β promotes Axin1-mediated β-catenin degradation and thereby negatively regulates the Wnt/β-catenin pathway.CCAAT/enhancer-binding protein-β (C/EBP-β), also known as NF-IL6 (nuclear factor for interleukin-6) or TCF5 (transcription factor 5), is a member of C/EBP family of transcription factors11. Inversely, we observed that adipogenic stimulation with MDI  led to a reduction in the level of β-catenin without altering the β-catenin messenger RNA (mRNA) level  of AXINI across various species for the presence of shared motifs in their regulatory sequences. Accordingly, cis-regulatory elements that were conserved in human, mouse, rat, and zebrafish were identified. The most enriched cis-regulatory element was identified for C/EBP-β , a bZIP transcription factor . As shown in Fig. TTTCCTAATCC) into the mutant-type (ACCGGTCCTAATCC) blocked the activation of the hAxin1 promoter in the presence of ectopic C/EBP-β expression  reporter plasmid. β-Catenin response transcription that had increased due to β-catenin expression was down-regulated upon transfection of HEK293-FL reporter cells with the C/EBP-β expression plasmid  and patterning the embryonic epidermis14, we investigated the function of C/EBP-β as a negative regulator of the Wnt/β-catenin pathway in Drosophila. To this end, we used GAL4/UAS system to overexpress slbo, a Drosophila ortholog of C/EPB-β, in fly wings and compared the resulting phenotypes to known effects of Wg mutations. As shown in Fig. slbo under the control of MS1096-GAL4 led to a reduction in the size of the wing, mimicking the phenotype of Wg loss-of-function mutation. Since epidermal cells with active Wg signaling secrete naked cuticle, we examined the effect of slbo on the Wg pathway in slbonull mutant flies. Compared to wild-type flies, elimination of slbo gene resulted in the expansion of Wnt signaling as evidenced by increasing the secretion of naked cuticle , the cle Fig. , suggestc/ebp-β transcripts by using whole-mount in situ hybridization starting from the two-cell stage to 48 h post fertilization (hpf). c/ebp-β is maternally provided and expressed ubiquitously throughout early development with higher levels being observed in the yolk syncytial layer and the liver  because c/ebp-β MO may not be able to completely deplete endogenous C/EPB-β or functional isoforms. Taken together, these results indicate that C/EPB-β negatively regulates Wnt/β-catenin signaling in zebrafish and Drosophila.We next tested whether C/EBP-β negatively regulates the Wnt/β-catenin pathway in zebrafish. To this end, we first analyzed the distribution of ver Fig. . Furtherryo Fig. . Additioage Fig. . In caseryo Fig. . MoreoveICM Fig. . myoD exryo Fig. . We alsoyos Fig. . The oveAXIN1 gene expression and the Wnt/β-catenin pathway. Consistently, the overexpression of C/EPB-β, which controls hepatocyte differentiation and their characteristic quiescent state17, led to increased Axin1 promoter activity in Huh-7 hepatoma cells -mediated activation of AXIN1 expression, and subsequent regulation of Wnt/β-catenin signaling , cells were incubated with 10% FBS, 0.5 mM of 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μg/mL insulin for 24 h.HEK293, SNU475, Huh-7, and 3T3-L1 cells were obtained from the American Type Culture Collection . HEK293 was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 120 g/mL penicillin, and 200 g/mL streptomycin. 3T3-L1 cells were maintained in DMEM supplemented with 10% FCS, 120 g/mL penicillin, and 200 g/mL streptomycin. SNU475 and Huh-7 were maintained in Roswell Park Memorial Institute 1640 (RPMI 1640) supplemented with 10% FBS, 120 g/mL penicillin, and 200 g/mL streptomycin. HEK293-FL reporter (TOPFlash) and control (FOPFlash) cells were established as previously describedTM Site-Directed Mutagenesis Kit . All clones and mutants were verified by sequencing. The pTOPflash reporter plasmid was obtained from Upstate Biotechnology . pCMV-RL plasmid was purchased from Promega . Transfections were performed using Lipofectamine 2000 , according to the manufacturer’s instructions. Luciferase assay was performed using the Luciferase Assay System .A bacterial artificial chromosome clone containing the region corresponding to Axin1 was used as a PCR template for cloning the promoter region of the human and mouse Axin1 genes. The amplified DNA fragments were sub-cloned into the luciferase reporter vector, pGL3-basic . Site-directed mutagenesis of the C/EBP-β-binding site was performed according to the QuikChange30. Proteins were separated by SDS-PAGE in a 4–12% gradient gel  and transferred to a nitrocellulose membrane . The membrane was blocked with 5% nonfat milk and probed with anti-β-catenin , anti-phospho-β-catenin (Ser33/37/Thr41) , anti-phospho-β-catenin (Thr41/Ser45) , anti-Axin1 , anti-Axin2 , anti-C/EBPβ , anti-cyclinD1 , anti-c-myc , anti-GFP , and anti-actin  antibodies. The membranes were then incubated with horseradish-peroxidase-conjugated anti-mouse IgG  or anti-rabbit IgG  and visualized using the ECL system .Cytosolic fractions were prepared as previously described31 The amplified DNA was separated on 2% agarose gels and stained with ethidium bromide. For real-time PCR, the CFX96 qPCR system  was used. Reactions were carried out in 96-well optical reaction plates in a 20 μl final volume containing 10 μl of the 2X SsoFast™ EvaGreen® Supermixes , 1 μl of each gene-specific forward primer, 1 μl of each gene-specific reverse primer, 1 μl of diluted cDNA sample, and 7 μl of water. After an initial denaturing step for 10 min at 95 °C, conditions for cycling were set to 40 cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C. The changes in gene expression were quantified by the comparative Ct method by calculating the relative fold changes normalized against β-actin expression.Total RNA was isolated using the TRIzol reagent  according to the manufacturer’s instructions. Complementary DNA (cDNA) synthesis, reverse transcription, and PCR were performed as previously describedSynthetic siRNAs targeting C/EBP-β were purchased from Applied Biosystems/Ambion . Targeted sequences were as follows: C/EBP-β sense, 5′-GGCCCUGAGUAAUCGCUUAtt-3′ and antisense, 5′-UAAGCGAUUACUCAGGGCCcg-3′. AccuTarget™ Negative Control siRNA  was used as a control. shRNA lentivirus plasmids specific for C/EBP-β were purchased from Sigma-Aldrich Cells were fixed with 1% formaldehyde for 10 min at room temperature. ChIP assays were performed using the Chromatin Immunoprecipitation (ChIP) Assay Kit  according to the manufacturer’s instructions. Chromatin extracts were immunoprecipitated with IgG  or anti-C/EBP-β antibodies . DNA-associated protein was analyzed with real-time PCR using primers targeting Axin1-binding site (−112/104). Primers used PCR were: forward, 5′-GGCCAAATATGTCTCTGCTGG-3′ and reverse, 5′-GCGCTGCACCCTAATACCTC-3′ (for mouse Axin1-binding site), and forward, 5′-GGGCATCCCTTCTTAGCATGAG-3′ and reverse, 5′-CGCTGCACCCTAATA CATCAGT AC-3′ (for human Axin1-binding site).2, 5 mM HEPES, pH 7.2) and treated with PTU  to suppress pigmentation. Embryonic stages were determined by the hpf and microscopic observation of gross morphology.Zebrafish were maintained at 28.5 °C on a 14 h light/10 h dark cycle. Wild-type embryos were cultured in Ringer’s solution , linearized with NcoІ, and transcribed in vitro by using SP6 polymerase and digoxigenin-labeled UTP (Roche). Whole-mount in situ hybridization was performed using standard protocols.To make an antisense RNA probe, the amplified zebrafish c/ebp-β was synthesized with SP6 RNA polymerase  after linearization of pCS2 + c/ebp-β. The synthesized mRNA capped RNA was dissolved in nuclease-free water containing 0.2% phenol red as a tracking dye. Microinjections were performed in embryos at one-cell to four-cell embryos by using a microinjector (WPI).Capped sense RNA encoding w1118. We obtained slboe7b from the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/).Put flies into egg-laying cages with apple juice agar plates and incubate at 25 °C in the dark until 50–100 eggs have been laid, and then allow embryos to age at 25 °C until before they hatch. After unhatched eggs were dechorionated with breach (8% sodium hypochlorite solution), rinse one more time with distilled water. Remove the vitelline membrane using hypodermic needle, and transfer to a drop of 1:1 lactic acid:Hoyer’s-based medium (Hoyer’s-based mountant) on a glass microscope slide. After that place a coverslip onto the slide, and then incubate the slide at approx. 60–65 °C overnight in an oven to allow digestion of internal tissues and clearing of the larvae. The control strain used in this study was Wild-type embryos were treated with 1 μM of thapsigargin from the 50% epiboly stage to 28 hpf, while control embryos were treated with DMSO.t tests were used to compare means between control and experimental groups. All experiments were performed three times. Statistical significance was set at P < 0.05. Results are presented as the mean ± SD.Student’s Supplemental Figures