Alisma plantago-aquatica Linn which has an antitumor effect. In this study, we aimed to explore whether alisol B 23-acetate could increase the antitumor effect of bufalin on liver cancer.  Liver cancer seriously threatens the health of people. Meanwhile, it has been reported that bufalin could act as an inhibitor in liver cancer. In addition, alisol B 23-acetate is a natural product derived from  In order to detect the effect of alisol B 23-acetate in combination with bufalin on liver cancer, human liver cancer SMMC-7721 and MHCC97 cells were used as subjects. Bufalin and alisol B 23-acetate were performed on cells. Cell viability was tested by MTT assay. In addition, flow cytometry was performed to assess the cell apoptosis. Autophagy-related protein levels were tested by western blotting. β and increased the expression of β-catenin in liver cancer cells.  The data revealed that bufalin significantly decreased the viability of liver cancer cells, and the inhibitory effect was further increased by alisol B 23-acetate. In addition, alisol B 23-acetate notably enhanced the apoptotic effect of bufalin on liver cancer cells through mediation of Mcl-1, Bax, Bcl-2, and cleaved caspase-3. Meanwhile, alisol B 23-acetate in combination with bufalin induced the autophagy in liver cancer cells through mediation of Beclin-1 and p62. Furthermore, alisol B 23-acetate in combination with bufalin significantly downregulated the level of GSK-3β-catenin axis, and these outcomes might shed new lights on exploring the new methods against liver cancer. In summary, these findings provide the first evidence that alisol B 23-acetate improves the anticancer activity of bufalin on liver cancer through activation of the Wnt/ Liver cancer (LC) is a frequent malignancy which has high incidence and mortality according to the previous report . In addiAlisol B 23-acetate (AB23A) is a tetracyclic triterpenoid isolated from the rhizomes of Alisma orientalis which is a medicinal plant formulated into different traditional Chinese herbal medicine formulas . In receβ-catenin axis in tumor progression has been well documented [β-catenin axis activation is reported to increase PTEN levels in bladder cancer in vivo, which leads to tumorigenesis when PTEN is genetically lost [β-catenin axis has been demonstrated to have a close connection with the biological processes involved in cancer [β-catenin signal activation [β-catenin axis remains scarce. Based on the above backgrounds, the novelty and motivation of the study is to investigate whether AB23A in combination with bufalin can be an efficient treatment approach for LC and is the effect mechanism through Wnt/β-catenin signal activation. This study probes into the impact of AB23A in combination with bufalin on LC, in the hope of shedding new lights on exploring new treatments for LC.The significant regulatory role of the Wnt/cumented , 16. Thelly lost . Furthern cancer , 19. Rectivation . However2 incubator at 37°C in a humidified atmosphere.Supplied and authenticated by the Chinese Academy of Sciences, Shanghai, human LC cell strains SMMC-7721 and MHCC97 were cultivated in the 1640 complete medium  comprising 1% penicillin-streptomycin solution +10% fetal bovine serum. The incubation was performed in a 5% COAfter solubilization, bufalin  was stored at a concentration of 10 mM in 13% ethanol at 4°C, followed by dilution in the medium for use. The final concentration of ethanol was made <0.01%, so as not to influence cell growth or viability. Similarly, AB23A  was subjected to solubilization, storage at a concentration of 25 mM in sterile water at -20°C, and subsequent dilution in the medium before use. SMMC-7721 and MHCC97 cells were treated with serial dilutions of bufalin and/or AB23A and incubated for 24 and 48 h.μM) [μM) [Cell viability measurement was performed using the colorimetric MTT (Sigma) method, which quantified the reduction of yellow tetrazolium salt to purple formazan crystals by the mitochondria of live cells . Cells aμM)  and/or bμM) [μM)  for 24, μL FITC and 5 μL propidium (PI) in the darkness. Annexin V-FITC/PI kit was purchased from Procell . The cell apoptosis rate was determined with the use of a flow cytometer .After trypsinization, LC cells were treated with rinsing by phosphate-buffered saline and resuspension in Annexin V Binding Buffer. Then, they were subjected to 15 min of staining with 5 5 cells/well) were planted onto 6-well plates and cultivated for 24 h with AB23A and/or bufalin. After treatment, they were treated with PBS washing and 30 min of paraformaldehyde (4%) fixation, followed by another PBS rinsing and nucleus staining with DAPI (2.5 μg/mL). Fluorescence signals were photographed using an inverted Olympus IX71 microscope (Japan).To assess the extent of apoptosis, nuclear morphology of SMMC-7721 and MHCC97 cells was evaluated using DAPI staining and observed using fluorescence microscopy . Both cell lines (3 × 10μg/lane) were then treated with 10% SDS-PAGE isolation and subsequent transfer onto polyvinylidene fluoride (Thermo Fisher Scientific) membranes. After a 1-hour blocking with 5% nonfat-dried milk, the membranes were immersed in Ι antibodies for overnight cultivation (4°C). Thereafter, they were immersed in a II anti-rabbit antibody  for another 1 h of cultivation at ambient temperature. Membrane scanning was performed via an Odyssey Imaging System and analyzed using Odyssey v2.0 . The Ι antibodies included the following : anti-Mcl-1 (#94296), anti-Bcl-2 (#15071), anti-caspase-3 (#9662), anti-Bax (#14796), anti-Beclin-1 (#4122), anti-p62 (#8025), anti-LC3 (#3868), anti-GSK-3β (#12456), anti-β-catenin (#8480), and anti-β-actin (#3700). β-Actin was considered an internal control.RIPA-extracted total protein from cell lysates or tissues was subjected to concentration determination with a BCA protein kit (Thermo Fisher Scientific). Proteins . One-way or two-way ANOVA was responsible for all the statistical analyses in this research, followed by Dunnett's multiple comparisons test, with the difference considered significant at a p value of 0.05.All experimental data were the average of at least 3 independent tests. Analysis of experimental data (mean ± SD) was done by GraphPad Prism To test the impacts of AB23A or/and bufalin on LC cell viability, SMMC-7721 and MHCC97 cell viabilities were determined by MTT assay after treatment with AB23A or bufalin of the above indicated concentrations. As shown in Figures Flow cytometry was utilized for the assessment of the impacts of AB23A/bufalin on LC cell apoptosis. As indicated in Figures To investigate the effect of AB23A/bufalin on LC cell autophagy, we investigated autophagy-related proteins  using western blotting. The data revealed that AB23A or bufalin significantly upregulated Beclin-1 and LC3 II/I and declined p62 in LC cells Figures . Furtherβ-catenin axis plays a similar role in AB23A/bufalin-induced apoptosis in LC. As presented in Figures β but increased β-catenin in LC cells , suggesting the ability of AB23A and bufalin to activate the Wnt/β-catenin axis. The results were similar to the previous studies that AB23A activates the Wnt/β-catenin axis in human osteosarcoma cells by downregulating GSK-3β and upregulating β-catenin [The precise molecular mechanism by which AB23A potentiates bufalin remains to be elucidated. Abnormal activation of the Wnt/e cancer , 44. Ma tivation . Howeverget gene , 46. Thi-catenin .trans-membrane potential (delta psim). In addition, animal studies are needed for investigating the potential of AB23A/bufalin in vivo. The connection between drug(s)-induced apoptosis or autophagy and Wnt/β-catenin signal activation remains to be investigated. Thus, the planning of our future research is to validate the mutual regulation of apoptosis and autophagy and its association with the Wnt/β-catenin axis. The impacts of AB23A and bufalin on LC cell stains SMMC-7721 and MHCC97 were discussed in the present work. Our data indicate that AB23A significantly potentiates bufalin in treating LC via Wnt/β-catenin signal inactivation, which provides novel ideas for LC treatment. Although our results were performed in vitro, the prospect of our study is to further conduct it to confirm the effectiveness and safety of AB23A in combination with bufalin in vivo and apply it to the clinical trials to provide a treatment approach in liver cancer.Our study presents some limitations. First, since caspase-dependent apoptosis is involved, the mitochondrial dysfunction should be confirmed using appropriate experiments such as reduction of mitochondrial


Gastric cancer is a common and lethal human malignancy worldwide and cancer cell metastasis is the leading cause of cancer-related mortality. MICAL2, a flavoprotein monooxygenase, is an important regulator of epithelial-to-mesenchymal transition. The aim of this study was to explore the effects of MICAL2 on gastric cancer cell migration and determine the underlying molecular mechanisms.Cell migration was examined by wound healing and transwell assays. Changes in E-cadherin/β-catenin signaling were determined by qPCR and analysis of cytoplasmic and nuclear protein fractions. E-cadherin/β-catenin binding was determined by co-immunoprecipitation assays. Cdc42 activity was examined by pulldown assay.MICAL2 was highly expressed in gastric cancer tissues. The knockdown of MICAL2 significantly attenuated migratory ability and β-catenin nuclear translocation in gastric cancer cells while LiCl treatment, an inhibitor of GSK3β, reversed these MICAL2 knockdown-induced effects. Meanwhile, E-cadherin expression was markedly enhanced in MICAL2-depleted cells. MICAL2 knockdown led to a significant attenuation of E-cadherin ubiquitination and degradation in a Cdc42-dependent manner, then enhanced E-cadherin/β-catenin binding, and reduced β-catenin nuclear translocation.Together, our results indicated that MICAL2 promotes E-cadherin ubiquitination and degradation, leading to enhanced β-catenin signaling via the disruption of the E-cadherin/β-catenin complex and, consequently, the promotion of gastric cell migration.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-022-00952-x. Gastric cancer is among the most commonly diagnosed malignancies worldwide. There is a correlation between the severity of tumor metastasis and mortality in gastric cancer patients . The migThe molecules interacting with CasL (MICALs) comprise a family of conserved flavoprotein oxidoreductases known to drive F-actin oxidization and participate in multiple cellular functions related to cytoskeleton dynamics, including vesicle transportation, mitosis, and the development of hippocampal mossy fiber connections –7. In huβ-catenin is a dual function protein, both coordinating intercellular adhesion at adherens junctions and regulating the transcription of target genes. β-catenin forms a complex with E-cadherin, which, in turn, can function as an anchoring junction and act to stabilize cell adhesion . When a All immunohistochemical assays involving human tumor specimens were conducted according to the institutional guidelines of Jiangsu Province.2. Cells were grown on coverslips for immunofluorescence staining and in six-well plates  for RNA isolation and protein extraction.The human gastric cancer cell lines BGC-823, MGC-803, and SGC-7901 and the normal gastric epithelial cell line GES-1 were purchased from the Cell Biology Institute of the Chinese Academy of Science . All cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) . Cells were maintained in a humidified incubator at 37 °C with 5% COThe empty vector control pcDNA-3.1-HA-C and full-length human MICAL2 cDNA were purchased from YouBio . The pEGFP-N1 vector containing the full-length Cdc42-Q61L (CA) insert was kept in this laboratory. The siRNAs (control siRNA or siRNA targeting MICAL2 [siMICAL2]) used in this study were synthesized and purified by GenePharma . The sequences of the siRNAs targeting MICAL2 were as follows: siMICAL2 #1, 5′-GAGAACGUGAACCAAGACATT-3′; siMICAL2 #2, 5′-GCAUAGAUCUUGAGAACAUTT-3′; siMICAL2 #3, 5′-GCAGCGACACGUGUUACUUTT-3′. Transfection (plasmids or siRNA) was performed using Lipofectamine 2000 following the manufacturer’s instructions. After 24 h of transfection, the cells were cultured in starvation medium overnight and then treated with cycloheximide (CHX) , LiCl (Sigma-Aldrich), MG-132 , or chloroquine diphosphate (Chlq)  at the indicated time points.For the wound healing assay, cells were seeded in six-well plates until confluence. A wound was then made in the cell monolayer by scratching with a 10-µL pipette tip. After rinsing with PBS, the cells were treated with the indicated stimulator and allowed to migrate for 24 h. Images of wound areas were captured using an inverted phase-contrast microscope  at 0 h and 24 h.4 cells were seeded in the upper chamber in serum-free medium while medium containing 10% FBS was added to the lower chamber. After incubation at 37 °C for 24 h, the cells on the upper side of the membrane (non-migrated cells) were removed while those that had migrated to the underside of the membrane were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The average number of cells was determined and scored under a fluorescence microscope (Carl Zeiss Meditec).The transwell assay was performed using a 24-well cell culture insert with 8-μm pores. A total of 1 × 10Cell lysates were prepared using RIPA lysis buffer . Cytoplasmic and nuclear protein fractions were obtained using a nuclear protein extraction kit from Beyotime following the manufacturer’s instructions. Protein concentrations were assessed using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of cellular protein were separated by SDS–PAGE, transferred to a pure nitrocellulose membrane, blocked with 5% skimmed milk, and then incubated with primary antibodies targeting MICAL2 , GAPDH , E-cadherin , N-cadherin , β-catenin, vimentin, Cdc42, and HA-tag  overnight at 4 °C. After washing and incubation with secondary antibody , the bands were visualized using enhanced chemiluminescence reagent  and analyzed using Quantity One software .−ΔΔCt method and StepOne Software v2.1 (Applied Biosystems).Total cellular RNA was isolated using Trizol reagent (Invitrogen) and reverse-transcribed with HiScript Q RT SuperMix for qPCR . The sequences of the primers used were GAPDH: 5′-TCGGATCAACGGATTTGGT-3′ (sense) and 5′-TTCCCGTTCTCAGCCTTGAC-3′ (antisense); MICAL2: 5′-GGGGATTTCCCGCAGAATAAAC-3′ (sense) and 5′-GGCTGGGATGAAAATGGAACC-3′ (antisense); vimentin: 5′-AGTCCACTGAGTACCGGAGAC-3′ (sense) and 5′-CATTTCACGCATCTGGCGTTC-3′ (antisense); E-cadherin: 5′-ATTTTTCCCTCGACACCCGAT-3′ (sense) and 5′-TCCCAGGCGTAGACCAAGA-3′ (antisense); N-cadherin: 5′-AGCCAACCTTAACTGAGGAGT-3′ (sense) and 5′-GGCAAGTTGATTGGAGGGATG-3′ (antisense). Real-time PCR was carried out using AceQ qPCR SYBR Green Master Mix (High ROX Premixed) (Vazyme) in an ABI StepOne Real-Time PCR System . Relative mRNA expression levels were calculated using the 2Cells were placed on a glass coverslip, fixed in 4% paraformaldehyde for 20 min, treated with 0.2% Triton X-100 for 5 min, blocked with 1% BSA for 1.5 h, and then incubated with primary antibodies against β-catenin, E-cadherin, EEA1, Rab7 , and LAPM1 (Proteintech) overnight. After washing three times with PBS, the cells were incubated with Alexa-conjugated species-matched secondary antibody or TRITC-conjugated secondary antibody for 1 h. The nuclei were counterstained with DAPI (Beyotime). Images were captured using an Olympus BX51 microscope fitted with an Olympus DP70 digital camera .Rho GTPase pulldown assays were performed as previously described . Active Co-IP was conducted as previously described . In brieThe Oncomine and UALCAN databases were used to analyze the association between the mRNA or protein expression of MICAL2 and clinical parameters as well as the prognostic value of mRNA expression of MICAL2 in gastric cancer tissue. Patients with gastric cancer were divided into high and low MICAL2 expression groups based on the median values of MICAL2 mRNA expression.t-test was used for comparisons between two groups. One-way ANOVA was utilized for comparisons among three or more groups. A P-value < 0.05 was considered significant. All experiments were repeated independently at least three times.Data were analyzed using SPSS version 19.0  and are reported as means ± standard error of the mean. The Student’s www.proteinatlas.org), we confirmed that MICAL2 was strongly expressed in the cytoplasm/membrane of gastric cancer samples  dataset, and found that MICAL2 expression was significantly higher in tumor tissues than in normal tissues Fig. A. We furles Fig. D. Kaplanles Fig. E. Next, les Fig. F. OveralTo confirm the role of MICAL2 in the regulation of cell migration, we performed MICAL2 loss-of-function assays in gastric cancer cells. First, we silenced MICAL2 expression in BGC-823 and MGC-803 cells using siMICAL2. The knockdown efficiency was determined by qPCR and western blotting. As shown in Fig. We next tested whether MICAL2 modulates the expression of EMT markers in gastric cancer cells. BGC-823 and MGC-803 cells were transfected with siMICAL2 #2 and #3, following which vimentin, N-cadherin, and E-cadherin mRNA and protein levels were evaluated by qPCR and western blotting, respectively. Whereas BGC-823 and MGC-803 cells underwent markable reduction of MICAL2, no significant increase in the abundance of E-cadherin mRNA was observed Fig. A led to To uncover the potential mechanism underlying how the silencing of MICAL2 induces E-cadherin protein expression, we evaluated E-cadherin distribution in the cytoplasm using an immunofluorescence assay. Normally, after binding to its ligands, E-cadherin undergoes dimerization and autophosphorylation. Phosphorylated E-cadherin is then ubiquitinated and enters early and late endosomes in the cytoplasm. Finally, it is degraded in lysosomes, resulting in reduced recycling of E-cadherin to the lateral membrane . To furtAs shown in Fig. Wnt/β-catenin signaling has been reported to play an important role in MICAL2-induced EMT in ovarian cancer cells . To uncoβ-catenin nuclear translocation can lead to the activation of the Wnt/β-catenin signaling pathway . To explIt has been reported that Cdc42 plays an important role in the internalization and degradation of receptors . To furtThe results of numerous studies have demonstrated that β-catenin is a major risk factor for gastric tumor initiation, growth, metastasis, and resistance to therapy . TherefoWnt/β-catenin signaling activation is known to be associated with β-catenin nuclear translocation and negatively regulated through the degradation of β-catenin via the β-catenin destruction complex  in the cytoplasm. Here, we found that total β-catenin levels were not affected by the knockdown of MICAL2. Instead, siRNA-mediated depletion of MICAL2 led to a significant reduction in β-catenin nuclear content and a decrease in the migratory ability of gastric cancer cells, effects that were significantly attenuated by pre-treatment with LiCl, which is known to activate Wnt signaling by inhibiting GSK3β and consequently stabilizing free cytosolic β-catenin. These data suggested that MICAL2 promotes cell migration-related signaling in gastric cancer cells through regulating the cytosolic-nuclear shuttling of β-catenin. However, relatively little is known of the mechanism underlying how MICAL2 influences β-catenin translocation.E-cadherin is a transmembrane protein expressed in almost all epithelial cells. The extracellular region of E-cadherin binds to cadherins present on adjacent cells, whereas its intracellular region contains binding sites for catenins, including β-catenin, which binds to the C-terminal of the E-cadherin cytoplasmic region. E-cadherin forms a complex with β-catenin, which, in turn, serves as an anchoring junction and acts to stabilize cell adhesion . In thisIn this study, we observed that E-cadherin protein was diffusely distributed near the cytoplasmic membrane of gastric cancer cells. This suggested that continuous E-cadherin endocytosis was already underway in these cells, thus facilitating E-cadherin degradation. The lysosomal and proteasome-based proteolytic pathways represent the two main cellular protein degradation pathways. Proteins in the MICAL family are key regulators of membrane trafficking during cell division . For insThe most significant finding in this study was that the molecular mechanism involved in the regulation of E-cadherin degradation was largely dependent on the induction of Cdc42 activation by MICAL2. Cdc42 is known to control a wide variety of biological responses in metastatic cells, both directly and indirectly. Additionally, Cdc42 has been identified as a potential positive regulator of E-cadherin ubiquitination and degradation . In the In conclusion, we identified a novel role for MICAL2 in the regulation of gastric cancer cell migration, and showed that the underlying mechanism involved the impaired binding of E-cadherin to β-catenin in the cytoplasm, subsequently leading to the accumulation of β-catenin in the nucleus. We further showed that this effect of MICAL2 was likely mediated via the activation of Cdc42, which led to increased E-cadherin ubiquitination and degradation. To the best of our knowledge, this was the first study to report the degradation of E-cadherin mediated by MICAL2. Further advances in our understanding of the function of MICAL2 will likely have important implications for the treatment of gastric cancer.


Both PAR4 and PAR2 are able to drive the association of methyltransferase EZH2 with β-catenin, culminating in β-catenin methylation. This methylation on a lysine residue at the N-terminal portion of β-catenin suppresses the ubiquitination of β-catenin, thereby promoting PAR-induced β-catenin stability and transcriptional activity. Indeed, EZH2 is found to be directly correlated with high PAR4-driven tumors, and is abundantly expressed in large tumors, whereas very little to almost none is expressed in small tumors. A truncated form of β-catenin, ∆N133β-catenin, devoid of lysine, as well as serine/threonine residues, exhibits low levels of β-catenin and a markedly reduced transcriptional activity following PAR4 activation, in contrast to wt β-catenin. Our study demonstrates the importance of β-catenin lysine methylation in terms of its sustained expression and function. Taken together, we reveal that PAR-induced post-transcriptional regulation of β-catenin is centrally involved in colon cancer.G-protein-coupled receptors (GPCRs) are involved in a wide array of physiological and disease functions, yet knowledge of their role in colon cancer stem cell maintenance is still lacking. In addition, the molecular mechanisms underlying GPCR-induced post-translational signaling regulation are poorly understood. Here, we find that protease-activated receptor 4 (PAR Despite the emerging role of G-protein-coupled receptors (GPCRs) in a wide array of physiological and disease functions, knowledge of their regulation of post-translational signaling is still incomplete ,2,3,4. WGPCRs control many aspects of tumorigenesis, including proliferation, invasion, survival at secondary sites, and several cancer-associated signaling pathways . As a suhi and Wntlo, respectively [It has been well established that the main resistance to standard of care chemotherapy in colorectal cancer (CRC) cannot be explained only on the basis of genetic mutations. Evidence supports the notion that the majority of CRCs possesses colorectal cancer stem cells (CSCs) that drive tumor growth and play a central role in chemoresistance. CSCs have been identified on the basis of nuclear β-catenin activity, where CSCs and non-CSCs are Wntectively . Therefo1–4 [1 or PAR2 oncogenes are effective inducers of β-catenin stabilization, acting via recruitment of LRP5/6 coreceptors in both the malignant and the physiological invasion of placenta anchorage to the uterus decidua [2 was attributed a dominant role over PAR1 [3 functions mainly as a coreceptor. PAR4 is a receptor for thrombin-induced human platelets along with PAR1 [Par4-deficient mice display a normal phenotype; however, hemostasis is diminished, because platelets from these mice no longer respond to thrombin stimulation [1 and PAR4 [4 is critical for leukocyte recruitment and migration to sites of injury, as well as for inflammation [4 decreased leukocyte infiltration and cardiomyocyte apoptosis while improving other cardiac tasks following acute ischemia/reperfusion wounds [4 as an oncogene [4 (f2rl3) and PAR2 (f2rl1). As such, they are considered to be part of the CSC niche compartment.Within the dynamic and flexible tumor microenvironment, both matrix-immobilized and soluble proteases are engaged in order to maintain tumor progression . A good 1–4 ,14,15. W decidua ,18,19,20ver PAR1 , while Pith PAR1 ,23. Par4mulation ,25. The and PAR4 . On the ammation ,27. In tn wounds ,29. Addioncogene ,29,30,31oncogene . Among tThe Polycomb complex is an important effector linking cancer and stem cells ,34. It r2 or PAR4, which subsequently endows β-catenin with enhanced stability. EZH2 is upregulated in PAR-driven tumors as well as in aggressive colon cancer cell lines overexpressing PAR2 and PAR4. In contrast, no expression of EZH2 was observed in the non-aggressive colon cancer cells, which exhibit very little to nearly null PAR expression. Activation of PAR2 or PAR4 induces the association of EZH2 with β-catenin and methylation on lysine/s (K) residue/s. Consequently, it prolongs the stabilization of β-catenin and enhances its transcriptional activity. PAR-induced β-catenin contributes centrally to colorectal cancer growth.Here, we demonstrate novel post-translational regulation of β-catenin induced by either PAR4 induces β-catenin stabilizationPAR. Previously, we have demonstrated the stabilization and TOPflash transcriptional activity of β-catenin induced either by PAR1 or PAR2 [4, we set out to study the PAR4-induced β-catenin stabilization path. For this purpose, HEK293 cells were transiently transfected with flg-β-catenin and Par4, followed by the addition of AYPGKF, a synthetic hexapeptide exhibiting the internal PAR4 ligand sequence for activation. A marked increase in β-catenin levels was observed after 4–5 h of PAR4 activation  HCT-116 cells . These clones were named RKO/Par4a–c. The levels of Par4 mRNA in these cells are shown for a representative clone, RKO/Par4a (s.c.), large tumors were generated compared to the mice injected with parental RKO cells lacking Par4 expression . Overall, this points to the powerful role played by PAR4 as a member of the colon cancer stem cell compartment. be seen C, all ge4 or PAR2 promotes the binding of EZH2 to β-cateninActivation of PAR. We next examined whether EZH2 interacts with β-catenin following the activation of PAR4 or PAR2. To this end, immunoprecipitation (IP) analysis between EZH2 and β-catenin was carried out following application of either SLIGKV (for PAR2 activation) or AYPGKF (for PAR4). Briefly, HEK293 cells were transfected with flg-β-catenin, ezh2 and Par4 plasmids. Cells were activated for the indicated periods of time, which were between 15 min and 4 h. High levels of β-catenin were obtained within the immune complex of EZH2 following 15 min of PAR4 (or PAR2) activation, which subsequently decreased. It was concluded that the EZH2–β-catenin association takes place early on, prior to β-catenin stabilization. Concomitantly, lysine (K) methylation of β-catenin was obtained at the same immune complex . This is indicative of the important role of EZH2 in mediating β-catenin methylation followed by PAR4 (or PAR2)-induced stability of β-catenin  pulse-chase assay. CHX is a known inhibitor of eukaryotic protein synthesis. In the presence of CHX, levels of unstable proteins will decrease, whereas relatively stable proteins will show little change over time [β-catenin and Par4 plasmids alone, or in combination with ezh2. Next, cells were activated with AYPGKF for PAR4 activation to stabilize β-catenin, then CHX was added so that no additional proteins could be synthesized. In the presence of ezh2, β-catenin expression levels can be seen for longer and sustained periods of time compared to cells transfected with β-catenin and Par4 alone  49. Consequently β-catenin is stabilized and enters the nuclei, where it acts as a co-transcription factor and generates tumors  residues: K19, K49 and K133. The truncated form of β-catenin exhibits low levels of expression and no transcriptional activity. It is proposed that, early on, methylation of β-catenin is a prerequisite for the stabilization and transcriptional activity of β-catenin. PAR-induced β-catenin stabilization plays a central role in colon cancer growth.Truncated N133 β-catenin, in addition to lysine amino acids, is devoid of serine/threonine (S/T) residues for phosphorylation. The phosphorylation of β-catenin  assigns it to proteasomal degradation ,47. SpecZhu P et al. discovered a long non-coding RNA called lnc-β-Catm (lncRNA for β-catenin methylation) that associates with β-catenin and EZH2, thereby promoting the methylation of β-catenin on lysine 49 (K49) by EZH2. This methylation inhibits β-catenin ubiquitination by the E3 ligase and promotes its stability, thus allowing β-catenin to activate Wnt-β-catenin signaling for a longer period of time, contributing to the sustained stemness of liver CSCs [EZH2 also functions in a PCR2-independent manner. Emerging research has shown that EZH2 methylates non-histone targets interacting with other proteins to activate downstream genes. For example, Kim E et al. discovered that EZH2 binds to and methylates STAT3, leading to its enhanced activity through the increased tyrosine phosphorylation of STAT3 . AnotherMutations of EZH2 are found in hematological malignancies . They prIn fact, EZH2 has been shown to be overexpressed in many cancers, including hepatocellular carcinoma, breast , bladderAlthough EZH2 works in a variety of ways, including the canonical pathway of epigenetic transcription  and geneezh2 expression by targeting miRNA [4 cells are highly expressed, higher levels of EZH2 expression are exhibited, as indicated in our data. Along this line of evidence, we also showed that the EZH2 inhibitor GSK126 impairs PAR4-induced spheroid growth, as well as the growth of stem cell markers that are otherwise markedly elevated by PAR4 activation. Taken together, these results emphasize the central role played by EZH2 in PAR4-induced malignancy. Elevated levels of several Lef/Tcf transcription targets, such as lgr5, cd44 and oct4, were observed in these spheroids. The expression of these genes is then quelled by inhibition of EZH2 with GSK126.EZH2 has been shown to bind to and transactivate β-catenin, leading to increased expression of the target genes c-myc and cyclin D1 . C-myc eng miRNA . Aberranng miRNA . Therefo4 and PAR2 potently induce lysine methylation of β-catenin by EZH2, promoting its sustained stability levels in colon cancer.Overall, it is proposed herein that activation of the GPCR oncogenes PARThe animals used in the experiments were treated in accordance with the guidelines of the institution ethics committee (AAALAC standard). Mice (HSD: Athymic nude-Foxn1Nu Nu/Nu mice) were kept under specific pathogen-free (SPF) conditions at the Hadassah Medical Center animal facility unit of the Hebrew University and were regularly screened for standard pathogens. All animal experiments were approved by the animal committee of the Hebrew University (MD-20-15924-5).2 at 37 °C. To generate spheroids, HT29 cells were cultured for 10–14 days in spheroid media  with 50% Matrigel matrix  at 37 °C under 8% CO2 in a humidified incubator. Spheroid growth was microscopically monitored daily, and the incubation medium was replaced every 3 days.HCT-116, HT29, RKO and HEK293  were grown in DMEM, supplemented with 1 mM L-glutamine, 50 µg/mL streptomycin, 50 U/mL penicillin  and 10% fetal calf serum . Cells were maintained in a humidified incubator with 8% COPar4 (cat #53231), pCMVHA hEZH2 (cat #24230), pcDNA3-mRFP (cat #13032), pCMV-VSV-G (cat #8454) and pCMV-dR8.2 dvpr (cat #8455) plasmids were purchased from Addgene. Human PAR2 (hPar2/ f2rl1) plasmid was kindly provided by Dr. Morley D. Hollenberg . flg-β-catenin and flg-Axin were kindly provided by Dr. Ben-Neriah . All of the mentioned plasmids were sequenced to confirm the absence of undesirable mutations. Details of the plasmids are available on request.pBJ-FLAG-hCells grown to 70–80% confluency were transfected with 0.5–1 µg amounts of plasmid DNA using PEI transfection reagent  according to the manufacturer’s instructions. Cells were collected 48 h after transfection, and protein lysates/RNA were prepared. To activate PAR2, a synthetic peptide “SLIGKV” was employed, while to activate PAR4, a synthetic peptide “AYPGKF”  was used. To inhibit EZH2 activity, we used between 2 µM and 10 µM of GSK126  or 4.6 µM of GSK343.g rpm.To prepare sh-RNA of PAR4, the sequence was successfully cloned into the plentilox3.7 (pLL3.7) lentiviral vector following the protocol provided by the website of Addgene (Cat#11795). The target sequence was as follows: sh-Par4# (5′- ATGACAGCACGCCCTCAAT′-3). For the generation of shPar4 viral particles, HEK 293 cells were transfected with a three-plasmid system that included packaging CMVD R8.91, envelope (CMV-VSV-G), and shPar4-pLL3.7, using PEI as a transfection reagent. The medium was replaced with fresh medium 24 h later. On day 3 after transfection, the medium was collected, and the viral particles were concentrated 10-fold by centrifuging for 1 h at 40,000× CTNNB1 gene was prepared that was devoid of 133 a.a at its N-terminus. In brief, CTNNB1 wild-type plasmid template was amplified with Q5 high-fidelity polymerase , using forward primer (5′ CATGCAGTTGTAAACTTGATTAA 3′) with a BamH1 restriction site and reverse primer (5′ CAGGTCAGTATCAAACCAG 3′) with a Not1 restriction site. The amplified product was purified, digested, and cloned into the pcDNA3-RFP vector between the BamH1 and EcoRI sites. Sequences of the plasmid constructs ΔN133-pcDNA-RFP were confirmed by Sanger sequencing.To eliminate the methylation of β-catenin, a truncated construct of the h)Par4 gene was amplified with Q5 high-fidelity polymerase  using forward primer (5′GAATTCGCCGCCACCATG TGGGGGCG ACTGCTCC′3) with an EcoR1 restriction site (underlined) with Kozak sequences (bold) and the reverse primer (5′ACTAGTTCACTGGAGCAAAGA GGAGTGGG′3) with an Spe1 restriction site, and cloned into the pLVX- EF1 α-IRES-Puro viral vector. For the generation of viral particles, the same procedure was applied as that described earlier.To prepare stable clones of PAR4, the human (RKO (0.5 × 106) cells were infected with hPar4 10× viral particles along with Polybrene infection reagent. At 72 h post transduction, cells were subjected to puromycin selection (0.5 μg/mL). Cells with puromycin resistance were grown and collected, and were either used to isolate RNA or to perform protein lysate preparation.−ΔΔCt method as described in the manufacturer’s instructions, and were expressed as fold change over the indicated controls.RNA was extracted from cells using GenElute RNA kit . To prepare the cDNA, 1 µg of RNA was reverse transcribed using reverse transcriptase . qRT PCR was conducted using specific forward and reverse primers for each gene listed in sh-Par4, RT-PCR was performed. The PCR conditions were an initial denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing for 60 s at 60 °C, and extension for 1 min at 72 °C (31 cycles of amplification). The GAPDH gene was used as an internal control.To compare expression of Par4 in HCT116 vs. HCT116 TMM buffer , while for the rest of the protein cell lysate preparation, we used lysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% triton X-100. All of the mentioned lysis buffers were supplemented with protease inhibitor cocktail, 1 mM phenylmethylsulfonylfluoride, PMSF, and 1 mM Na-orthovanadate  to prevent protein degradation. The protein cell lysate used for IP and BA were incubated at 4 °C for 20 min and then disrupted by sonication. Finally, soluble supernatant was collected after centrifugation at 12,000× g rpm for 20 min at 4 °C.To prepare protein cell lysate for immunoprecipitation, cells were solubilized in CelLyticProtein cell lysates were separated on 8–10% SDS-PAGE followed by transfer to Immobilon-P membrane . Membranes were accordingly blocked and probed with the appropriate primary antibodies. Primary antibodies, including anti-flag , anti-β-actin , anti-KMT6/EZH2 , anti-Methylated Lysine , anti-HA  and anti-beta Catenin antibodies , were suspended in 3% BSA in 10 mM Tris-HCl pH 7.5, 100 mM NaCl and 0.1% Tween-20. After washing, blots were incubated with secondary antibodies conjugated to horseradish-peroxidase. Immunoreactive bands were detected by the enhanced chemiluminescence (ECL) reagent . Protein cell lysates (400–800 μg) were used for immunoprecipitation analysis. Anti-HA  was added to the cell lysates and processed as previously described .6) were seeded in 6-well plates and incubated overnight at 37 °C. The cells were transfected with the desired target plasmids (PAR2/PAR4/EZH2/β-catenin wt or ΔN133) along with human Lef-1 TOPflash  reporter plasmid containing two sets (the second set in reverse orientation) of three copies of the Tcf binding site upstream of the thymidine kinase (TK) minimal promoter and luciferase open reading frame using PEI transfection reagent (Boehringer-Mannheim). CMV/β-gal plasmid was co-transfected as an internal control for transfection efficiency. After 48 h transfection, the cells were washed and lysed, and then luciferase assay was performed with the Luciferase Reporter System  according to the manufacturer’s instructions, and luminescence was detected on a Tecan SparkTM10M multimode microplate detection system (Switzerland).HEK-293T cells  for 2 h and then exposed to cycloheximide (100 µg/mL) for 5 min to 2 h. Cells were then harvested at regular intervals, and lysates were prepared as mentioned above.f2rl3 gene, an ectopic tumor xenograft mouse study was performed. In brief, RKO/PAR4-RKO stable clones or HCT116/HCT116 shPAR4 cells were starved O/N, and the next day were treated with AYPGKF (200 µM) for 4 h. After washing off the cells, 1 × 106 cells were injected subcutaneously into the right flank of groups (n = 6) of six- to eight-week-old Hsd: Athymic Nude-Foxn1nu mice (referred to as nude mice). Tumor volumes were monitored twice a week by caliper measurements of each dimension and calculated using the following formula: V = 4/3 π (length/2) (width/2) (depth/2). Mice were terminated by cervical dislocation under aesthetic conditions when the tumor volumes reached the volume stipulated in the Institutional Animal Committee’s approval or when the animals showed distress, in order to avoid unnecessary suffering.To determine the in vivo tumorigenicity of the PAR4/3) were grown in sphere formation medium  over a layer of 50% Matrigel. Two weeks later, spheres larger than 100 μm were counted, and photographs were taken. Spheroid lysates were prepared in the same manner as that described above (solubilized in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% triton X-100).HT29 cells  in a microwave oven with 1× antigen retrieval citrate buffer . After blocking with CAS-Block , the slides were incubated with EZH2 antibody . Next, following washing, the slides were incubated with peroxidase-conjugated antibody . Color was developed using the DAB substrate kit , followed by counter staining with Mayer’s hematoxylin . Controls using only secondary antibodies (with no primary antibodies) showed low to background staining in all cases.t test or analysis of variance (ANOVA) with Tukey’s multiple comparison post test , wherever required. The criterion for statistical significance was as follows: p < 0.05 was considered significant (*), p < 0.01 as highly significant (**), and p < 0.001 as very highly significant (***).All of the experiments were carried out in triplicate, whereby the data are represented as mean ± SD. Significant differences in the tested samples in comparison to control were determined by performing either Student’s


This cross-sectional study aims to explore the prevalence of protein-energy wasting (PEW) in dialysis patients in Catalonia, Spain, using a new and practical online tool which enables rapid calculation and comparison with other nutritional scores. Methods: A web tool  was created to introduce different variables and automatically calculate PEW, Malnutrition inflammation Score (MIS) and Subjective Global Assessment (SGA) in 1389 patients ), 12% in peritoneal dialysis (PD) from different regions of Catalonia. Results: A prevalence of 23.3%  of PEW was found, with a mean MIS score of 6 and SGA score of C in 7% of the patients. ROC analysis showed MIS as the best nutritional score to diagnose PEW (AUC 0.85). Albumin delivered lower diagnostic precision (AUC 0.77) and sensitivity (66%). A cut off point of 7 (86% sensitivity and 75% specificity) for MIS and 3.7 mg/dL for albumin were found to predict the appearance of PEW in this population. SGA B or C showed an 87% sensitivity and 55% specificity to diagnose PEW. Very low nutritional intervention (14%) was recorded with this tool in patients with PEW. Conclusions: This new online tool facilitated the calculation of PEW, enabling different professionals—including nephrologists, dieticians and nurses—to efficiently obtain insights into the nutritional status of the Catalonian dialysis population and implement the required nutritional interventions. MIS is the score with more sensitivity to diagnose PEW. Protein-energy wasting (PEW)  is the tPEW is much more prevalent in dialysis than in pre-dialysis phases  because In Catalonia, a registry to evaluate the malnutrition–inflammation status of our patients in chronic renal replacement therapy does not exist, so the exact prevalence of malnutrition is unknown, and nutrition intervention methods differ between centers. The objective of this study is to evaluate the prevalence of malnutrition in CKD patients in dialysis in Catalonia, using a new practical online tool to easily calculate the prevalence of PEW and compare it to different scores (MIS and SGA).We designed a cross-sectional, observational and descriptive multi-center study, including patients from different geographical areas of Catalonia in renal replacement therapy: both hemodialysis (HD) and peritoneal dialysis (PD). This included all men and women over 18 years of age who started renal replacement therapy at least 12 weeks before the study began. The project was approved by the Ethics Committee of all participating centers, in accordance with the Declaration of Helsinki, as well as the Organic Law on Data Protection (15/1999), which aims to guarantee and protect public freedoms and fundamental rights of natural persons, and especially the personal and family privacy of all patients who participated in the study.Once the potential patients for this study were identified, their written consent was sought after having provided them with detailed information on the study characteristics; their duties, rights and the potential benefit that research would bring. Through this process, 1223 patients in HD and 166 in PD were recruited for the study.Different demographic and anthropometric variables, clinical history related to renal replacement therapy, functional capacity, dietary intake (according to MIS/SGA criteria) and gastrointestinal symptoms were collected at the time of inclusion. Major comorbidities (CCM) were identified as congestive heart failure III-IV, severe ischemic heart disease, AIDS, moderate-severe chronic obstructive pulmonary disease, major neurological sequelae, neoplasia with metastasis or recent chemotherapy. The patient was then classified as no comorbidity, without CCM, moderate CCM if one CCM was present and severe CCM if two or more were present. Fat and muscle reserves were assessed by physical examination and analytical variables with the aim of applying the diagnosis of PEW (currently defined as the gold-standard) and for calculating the MIS and SGA scores. To predict PEW, the Nutrendial web application applies an algorithm including the variables needed to calculate PEW according to the classical criteria  for eachFor the biochemical parameters section, albumin < 3.8 g/dL or cholesterol <100 mg/dL were used.2 or a weight loss > 10% in the last 6 months were used.For the body mass section, BMI < 23 kg/mFor the muscle mass section, creatinine < 8 mg/dL was used .For the dietary intake section, nPCR < 0.8 g/kg/day was used.The data were collected during the second HD session of the week or in the peritoneal dialysis control. A form was provided to facilitate data collection . In the www.nutrendial.cat) was created for the introduction of the different variables that were stored in a centralized database . Prevalence rates of malnutrition were determined according to the different scores. Descriptive statistics were carried out, defining the continuous variables by mean and standard deviation. A comparison of continuous variables was performed by using the Student’s 2, 3.70 ± 0.41 g/dL, 237.63 ± 52.06 mg/dL and 151.98 ± 37.77 mg/dL, respectively.A total of 1389 dialysis-dependent patients were enrolled in this study. The median age was 72 years (IR 60–81), 63.4% of patients were men and 36.1% had diabetes mellitus. A total of 88% of participants were on hemodialysis (76% online hemodiafiltration) and 12% on peritoneal dialysis (50% automated PD). The median of time on dialysis was 31 months (RI 13–66). Regarding nutrition parameters, the mean of BMI, albumin, TIBC and cholesterol was 25.72 ± 5.15 kg/mp < 0.001), but there were no differences in gender or time on dialysis. As expected, all the serum nutritional parameters analyzed were significantly lower in the PEW population, including the protein catabolic rate (nPNA). Compared to those on hemodialysis, patients on peritoneal dialysis had a lower percentage of PEW  and were also significantly younger, with less time on dialysis, greater body mass and with more comorbidities, but with better functional capacity (p < 0.05)  and 7% of patients with an SGA score of C. The baseline characteristics of the study population according to the PEW diagnosis are presented in  < 0.05) .p < 0.001). Related to functional capacity, 25.1% of the PEW patients were classified in the Nutrendial tool as “normal” in comparison with 59% of those without PEW. Patients with PEW presented significantly greater fatigue, experienced some difficulty walking and almost 17% reported not doing any type of physical activity (p < 0.001).Patients with PEW presented at least one CCM (40.2%) and had two or more CCMs (8.4%) compared to patients not classified as PEW, of which 61.5% had no moderate or major comorbidity registered, while 28.5% had one CCM . Moreover, gastrointestinal symptoms, such as occasional nausea, vomiting, diarrhea or anorexia were present in 61.5% of the patients diagnosed with PEW vs. 22.3% in patients without PEW. Physical examination showed that subcutaneous fat loss (moderate-severe in 47.6% vs. 9.1%) and signs of muscle loss (moderate-severe 48.3% vs. 9.5%) were higher in the PEW group .Nutritional intervention was also recorded in the Nutrendial online tool, and the results showed that patients with PEW received a higher volume of interventions (14% vs. 9.8%) . More spCross tabs were performed to evaluate the ability of the SGA score as a predictor of PEW, and the group classified as VGS C had a specificity to diagnose PEW of 100% and a sensitivity of 30%, but when we combined the groups classified as B/C, the sensitivity increased to 88%, although the specificity decreased to 55%.p < 0.001). Analysis of the MIS score enabled the identification of a cut off value after which the appearance of PEW became more probable. For this Catalonian dialysis population, the calculated value is seven, with a sensitivity of 86% and specificity of 75.3%.ROC curve analysis was also performed to evaluate the ability of MIS score as a predictor of PEW . The cal2  and a cut-off point for predicting the appearance of PEW in this population of 3.71 mg/dL, with lower sensitivity (66%). Body mass index (BMI) proved a more reliable indicator for the prediction of PEW with a value of 0.789 (95% CI: 0.760–0.818), greater sensitivity (88%) and a cut-off point for this population of 22 kg/m2 , Table 3www.nutrendial.cat) which offers an easy-to-manage method to calculate the prevalence of PEW  as well as nutritional scores, such as SGA and MIS, also allowing comparison between them. We observed that 23.3% of the patients on dialysis included in our study presented PEW, obtaining the best PEW diagnostic precision with MIS, with the albumin being far apart.This is the first study analyzing the nutritional status in the Catalonian dialysis population, using a new web tool  and in PD patients, between 32–49%  . In our Ashabi et al. were the first group to compare various scoring methods  and MIS) for the diagnosis of PEW in 291 HD patients  showing Like the Catalonian sample, in the Madrid cohort, the group did not observe significant differences by gender, and they detected less prevalence of PEW in the diabetic population . In the Among the biochemical markers available for the diagnosis of PEW in patients with CKD, it is widely known that there is no nutritional marker that meets all the requirements as such. Low serum albumin is a strong predictor of mortality, but this does not necessarily equate to a diagnosis of malnutrition. The cut-off points suggested are often based on survival analysis, and its levels can be altered by causes other than nutritional deficit . On the other hand, given its wide body distribution and half-life (15–20 days), it responds slowly to alterations in visceral protein reserves, so it would be a late marker of malnutrition and, therefore, not very practical for early nutritional interventions. In addition, studies in the older population without CKD have shown that serum albumin levels tend to be lower than in the younger population .2, obtaining very good sensitivity (88%) and a moderate specificity (81%); both very similar to the cut-off specified in the criteria for the clinical diagnosis of PEW  [2), while 14.3% were obese (BMI > 30 kg/m2) [2, very similar to the data previously reported.All the serum nutritional parameters in our study were significantly lower in the PEW group, but we were interested in evaluating these different serum parameters, as well as the BMI and MIS as possible predictors of PEW. We also calculated the cut-off values for our Catalonian dialysis population, based on the considerations of other authors who conveyed that the cut-off points of most malnutrition parameters proposed by ISNMR to calculate PEW derive from American populations, and it may be that they cannot be extrapolated to other geographic areas and lifestyles, such as Europe, Asia or, in our case, Mediterranean countries ,23. In o0 kg/m2) , confirmOnly 14.2% of the patients with PEW received nutritional intervention. Nutritional support is shown to improve survival markers, protein intake or promote changes in body composition with increased lean mass . Many ofPEW criteria are initially attractive due to their multifactorial scope , but implementation in clinical practice might be difficult and time consuming for health professionals, including dieticians. A recent study based on a survey related to kidney nutrition care, answered by key kidney care stakeholders in ISN-affiliated countries, revealed that only 48% of the 155 countries have dieticians/renal dieticians to provide this specialized service. Important deficiencies in interdisciplinary communication on kidney nutrition care delivery were also detected, especially in lower-income countries . IntegraEasy-to-use tools to identify patients at malnutrition risk are critically needed by the renal community. Some time ago, we suggested bioimpedance spectroscopy as a practical tool for early detection and prevention of PEW in hemodialysis patients , and witWe did not compare the data with bioimpedance as it was not available for all dialysis centers at the start of the study.This study demonstrates the strength of the Nutrendial online tool as a streamlined means of obtaining a final nutritional result. The study identifies a good correlation between other widely used scores to detect the PEW status. The study uses a sample size of 1389 dialysis patients, representative in age and etiology of CKD patients in dialysis as shown in The assessment of nutritional status for the detection and management of PEW is strongly recommended in CKD patients and should be included within the usual practices of a dialysis unit. We have developed an effective online tool that enhances the ability of different healthcare professionals—nephrologists, nutritionists, nurses—to diagnose PEW, better enabling them to implement the required nutritional interventions which can improve quality of life and survival rates among dialysis patients.


In this work, a characterization study of the interfacial interaction between different types of graphene nanoplatelets and an epoxy matrix is computationally performed. To quantify the discrete mutual graphene–epoxy “interfacial interaction energy” (IIE) within the nanocomposite, molecular dynamics simulations with a reactive force field are performed on a localized model of the suggested nanocomposite. Pull-out molecular dynamics simulations are also performed to predict the interfacial shear strength between the two constituents. The results indicate a significant increase in interfacial adhesion of functionalized nanoplatelets with the hosting epoxy matrix relative to virgin graphene nanoplatelets. The obtained results also demonstrate a dramatic increase in the interfacial interaction energy (IIE) (up to 570.0%) of the functionalized graphene/epoxy nanocomposites relative to the unmodified graphene/epoxy nanocomposites. In the same context, the surface functionalization of graphene nanoplatelets with the polymer matrix leads to a significant increase in the interfacial shear strength (ISS) (up to 750 times). The reported findings in this paper are essential and critical to producing the next generation of lightweight and ultra-strong polymer-based nanocomposite structural materials. The development of aerospace structural materials has been going through a determined research effort for the last two decades ,2,3. IncGraphene nanoplatelets (GNP) are widely utilized to reinforce polymer structures used in many engineering applications because of their remarkable mechanical, thermal, and electrical properties. The synthesizing process of GNP/epoxy nanocomposite materials requires professionality and a deep understanding of their molecular structure, especially when they are classified as advanced composites used in manufacturing aerospace and aeronautics structural components. Dealing with a wide range of key processing parameters in these nanocomposites, such as the type and curing degree of the epoxy resins, in addition to the size, concentration, and dispersion of the GNPs, is essential to optimize the mechanical properties and integrity of the bulk nanocomposite. The mutual interfacial interaction energy (IIE) is one of the most important factors used in determining the load transfer between nanocomposite constituents at the nanoscale. Fortunately, polymer matrices have shown a natural affinity to the reinforcements of carbon allotropes, including GNP. What is more, chemical modification  of the GNP surface can further improve the mutual affinity with the hosting matrix ,16,17.2 > -COOH > -OH > -F > -CH3. Finally, the study was concluded by specifying several factors which affect the mutual interaction energy between the constituents in the nanocomposite. These factors were identified by the used functional group types or their chemical structure, concentration, polarity, and their distribution on the nanoplatelet surface. However, the mutual shear stress level between the functionalized graphene nanoplatelets and polymer matrices was affected by another factor, which is referred to as the mechanical interlocking between them.The pull-out energy can be utilized to determine the shear strength or interfacial adhesion between two surfaces. Due to the difficulty and limitations of using standard experimental tools to fully access the pull-out energy, a reliable molecular dynamics (MD) computational tool called “pull-out MD” (POMD) simulations has shown its validity in addressing the interfacial shear strength (ISS) between nano-reinforcements (such as GNP and carbon nanotubes) and the hosting polymer matrix ,20,21,226H13, and -C2H4(C2H5)2. The results indicated that the largest functional group type of -C6H13 produced the highest interfacial bonding energy between the constituents. This is because they were relatively well-embedded within the polymer matrix. However, the complex configuration of the -C2H4(C2H5)2 functional groups provided better mechanical interlocking, and, thus, the strongest shear force. This concludes that the size and molecular configuration of the functional group play an important role in controlling the interlocking mechanism and interfacial load transfer.In another study, Lv et al.  investig2, -CH3. The reactive force field (ReaxFF), developed by Chenoweth et al. [2 functional groups can produce higher electrostatic (Coulomb) energy relative to the -H and -CH3 functional groups, which are non-polar. Due to their high polarity, the epoxide -O- groups provided the highest level of interfacial binding energy. Thus, the epoxide -O- groups exhibited a traction effect on the PE chains that was comparable to the -CH3 functional groups, regardless of their poor interfacial binding.The POMD simulations were utilized by Jin et al.  to exploh et al. , was useThe use of reactive force fields is critical for most MD simulations, and it could have benefits over using fixed-bond force fields, as it can efficiently capture the interactions of the intermolecular covalent bonding process occurring at the interface between the matrix molecules and the reactive functional groups in the nanofillers. In this context, Hou et al.  employedWith the capabilities of POMD simulations to capture the characteristics of interfacial regions between nanofillers and polymer matrices, which are briefly presented above in addition to other relevant computational results in the literature ,32,33,342 and Amide -C(=O)-NH2) to represent the FGO/epoxy nanocomposite. The chemical compositions of these two case studies were modeled based on the data presented by Park et al. [This work utilizes MD samples for the interphase region of GNP/epoxy nanocomposites, which were previously modeled by Al Mahmud et al. . Three ik et al. .Five replicates of the MD models were established for every case study to consider the variation probability in the predicted mechanical properties. Representative models of the three nanoplatelet/epoxy  systems are shown in The atomistic data used in establishing the MD models are given in Here is addressed two important correlative interfacial characteristics at the nanoplatelet/epoxy interphase region, which are investigated in two separate subsections. First, the prediction of the IIE between GNP and epoxy is investigated and discussed. The prediction of the ISS between GNP and epoxy is subsequently discussed.NP), epoxy (PEepo), and the full nanocomposite MD model (PEMD) [The nanoplatelets/epoxy interfacial binding can be assessed using the IIE. The IIE for a nanoplatelet/epoxy nanocomposite can be estimated using Equation (1) using the potential energies of the nanoplatelets (PEl (PEMD) ,41:IIE =The levels of the estimated IIE for the GNP/epoxy, GO/epoxy, and FGO/epoxy systems are shown in As the IIE involves different types of energy terms, which are established depending on the molecular system type and configuration, an analysis of the contribution percentages of the energy terms in the predicted IIE for each nanocomposite system is shown in 3 to sp2 ratio  behavior of the applied pulling force whose profile is shown in pull depends only on one variable (tstep) and two constant values , it is highly critical to specify the optimum values for these three terms. To clarify that, the increment in tstep has to be as small as possible to capture the threshold value of the pulling force in addition to being able to distinguish the stages in the obtained slipping profile. The total time required for the simulation process  has to be as large as possible to satisfy the nanoplatelet slipping onset point and securely obtaining the entire slipping stage in the simulation process. To satisfy all of the above requirements, specifying the optimum value of Fmax has to be somehow compatible with the other terms in Equation (2). An additional hidden term has to be considered in Equation (2) represented by the computational cost, which depends on the available computational resources.The pulling process was proposed to be performed in a very steady slow manner. This is necessary to capture the critical value of the pulling force at which the relative interfacial slipping onset occurs between the nanoplatelet and the epoxy. Thus, the variable of the applied force was derived to be gradually increased with time in the MD simulation, which is obtained by using Equation (2):Two important aspects were considered in the simulation process of all the MD models. First, it is necessary to activate the periodic boundary condition settings for the MD models to mimic a real-sized system. Second, both the zigzag and armchair directions in the graphene structure, which are illustrated in The following results were obtained after performing the pull-out simulations for the three nanoplatelet/epoxy systems. For a representative GNP/epoxy MD model, a displacement profile of the GNP as a function of the pulling force on at each carbon atom in the GNP lattice is shown in −3 Kcal/mol-Å per carbon atom. On the other hand, the corresponding GNP displacement value was estimated to be around 25 Å, yet it was around 5 Å at the beginning of stick–slip behavior. Note that any of the two threshold values was obtained by estimating its average value over the MD replicate models and considering the two directional pulling axes in each replicate.Once the GNP starts its slipping after the initial sticking stage, pulling the GNP along the armchair direction demonstrates prominent stick-slip stages which form a stair-like profile. In contrast, a very short sticking stages can be observed once the GNP begins to slip along the zigzag axis. The relative difference in the stick–slip behavior is associated with the contrast in the slipping path along each of the two axes, which is governed by the energy surface topology of the aromatic lattice structure ,44,45. TTo assess the interfacial binding in the functionalized graphene nanoplatelet/epoxy systems, a quite similar MD simulation procedure was adopted. The results were also obtained and analyzed to be compared with the predictions above. As functionalized nanoplatelets exhibit a wrinkled structure and rough topology, in addition to the produced interfacial covalent and/or noncovalent bonding with the hosting epoxy matrix, both the GO and FGO were expected to yield larger pull-out resistance levels in comparison to that observed in the GNP/epoxy system. For the GO/epoxy system, From the visual perspective, For the FGO nanoplatelet shown in 10) of the applied pulling force per carbon in the nanoplatelet lattice and parallel to the two directional axes. Once again, the particular different behavior in the GNP slipping profile is recognized at the initial stick stage, which is highly sensitive to the pulling direction. In contrast, all the slipping profiles of GO and FGO are closely analogous by using the logarithmic scale and demonstrate less sensitivity to the pulling direction. More importantly, both the GO and FGO require larger magnitudes of the applied pulling force to fulfill their slipping onset, which is relatively much lower (in the range of two to three orders of magnitude) for the GNP. The nearly identical mechanical behavior observed in the slipping of the GO and FGO ensures that the nanoplatelet-matrix ISS is likely to be heavily influenced by the interlocking between the two constituents, which is established by the wrinkled and rough surfaces of the functionalized nanoplatelets.To accomplish analyzing the difference in the pulling response between the three nanoplatelet types, Given that ISS is the pulling force threshold registered at the onset of slipping, In addition to its importance in estimating the ISS, the pulling force threshold value is essential to assess the static interfacial friction between the nanoplatelet and the hosting epoxy matrix. The ISS can be basically determined by multiplying the overall applied pulling force threshold value averaged over the MD replicates of the nanocomposite system by the number of carbon atoms in the nanoplatelet. Thus, as the graphene nanoplatelet lattice was modeled with 836 carbon atoms, the ISS in the GNP/epoxy system is estimated to be about 0.4 kcal/mole-Å. This is significantly less than the ISS in the GO/epoxy system, where its average value was estimated to be about 250 kcal/mole-Å. The FGO/epoxy system, however, surpasses the other nanocomposite systems by registering the highest ISS with an average value estimated to be about 300 kcal/mole-Å. In other words, the surface functionalization of the GNP resulted in a significant enhancement in the produced ISS in the GO/epoxy and FGO/epoxy systems over the GNP/epoxy system by about 625 and 750 times, respectively. The surface functionalization of the GNP resulted in a tremendous enhancement in the produced ISS in the GO/epoxy and FGO/epoxy systems over the GNP/epoxy system, by about 625 and 750 times, respectively. Due to the covalent interfacial bonding involving the highly reactive amine/amide functional groups in the FGO and the hosting epoxy, the ISS of the FGO/epoxy system is larger than that of the GO/epoxy system by about 20%.Based on the ISS values, which are estimated for the three nanocomposite systems, it is clear that the functionalization of graphene nanoplatelets can substantially enhance adhesion at the nanoplatelet–matrix interface. Thus, the bulk material strength and toughness are implicitly promoted. The predicted results confirm that the degree of improvement in the material ISS is highly reliant on the composition and quantity of the functional groups. This can be logically recognized by the additional improvement in the ISS of the FGO/epoxy system over the GO/epoxy system, which is attributed to the interfacial covalent bonds established between the amine/amide functional groups and the epoxy matrix. In general, producing a nanocomposite with a robust molecular structure is governed by establishing an interface with covalent and noncovalent bonding. The interface interlocking is stimulated by the wrinkled and rough topology of the GO and FGO, and results in improvement of the material robustness. These factors qualify the localized interphase regions of the functionalized nanoplatelet/epoxy to serve as strong toughening zones within the bulk composite material.This computational study utilizes experimentally informed nanocomposite MD models to address the effect of surface functionalization on the graphene/epoxy interfacial binding using POMD simulations. It has been established that introducing functional groups to the graphene surface can significantly improve both IIE and the ISS for the simulated nanocomposites. The outcomes from the simulated nanocomposite MD models refer to the merit of using GO rather than GNP reinforcements which is demonstrated by increasing the IIE up to 215% and increasing the IIE up to 625 times in the nanocomposite. This significant promotion in the material interfacial properties was primarily originated due to the high concentration of oxygen groups bonded to the graphene nanoplatelet. Substituting some of the oxygen functional groups with highly reactive amine and amide functional groups resulted in further increasing the IIE up to 570% and the ISS up to 750 times in FGO/epoxy relative to GNP/epoxy system. Such reactive functional groups have been found to promote the nanoplatelet-matrix interfacial adhesion by establishing supplementary covalent and noncovalent interaction energies. Other additional factors represented by the nanoplatelets wrinkling and rough topology of GO and FGO have been found to enhance the interference between the functionalized nanoplatelets and the hosting matrix. This, in turn, promotes the interlocking mechanism between the two constituents at the interphase region. Consequently, locally promoting the nanoplatelet-matrix interfacial binding is implicitly essential in establishing the bulk-level strength and toughness of the nanocomposite material.


Aberrant expression of the transcription factor DUX4 from D4Z4 macrosatellite repeats on chromosome 4q35, and its transcriptome, associate with pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Forced DUX4 expression halts skeletal muscle cell proliferation and induces cell death. DUX4 binds DNA via two homeodomains that are identical in sequence to those of DUX4c (DUX4L9): a closely related transcriptional regulator encoded by a single, inverted, mutated D4Z4 unit located centromeric to the D4Z4 macrosatellite array on chromosome 4. However, the function and contribution of DUX4c to FSHD pathogenesis are unclear. To explore interplay between DUX4, DUX4c, and the DUX4-induced phenotype, we investigated whether DUX4c interferes with DUX4 function in human myogenesis. Constitutive expression of DUX4c rescued the DUX4-induced inhibition of proliferation and reduced cell death in human myoblasts. Functionally, DUX4 promotes nuclear translocation of β-CATENIN and increases canonical WNT signalling. Concomitant constitutive expression of DUX4c prevents β-CATENIN nuclear accumulation and the downstream transcriptional program. DUX4 reduces endogenous DUX4c levels, whereas constitutive expression of DUX4c robustly suppresses expression of DUX4 target genes, suggesting molecular antagonism. In line, DUX4 expression in FSHD myoblasts correlates with reduced DUX4c levels. Addressing the mechanism, we identified a subset of genes involved in the WNT/β-CATENIN pathway that are differentially regulated between DUX4 and DUX4c, whose expression pattern can separate muscle biopsies from severely affected FSHD patients from healthy. Finally, blockade of WNT/β-CATENIN signalling rescues viability of FSHD myoblasts. Together, our study highlights an antagonistic interplay whereby DUX4 alters cell viability via β-CATENIN signalling and DUX4c counteracts aspects of DUX4-mediated toxicity in human muscle cells, potentially acting as a gene modifier for FSHD severity. Importantly, direct DUX4 regulation of the WNT/β-CATENIN pathway informs future therapeutic interventions to ameliorate FSHD pathology. Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular dystrophy, manifesting as a descending left/right asymmetric muscular weakness and wasting. FSHD is slowly progressive, initially generally affecting facial, shoulder, and proximal upper limb muscles and continuing to lower limb muscles . In addide novo cases less frequent. FSHD is associated with epigenetic derepression occurring at the subtelomeric region of chromosome 4 (4q35), covering a large macrosatellite array of D4Z4 repeats that are normally transcriptionally silenced through epigenetic mechanisms (158900) is the more prominent (95% of cases) where the 4q35 locus has a reduction of D4Z4 units from the usual ≥11–100+ copies found in non-affected individuals, to only 1–10 repeats on at least one allele in FSHD1 patients (158901), where the residual number of D4Z4 units is usually within the lower end of the ‘normal’ range, and epigenetic derepression is primarily caused by mutations in the chromatin remodelling protein SMCHD1  (FRG1 (FRG2 (TUBB4Q (ANT1 (SLC25A4)  , FRG1 (G9)  .DUX4c is encoded by a single truncated and inverted D4Z4 unit located 42 kb centromeric to the D4Z4 array on chromosome 4 , whereas expression of DUX4 target genes TRIM43, PRAMEF1, ZSCAN4, and KHDC1L peaked between 16–24 h of DOX treatment, when DUX4 mRNA levels were reduced compared with the 7 h time point. Thus, robust activation of DUX4-target genes starts after approximately 9–10 h of DOX induction (To standardise DUX4 induction in human LHCN-M2-iDUX (iDUX4) myoblasts , we adminduction . We seleiDUX4 cell number was significantly decreased upon DUX4 induction using 62.5 ng/ml DOX or higher , suggestTo explore proliferation dynamics upon DUX4 expression, iDUX4 myoblasts were pulsed with EdU for 2 h after 24 h in growth medium supplemented with DOX, and EdU incorporation compared with non-induced iDUX4 . The proDUX4 accumulation caused altered cell morphology, inducing a smaller and irregular cell shape phenotype, likely indicating the onset of programmed cell death  in line DUX4c mRNA in iDUX4/DUX4c non-induced myoblasts, compared with iDUX4/Ctrl  . IntriguTo better investigate the candidate biomarker signature, we then interrogated the 41 Up in DUX4/Down in DUX4c and 47 Down in DUX4/Up in DUX4c target gene subsets using Gene Ontology (GO) analysis to evaluate their contribution to specific biological processes. The two gene subsets retrieved the common processes ‘Cell Differentiation and Development’ and ‘Cell Response to Stimuli’. However, the Up in DUX4/Down in DUX4c geneset-specific biological processes included ‘WNT/β-CATENIN’, ‘Immune Response’, and ‘Cell Death’ , while tOur transcriptomic analysis retrieved the biological process WNT/β-CATENIN within genes up-regulated by DUX4 and down-regulated by DUX4c . WesternLGR5 but did not alter DUX4 accumulation and expression of its target gene PRAMEF1 compared with non-induced iDUX4 myoblasts  and exit the cell cycle  displayed significantly higher levels of DUX4 compared to their D4Z4 non-contracted control clone (54-6) or sibling-matched unaffected controls (12U and 16U)  . In contcontrols , resemblcontrols  and suggWNT/β-CATENIN has previously been implicated in FSHD pathology and positively correlates with the regulation of CASP3-mediated apoptosis in FSHD muscle biopsies . InteresFSHD myoblasts have a reduced proliferation rate compared to matched unaffected lines . Since DDUX4c expression in myoblasts. ii) DUX4 and DUX4c differentially regulate a subset of genes in opposite directions, defining a transcriptomic signature exploitable as a FSHD biomarker. iii) DUX4c rescues the anti-proliferative effects of DUX4 and attenuates DUX4-induced CASP3-mediated apoptosis. iv) DUX4 causes apoptosis through nuclear accumulation of β-CATENIN, along with the activation of the β-CATENIN-mediated signalling program. v) DUX4c blunts DUX4-mediated activation of β-CATENIN signalling. vi) A minimal transcriptomic signature that highlights DUX4/DUX4c convergence on the β-CATENIN signature efficiently separates severe FSHD from healthy control muscle biopsies. vii) β-CATENIN inhibition efficiently rescues proliferation of FSHD myoblasts.We report that DUX4 affects WNT/β-CATENIN signalling to cause deleterious effects in FSHD and that molecular antagonism between DUX4 and DUX4c moderates FSHD pathomechanisms through seven major findings. i) DUX4c robustly reduces expression of DUX4 target genes, while DUX4 induction decreases endogenous Together, our observations suggest that antagonism between DUX4 and DUX4c contributes to moderating FSHD pathogenesis via balancing WNT/β-CATENIN signalling.In human rhabdomyosarcoma cell lines, DUX4c is reported to contribute to cell viability  implyingDUX4c and FRG2 loci, was reported in two FSHD families  allele termed the “D4F104S1 genomic deletion”, which extends from the D4Z4 repeat array to include the families . This imfamilies  suggestitoxicity . However by DUX4  and FRG2orrelate , so it i lineage , further lineage , so compGiven the complete sequence homology between the two DNA-binding homeodomains of DUX4 and DUX4c, it is unsurprising that over 75% (268/356) of human gene orthologs differentially regulated by DUX4 and DUX4c in mouse are either up- or down-regulated by both DUX4 and DUX4c, although differences in the magnitude of change are likely due to the strong C-terminal transactivation domain of DUX4 . The proWe previously reported a positive correlation between CASPASE signalling and β-CATENIN in the FSHD interactome , indicatUncontrolled activation of WNT/β-CATENIN signalling causes increased mitochondrial-derived reactive oxygen species (ROS) production and induces oxidative damage in murine muscle cells . In turnWe found that a subset of 17 of the 88 (19%) genes differentially regulated upon DUX4 or DUX4c expression refer to WNT/β-CATENIN signalling. This 17-gene signature also efficiently separates severe FSHD patients from healthy controls, providing a minimal transcriptomic signature suitable for molecular profiling of severe FSHD patients. Importantly, previous transcriptomic analysis also highlighted an enrichment for altered mitochondrial pathways in the same dataset  and in Fin vitro  , DUX4c d deleted . This in deleted . This stown mRNA . FinallyMYOG (highly expressed in rhabdomyosarcoma) was modified to enhance rhabdomyosarcoma-specificity. Using this modified MYOG promoter to drive expression of the HSV-TK suicide gene resulted in specific targeting of tumour cells both in vitro and in vivo , but th in vivo . Such a Together, our results highlight alternative potential interventions to suppress DUX4 toxicity, rather than attempting to further reduce levels of an already difficult-to-detect protein. DUX4 expression and its signalling cascade appear to be the root cause of FSHD, but the detrimental effects of the DUX4-driven molecular cascade extend beyond muscle. For example, immortalized FSHD lymphoblastoid cells display robust DUX4 expression and a DUX4-signature comparable to muscle, indicating an immune contribution in FSHD pathogenesis . In lineIn summary, we found that DUX4 operates in part through a WNT/β-CATENIN pathomechanism to reduce myoblast proliferation and cause apoptosis. Molecular antagonism between DUX4 and DUX4c indicates that DUX4c is a genetic modifier of FSHD pathology.pMSCV-puro vector (Clontech), in which the puromycin resistance gene has been replaced with an internal ribosomal entry site (IRES) preceding the coding sequence for enhanced green fluorescent protein (eGFP) to obtain pMSCV-IRES-eGFP (pMIG), as previously described (pMSCV-DUX4c-IRES-eGFP (RV_DUX4c-IRES-eGFP) or the empty control pMSCV-IRES-eGFP (RV_-IRES-eGFP) constructs were transfected in HEK293T cells, as previously described  for the indicated period of time. For proliferation assays, iDUX4 myoblasts in proliferation medium were pulsed with 10 mM EdU (Invitrogen) for 2 h immediately prior to fixation. Incorporated EdU was detected using the click-iT EdU AlexaFluor Kit (Invitrogen) according to the manufacturer’s instructions.LHCN-M2 Myoblasts were previously engineered to express DUX4 under DOX control to generate LHCN-M2-iDUX (iDUX4) myoblasts . RetroviThe three immortalised FSHD patient-derived cellular models were the isogenic ‘54’ series derived from the biceps of a male mosaic FSHD1 patient , where 5β-CATENIN inhibitors were selected on the basis of reported effectiveness. IWR-1 and XAV939 (Generon) inhibit Tankyrases, thereby promoting degradation of β-CATENIN . For assFor immunolabelling, iDUX4 myoblasts were fixed in 4% paraformaldehyde/PBS for 10 min, washed in 3 X PBS for 5 min and permeabilised for 5 min with 0.5% triton X100/PBS. Subsequently, cells were blocked for 1 h using 5% goat serum/PBS (blocking buffer). Primary antibodies were added in PBS and incubated overnight at 4°C. Primary antibodies were: rabbit polyclonal anti-Cleaved-CASPASE3 , mouse monoclonal anti-total β-CATENIN , rabbit monoclonal anti-Non phospho(Active)β-CATENIN , chicken polyclonal anti-GFP , and mouse monoclonal anti-DUX4 . DUX4 antibody 9A12 recognises a region common to both DUX4 and DUX4c, so was also used to detect DUX4c expression as previously described . Cells wwww.Fiji.sc). Uncropped scans of Western blots shown in this work are reported in Western blot was performed as described previously . Briefly3 cells/well in flat-bottomed 96-well plates. After 16 h, the medium was replaced with a 1:1 solution containing 2X RealTime-Glo MT Cell Viability reagent mixture (Promega) supplemented with DOX at the desired final concentration in a fresh proliferation medium following the manufacturer’s instructions. Luciferase signal was measured at 16, 24, 40, and 48 h of DOX treatment and displayed as fold change to the 16 h normalised for background.For viability assay, iDUX4 myoblasts were plated at a density of 5 × 104 cells/well in flat-bottomed 96-well plates. After 16 h, the medium was replaced with a solution containing 1X RealTime-Glo Annexin V Apoptosis and Necrosis assay reagent mixture (Promega) supplemented with DOX, and with or without β-CATENIN inhibitors or DMSO vehicle, at the indicated final concentration in fresh proliferation medium following the supplier’s instructions. Apoptosis and necrosis were measured after 16 and 24 h, or as indicated, of DOX treatment and considered detectable when the signal increased over background levels.For apoptosis/necrosis assays, cells were plated at a density of 1 × 104 cells/well in flat-bottomed 96-well plates. After 16 h, the medium was replaced with a solution containing 1X Caspase-Glo3/7 Assay System reagent mixture (Promega) supplemented with DOX at the indicated final concentration in fresh proliferation medium following the supplier’s instructions. Activity of Caspase was measured at the indicated time points of DOX treatment and considered detectable when the signal increased over background levels.For Caspase activity assay, cells were plated at a density of 1 × 105 cells/well in 24-well plates. For β-CATENIN inhibition after 24 h, the medium was replaced with fresh medium containing either 5 μM of IWR-1 or 4 μM of XAV939. At indicated time points, cells were harvested, and the number of viable cells was counted with a haemocytometer upon Trypan Blue staining.For growth curve and cell count, 16A and 16U myoblasts were plated at a density of 2 × 10TBP or RPLP0 and fold changes were calculated using the ΔΔCt method  and quantified using a NanoDrop before being retrotranscribed with QuantiTect Reverse Transcription Kit (Qiagen) or SuperScript First-Strand Synthesis System (Thermo Scientific). RT-qPCR was carried out using MasterMix solution (Qiagen) or Takyon Low ROX SYBR 2X MasterMix blue dTTP (Takyon) as per the manufacturer’s instructions on a ViiA7 thermal cycler (Applied Biosystems). RT-qPCR analyses were performed as previously described . Ct valut method . ResultsThree images were taken per well at ×10 magnification for each replicate and counted manually using ImageJ. For immunolabelling, data are presented as the mean proportion of total DAPI-positive cells ±SEM, N = 3 biological replicates. For RT-qPCR, data were presented as average relative expression ±SEM, N = 3. Data presented as mean ± SEM from N = 3/4 independently treated wells, considered biological independent replicates, from a representative experiment(s). Statistical significance was calculated in GraphPad Prism using unpaired t-test or one-way ANOVA followed by Tukey’s post hoc or Holm-Sidak tests. Paired t-test was used to account for DUX4/DUX4c variation across FSHD myoblast samples.ebi.ac.uk/Tool/msa/clustalo (ncbi.nlm.nih.gov/Structure) and validated using the Expasy SIM alignment tool (web.expasy.org/sim) both with default parameters.Alignment of human DUX4 (NP_001280727.1) and DUX4c (Q6RFH8.1) protein sequences, retrieved from clustalo ) with dehttps://www.ncbi.nlm.nih.gov/geo/geo2r/) with default settings (https://biit.cs.ut.ee/gprofiler/orth) and resulted in 356 human genes  with de v3.8.2; ) and cluhttps://software.broadinstitute.org/morpheus) and applying ‘One plus Log2’ and ‘Zscore’ adjustments to CPM values of selected genes. Morpheus hierarchical clustering was applied blindly to assess the goodness of the 88 (Gene counts of human severe FSHD (6 samples) and healthy muscle biopsies (9 samples) were derived from f the 88  or 17 gef the 88  to clust