Alnus japonica has shown anti-cancer effects against colon cancer cells by suppressing FOXM1. The present study hypothesized that DFS can have anti-cancer effects against glioblastoma (GBM) tumorspheres (TSs). Immunoprecipitation and luciferase reporter assays were performed to evaluate the ability of DFS to suppress nuclear translocation of β-catenin through β-catenin/FOXM1 binding. DFS-pretreated GBM TSs were evaluated to assess the ability of DFS to inhibit GBM TSs and their transcriptional profiles. The in vivo efficacy was examined in orthotopic xenograft models of GBM. Expression of FOXM1 was higher in GBM than in normal tissues. DFS-induced FOXM1 protein degradation blocked β-catenin translocation into the nucleus and consequently suppressed downstream target genes of FOXM1 pathways. DFS inhibited cell viability and ATP levels, while increasing apoptosis, and it reduced tumorsphere formation and the invasiveness of GBM TSs. And DFS reduced the activities of transcription factors related to tumorigenesis, stemness, and invasiveness. DFS significantly inhibited tumor growth and prolonged the survival rate of mice in orthotopic xenograft models of GBM. It suggests that DFS inhibits the proliferation of GBM TSs by suppressing FOXM1. DFS may be a potential therapeutic agent to treat GBM.Forkhead Box M1 (FOXM1) is known to regulate cell proliferation, apoptosis and tumorigenesis. The lignan, (−)--1,4-O-diferuloylsecoisolariciresinol (DFS), from  Despite intensive treatment with surgery, chemotherapy and radiotherapy, patients with GBM have a high mortality rate and poor prognosis3. To date, furthermore, relatively little is known about the cells from which GBM originates or the genetic mutations that accompany GBM tumorigenesis, so GBM still remains a difficult disease to conquer5. However, previous studies reported that GBM tumorsphere (TS), characterized by GBM resident cells in culture has a treatment refractory properties, as well as a stemness profile and an ability to form tumors, and it has been shown to have value for clinical use7.Glioblastoma (GBM), the most common primary malignant tumor in the brain, is highly aggressive8. Expression of FOXM1 is significantly elevated in many human tumors, including non-small cell lung cancer, breast cancer, basal cell carcinoma, hepatocellular carcinoma, pancreatic cancer, prostate cancer, colon cancer, medulloblastoma and GBM13. High expression of FOXM1 in glioma cells enhances tumorigenicity, invasiveness and angiogenesis in GBM animal models16. Furthermore, FOXM1 was found to regulate the Wnt/β-catenin signaling pathway by promoting the nuclear translocation of β-catenin 18.Forkhead Box M1 (FOXM1) is a member of the Forkhead box transcription factor family that has been shown to regulate DNA damage repair, cellular proliferation, apoptosis, angiogenesis and tumorigenesisAlnus japonica (Betulaceae) is a plant used as a medicinal herb in east Asia. This plant contains pharmacologically active compounds, including the isolated lignan, (−)--1,4-O-diferuloylsecoisolariciresinol (DFS), which was found to reduce the viability of colon cancer cells and to interrupt the cell cycle. We previously reported that DFS interfere with β-catenin translocation into the nucleus, and to subsequently reduce the levels of expression of β-catenin mediated genes through suppression of FOXM1 protein expression in colon cancer19. DFS also induced autophagy and endoplasmic reticulum (ER) stress in prostate and colon cancer cells20. FOXM1/β-catenin interactions can also regulate the stemness and tumorigenicity of glioma stem cells18. Because GBM TSs are resistant to conventional treatments such as radiotherapy and chemotherapy, they have been considered a good platform for testing the therapeutic efficacy of drug candidates. We have tried to find promising anti-GBM agent from medicinal plants such as A. japonica. Relatively little is known, however, about the effects of the DFS in GBM TSs.The present study hypothesized that DFS has the ability to block β-catenin translocation into the nucleus by suppressing the formation of FOXM1/β-catenin complex in GBM TSs. In addition, the anti-cancer potential of DFS against GBM was evaluated using GBM TSs and an orthotopic xenograft mouse model.GBM tissue specimens were obtained from 49 patients who underwent surgery for GBM from May 2009 to December 2016, and among 49 patients, 8 normal brain tissue specimens were also obtained. All the specimens were from the patients diagnosed with GBM for the first time, and tissues from the patients with recurred GBM were excluded.22, the levels of expression of FOXM1 and β-catenin (CTNNB1) mRNAs were significantly higher in 49 of GBM tissues than in 8 of normal tissues, and public datasets, Oncopression and REMBRANDT, showed similar results of FOXM1 and β-catenin (CTNNB1) expression level  using transcription factor target gene setsThe effects of DFS pretreatment of GBM TSs on tumor growth were evaluated in orthotopic xenograft models of GBM using TS13-64. Magnetic resonance imaging (MRI) showed that tumors in the control group were larger than those in the DFS-pretreated group, indicating that pretreatment with DFS reduced the ability of GBM TSs to promote tumor growth in a mouse orthotopic xenograft model Fig. A,B. KaplIn addition, immunohistochemistry (IHC) was performed on mouse brain tissue that had been tested for stemness and invasiveness of GBM TSs to determine how DFS affects local microenvironment cells Fig. D. In DFS3. It exhibits malignant progression, resistance to conventional treatment, and infiltrative features7. Numerous active researches are being conducted on the origin of GBM, from neural stem cells in the subventricular zone of the adult human brain, and its treatment methods4. Although multimodal therapeutic strategy has been suggested including surgery, chemotherapy, and radiotherapy, the overall survival of patients with GBM is still very low at 14.6 to 21.12. This study was designed to find a new treatment method for this challenging disease.GBM is the most common and malignant primary brain tumor in adults with an unfavorable prognosisA. japonica24. A. japonica is a plant widely used as both a food and a medicinal herb in east Asia24. It is effective in various medical conditions, such as fever, diarrhea and hemorrhage, because it contains diverse pharmacologically active components., In previous study, DFS interfered with β-catenin mediated genes through lysosomal-dependent degradation of FOXM1 protein in colon cancer19. However, there has been no study on whether DFS is effective in GBM. The present study hypothesized that DFS would exert a proliferation-inhibiting effect in GBM because of the fact that stemness and tumorigenicity are regulated by FOXM1/β-catenin interactions in glioma stem cells18.DFS, a lignan isolated from a methanol extract of stems of 6. In this study, GBM cells were isolated from a specimen derived from a patient diagnosed with GBM, and then TS was obtained through cell culture. Among the GBM TSs used in this experiment, the results were analyzed for two GBM TSs, denoted TS15-88 and TS13-64, which showed a clear response after DFS treatment. GBM TS itself can be considered to show the same treatment response as in the patient’s GBM tissue, and has the advantage of being suitable for applying manipulations for in vivo experiments by implantation in a mouse model.According to previous study, GBM TS is a prominent phenotype with features such as stemness and invasiveness, and it has been found to have value in clinical use28, including in the promotion of stem cell genes32, in tumor migration and invasion, and in the epithelial-to-mesenchymal transition (EMT)35. FOXM1, a key mediator of the Wnt/β-catenin pathway, is overexpressed in many human cancers, including glioma. The finding in the present study, that the level of FOXM1 mRNA was significantly higher in GBM than in normal brain tissue, suggests that targeting FOXM1 in GBM can suppress tumor progression. And this result suggests the possibility that DFS may also have a therapeutic effect on GBM, considering the mechanism that DFS exerts anti-cancer effects by acting on FOXM1.The Wnt/β-catenin signaling pathway is aberrantly activated during the development and progression of various human cancers. This pathway plays important roles in the tumorigenesis and progression of malignant astrocytic gliomas18. The present study found that DFS reduced interactions between FOXM1 and β-catenin in the nucleus, subsequently reducing the expression of proteins such as cyclin D1 and cMyc by suppressing formation of the complex with TCF424. Moreover, the levels of expression of mRNA associated with Wnt signaling pathway and the activation of β-catenin/TCF complex-related genes were downregulated in DFS treated GBM TSs. The fact that DFS inhibited the FOXM1/β-catenin interaction and subsequently suppressed the expression of β-catenin/TCF complex-related genes in GBM TSs suggests that DFS, as a consequence, can regulate TCF in transcription level. It is known that FOXM1 regulate transcription by the direct binding of small molecules such as FDI-6. And DFS-induced FOXM1 protein degradation is considered to be dependent on lysosome19. We confirmed that DFS significantly reduced the enrichment score of FOXM1 for oncogenic transcription factors which shows that the expression of target genes of FOXM1 were reduced overall as shown in Fig. The interaction between FOXM1 and β-catenin has been shown to promote the nuclear translocation of β-catenin and to increase the expression of β-catenin target genes, thereby promoting self-renewal and tumorigenesis of GBM initiating cells (GICs)19. In this study, we investigated the fraction of β-catenin in both the cytosol and nucleus to quantitatively confirm the subcellular localization of β-catenin  phenotype43. The present study investigated the prominent phenotypes of GBM TSs, which have stemness and invasiveness. Stem-like cells that induce GBM TS in tumors are known to affect treatment resistance and recurrence, making the inhibition of stemness and invasiveness a positive indicator for evaluating the potential efficacy of DFS.FOXM1 has been shown to promote the stemness of GBMs by regulating the master stem cell regulator SOX249. In order to overcome this limitation, research on injection methods other than pretreatment should be conducted in the next stage of DFS study which increase the utilization of this drug.We used two TSs, TS13-64 and TS15-88, for all other experiments except in in vivo mouse model which was performed only with TS13-64 since there was no significant difference in the effect of DFS between these two TSs. And TS13-64 had shorter mouse survival time and more overall experiment data was available compared to TS15-88. Moreover, unfortunately, in vivo experiments using a orthotopic xenograft mouse model were performed only as a pretreatment method, not in other methods such as intravenous, intraperitoneal, or local injection. We previously conducted an experiment to administer DFS to mice by intraperitoneal injection, however, the effect of DFS from the experiment was not seen. In addition, because DFS is a new drug that is still in the research stage, the mechanism of action in the brain has not been established. So we conducted an experiment using the pretreatment technique with reference to other literaturesIn summary, DFS can interfere with signaling steps of stemness, invasiveness and proliferation by suppression of FOXM1 Fig. F. DFS co46. Briefly, cells were cultured in TS complete medium, consisting of Dulbecco’s modified Eagle’s medium/F12 , 1 × B27 , 20 ng/mL basic fibroblast growth factor  and 20 ng/mL epidermal growth factor . All in vitro experiments were performed under the culture conditions for TSs. DFS, an anti-GBM lignan, was isolated from a chloroform soluble fraction of A. japonica extracts, as described19.GBM TSs were derived from two individual patients with GBM. These GBM TSs, denoted TS15-88 and TS13-64, were established from fresh GBM tissue specimens, as approved by the institutional review board of the Yonsei University College of Medicine. TSs were cultured according to standard methodsLysates of GBM TSs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on Tris–glycine gels. Cytosolic and nuclear fractions were prepared using cytoplasmic and nuclear extraction reagents , respectively, according to the manufacturer’s instructions. Proteins were transferred to nitrocellulose membranes and probed with antibodies against cleaved capase-3, CD133, PDPN, β-catenin, active-β-catenin, CD44, snail, Cyclin D1 and cMYC ; Sox2 ; Msi-1 ; nestin ; N-cadherin, Zeb1 ; Twist, Oct3/4, BAX, Bcl-2, PARP and GAPDH ; and FOXM1 , along with Western Lightning Plus-enhanced chemiluminescence reagent . Images were captured using an ImageQuant LAS 4000 mini . We expressed all the results of Western blot as a violin plot, and added this as a Supplementary To determine interactions between FOXM1 and β-catenin, GBM TSs were treated with DFS for 72 h, lysed with lysis buffer and incubated with anti-FOXM1 or anti-β-catenin antibody on a rotator overnight at 4 °C. Protein-antibody-protein A/G agarose complexes were prepared using protein A/G sepharose beads (Santa Cruz Biotechnology) for 4 h at 4 °C. After extensive washing with PBST, the immunoprecipitated complexes were eluted with 0.1 M glycine–HCl (pH 2.7), and neutralized with neutralizing buffer . The precipitates were then resuspended with SDS sample buffer and boiled for 10 min. The precipitated proteins were subjected to immunoblotting with the indicated antibodies.GST-tagged β-catenin was expressed in DH5α E. coli strain and purified by affinity chromatography on glutathione-Sepharose 4B beads . Flag-tagged FOXM1 was prepared by lysate by transfecting according to manufacturer's recommendation in TS15-88 and TS13-64, respectively. Confirming the purity of recombinant proteins was done with Coomassie brilliant blue staining by gel separation. For GST-pulldown assay, purified recombinant GST-β-catenin and GST control protein were co-incubated with 20 μl of 50% glutathione agarose beads slurry  in binding buffer at about 1 h at 4 °C. The bead elution process was done after washing the beads 3 times with a binding buffer. Thereafter, SDS sample buffer heating was assessed and immunoblotting was done with anti-Flag antibody.5 cells were seeded into each well of a 24-well plate for 1 day before transfection. The cells were transfected with 100 ng of firefly luciferase reporter plasmid and 10 ng of internal control plasmid pRL-TK  using Lipofectamine 3000 (Invitrogen) for 72 h according to the manufacturer's protocol. Samples were lysed with 1 × reporter lysis buffer for 15 min and centrifuged, and the supernatants were used for luciferase and β-galactosidase assays, according to the manufacturers' protocols. After confirming that the cells had been transfected using β-galactosidase assays, luciferase activity in 10 μg of protein lysate was quantified using the Dual Luciferase Reporter Assay System (Promega). Each sample was analyzed in triplicate.Canonical TCF/LEF signaling activity was measured using TOPflash/FOPflash reporter assays. Briefly, 1 × 104 single dissociated GBM TSs were seeded into each well of a 96-well plate. The plates were incubated at 37 °C for 24 h and treated with DFS for 72 h. WST reagent (10 μl/well) was added and absorbance was measured at 450 nm after incubating at 37 °C for 1 h. Each experiment was repeated three times in triplicate, and the results are expressed as the percentage of viable cells relative to controls.The effects of DFS on the survival of GBM TSs were determined using WST assays (Promega). Briefly, 1 × 104 cells per well. After incubating for 24 h, TSs were treated with DFS for 3 days, and then ATP levels were compared using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s protocol. Briefly, a volume of CellTiter-Glo Reagent equal to the volume of cell culture medium was added to each well, after which cells were incubated at room temperature for 10 min, and luminescence was measured.Dispersed GBM TSs were plated in 96-well plates at a density of 1 × 10To analyze the cell cycle, GBM TSs treated with DFS for 72 h were collected, fixed with 70% ethanol, dissociated with accutase (Sigma), and stained with 100 µg/ml of propidium iodide  at 4 °C for 15 min in the dark. To evaluate DFS-induced apoptosis in GBM TSs, GBM TSs were treated with the indicated concentration of DFS. Apoptotic cell death was detected by double staining with fluorescein-isothiocyanate (FITC)-conjugated annexin V  and PI. The stained cells were analyzed using an LSR II flow cytometer .Ten single dissociated GBM TSs were seeded in each well of a 96-well plate and treated with DFS after 24 h. The plates were incubated for 3 weeks in TS complete medium, with medium changed every week. The number of sphere-positive wells was counted, and the ratio of sphere-positive wells in the treated relative to the control group was calculated. The radius of spheres in each group was also measured. Images were captured and analyzed using ToupView software .Each well of a 96-well plate was filled with mixed matrix composed of matrigel , collagen type I (Corning Incorporated) and TS complete medium. Single spheroids had been seeded inside the matrix prior to gelation, followed by the addition of TS complete medium over the gelled matrix to prevent drying. The invaded area was quantified as occupied area at (72 h − 0 h)/0 h.GBM TSs were incubated by attaching them to coverslips in 24-well plates. After treatment for 48 h with or without treatment, cells were fixed with 3.8% formaldehyde for 10 min. The permeabilization process were done with 0.1% NP40/PBS and blocked with 1% BSA/PBS for 1 h. To simultaneously observe the subcellular localization of β-catenin and FOXM1, β–catenin (1:1000) and FOXM1 (1:1000) were incubated overnight at 4 °C. Cells then washed with PBS, and incubated with a secondary antibody conjugated with Alexa Fluor 488, 546 (1:1000) at room temperature for 1 h. Cells then washed with PBS and mounted with a mounting solution containing DAPI (Vetcached H-1200) and imaged with a confocal microscope . Acquired cell images were analyzed using ZEN 2009 software. The ImageJ software was used to calculate the relative fluorescence intensity of FOXM1 and β-catenin.47. Gene expression levels were measured with Cufflinks v2.1.148, using the gene annotation database of Ensembl release 7249. Non-coding regions were removed using the –mask option. Using GENE-E software, the average linkage of hierarchical clustering was determined with Pearson’s correction as a distance metric and expression levels depicted as heat maps.Total RNA was extracted from GBM TSs and used for RNA sequencing. The quality of the reads was checked using FastQC (v.0.10.1) and the sequencing adapters were removed using Trimmomatic (v. 0.38). Low quality reads were filtered according to the following criteria: reads containing more than 10% of skipped bases (marked as ‘N’s), reads containing more than 40% of bases with quality scores less than 20, and reads whose average quality scores of each read were less than 20. Filtered reads were mapped to the human reference genome using the aligner Tophat51. Cells were selected by trypan blue staining, and 5 × 105 viable cells were implanted into the right frontal lobe of each mouse at a depth of 4.5 mm using a guide-screw system52. Mice showing a > 15% reduction in body weight compared with the maximum were euthanized according to the approved protocol. For immunohistochemistry, 4-μm-thick sections were obtained with a microtome and transferred onto adhesive slides. Antigen retrieval and antibody attachment were performed using an automated instrument . Nestin and Zeb1 were detected using a peroxidase/3,3ʹ-diaminobenzidine staining system.Male athymic nude mice aged 4–8 weeks  were housed in micro-isolator cages under sterile conditions and monitored for at least one week before study initiation to ensure proper health. Lighting, temperature, and humidity were controlled centrally. Dissociated Luc-TS13-64 cells were pretreated with 10 µM of DFS for 72 h using the pretreatment protocolLevels of significance for comparisons among treatment groups were determined using one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Survival was analyzed using the Kaplan–Meier method and compared by log-rank tests. All the graphs and statistical analyses were performed using GraphPad Prism 9  software, with P-values < 0.05 considered statistically significant.All patients provided written informed consent, and permission for specimen sampling and evaluation was obtained from the Institutional Review Boards at our institutes (no. 4-2021-1319). This study was carried out in accordance with relevant guidelines and ethical regulations of the institutional and national research committee.All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures involving animals followed the ethical standards of the institution or practice at which the studies were conducted. All procedures related with the in vivo experiments and animal care were approved by the Committee for the Care and Use of Laboratory Animals at Yonsei University College of Medicine (no. 2020-0248) and were conducted according to the guidelines of the Care and Use of Laboratory Animals published by the US National Institutes of Health. We complied with the ARRIVE guideline for this reporting of animal experiment conducted in the present study.This article does not contain any studies involving human participants performed by any of the authors.The work described here has not been published before and is not under consideration for publication elsewhere. All authors have approved the manuscript.Supplementary Figures.


Background: The mortality of patients with non-small cell lung cancer (NSCLC) is rather high. This is largely because of the lack of specific targets and understanding of the molecular mechanism for early diagnosis. Dishevelled (Dvl) dysregulation leads to malignant progression. We confirmed that Dvl1 expression is associated with a poor prognosis of patients with NSCLC. However, how Dvl1 transmits signals through the Wnt/β-catenin pathway remains unknown.Methods: In this study, the expression levels of Dvl1 and β-catenin in resected NSCLC samples were immunohistochemically analysed. Dvl1 cDNA and small interfering RNA against β-catenin were transfected into NSCLC cells, and their effects on canonical Wnt signalling and biological behaviour of NSCLC cells were analysed. Using bioinformatics analyses, an interaction between microRNA (miR)-214 and β-catenin was identified; miR-214 expression was determined in NSCLC tissues using quantitative real-time polymerase chain reaction. An exogenous miR-214 (mimic) was used to analyse the biological behaviour of NSCLC cells and the effect of Dvl1 on canonical Wnt activation.Results: Dvl1 overexpression in NSCLC tissues as well as Dvl1 and β-catenin nuclear coexpression were significantly associated with poor prognosis of NSCLC (P < 0.05). Additionally, Dvl1 promoted Wnt/β-catenin signalling to enhance the malignant phenotype of NSCLC cells. Moreover, miR-214 directly targeted the 3′ untranslated region of β-catenin to inhibit the activation of canonical Wnt signalling induced by Dvl1.Conclusions: Our results suggest that Dvl1 is a potential therapeutic target for NSCLC and that miR-214 plays an inhibitory role in Dvl1-mediated activation of Wnt/β-catenin signalling in NSCLC cells, which could affect NSCLC progression. Lung cancer is a serious health problem worldwide MicroRNAs  are highly conserved, noncoding small RNAs that are 18-24-nucleotide long and play important regulatory roles in tumorigenesis. They interact with complementary target mRNAs to inhibit their translation. Several miRNAs, including miR-214 Wnt signalling regulates normal cellular development, but its dysregulation promotes tumorigenesis Dishevelled (Dvl), belonging to a family of phosphoproteins, relays signals that arise from all Wnt ligands to mediate tumour growth In this study, using TargetScan target prediction, we identified a potential interaction between miR-214 and the 3′-untranslated region (UTR) of β-catenin and elucidated the role of miR-214 in Dvl1-modulated Wnt/β-catenin signalling. The findings of this study might be valuable in the identification of new therapeutic targets for NSCLC.Twenty-four fresh NSCLC samples from patients who had undergone surgery were selected from the First Hospital of China Medical University in 2017. We excluded chemotherapy- and radiotherapy-treated patients. Corresponding paracancerous tissues (at least 5 cm from the lesion) were collected for comparison. The samples were frozen in liquid nitrogen and stored at -80 °C for quantitative real-time polymerase chain reaction (qRT-PCR) analysis.Tumour samples from 122 patients with NSCLC, who underwent surgical resection but did not undergo chemotherapy or radiotherapy prior to surgery, at the First Hospital of China Medical University between 2013 and 2015, were selected for immunohistochemical analysis.This study was conducted under the guidelines of the Institutional Review Board of China Medical University (No. 2016 [LS] 014). Written informed consent was obtained from all patients.Immunohistochemical procedures were the same as previously described 2 at 37 °C.The human NSCLC cell lines H1299, SPC, A549, and H157 and the human bronchial epithelial cell line HBE were obtained from the American Type Culture Collection . Short tandem repeat (STR) analysis was performed to test the authenticity of the cell lines. We selected A549 and H157, which have relatively low Dvl1 expression , for thiTotal RNA was extracted from NSCLC tissue specimens, paracancerous tissues, and NSCLC cell lines using TRIzol . qRT-PCR was performed as described previously CTNNB1 (accession no. NM_001098209.1) was amplified using PCR and subcloned into the pGL3 vector MCS cloning site . CTNNB1 was classified as either wild type (WT) or mutant , and pGL3-WT-CTNNB1-3′-UTR and pGL3-Mut-CTNNB1-3′-UTR plasmids were generated. The sequence for the CTNNB1 3′-UTR forward primer was 5′-TCTAGAATACAATGACTT TTTAGCTG-3′ and that for the reverse primer was 5′-TCTAGATTAGCCAAG-3′.The 3′-UTR of the β-catenin mRNA miR-214 mimic , mimic negative control , miR-214 inhibitor (5′-UGCCUGUCUGUGCCUGCUGUUU-3′), inhibitor negative control (5′-CAGUACUUUUGUGUAGUACAA-3′), and small interfering (si)RNA-β-catenin were obtained from Shanghai GenePharma Co., Ltd. . The Dvl1 expression vector pCS2-Myc-Dvl1 was provided by Dr. Zhenge Luo . NSCLC cells were transfected with the above plasmids or miRNAs  using Lipofectamine 2000 (Thermo Fisher). Transfected cells were incubated for 48 h at 4 °C for further experiments.Total protein extracted from NSCLC cells (50 μg) was quantified using the Bradford method and then separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After being transferred to polyvinylidene fluoride membranes, the membranes were blocked with 5% skim milk for 2 h at room temperature (24-26 °C) and then incubated overnight (4 °C) with primary antibodies against one of the following: c-Myc , Cyclin D1 , β-catenin , GAPDH , Myc tag , laminB1 , and α-tubulin . On the second day, we incubated the membranes with a horseradish peroxidase-labelled secondary antibody (Santa Cruz) for 2 h at room temperature. Subsequently, a BioImaging System  was used to analyse the protein bands densitometrically.The cells were fixed with 4% paraformaldehyde and incubated with primary antibodies against either Dvl1  or β-catenin  at 4 °C overnight and then incubated with goat anti-mouse IgG-FITC  at room temperature (24-26 °C) in the dark. We counterstained the nuclei with Hoechst 33258 (Thermo Fisher). The images were acquired using a confocal laser scanning microscope .5 cells/well) and then treated under specific conditions. We transiently co-transfected NSCLC cells with the TCF/LEF firefly luciferase construct (100 ng) and the Renilla luciferase vector (10 ng) . After incubation for 24 h at room temperature, luciferase activity was analysed using a Dual-Luciferase Reporter Assay System (Promega Corporation).NSCLC cells were seeded in 6-well plates  was used. Pearson correlation analysis was performed between the potential candidate miRNAs and CTNNB1.To predict the potential upstream miRNAs of t-test. Significant multiple-group differences were analysed using one-way analysis of variance (ANOVA). P < 0.05 was considered to indicate statistical significance.GraphPad 5.0 software  was used for statistical analysis. Results from at least three independent experiments were presented as mean ± standard deviation (SD). The correlation between miR-214 and β-catenin was analysed using Pearson's correlation analysis. Differences between two groups were assayed using Student's Dvl1 mRNA levels were significantly higher in NSCLC tissues than in paracancerous tissues (P < 0.05).Immunohistochemistry was applied to analyse Dvl1 expression in 122 NSCLC and 32 normal lung tissue samples. Bronchial and alveolar epithelial cells from normal lung tissue samples showed negative Dvl1 expression  but not significantly associated with sex, age, or lymphatic metastasis (P > 0.05). Dvl1 positivity was considerably higher in lung adenocarcinomas than in lung squamous cell carcinomas and significantly higher in stages II-III than in stage I (P < 0.05). Kaplan-Meier analysis suggested a notably lower survival period for patients with NSCLC with positive Dvl1 expression than for patients with negative Dvl1 expression  and nucleus . The coexpression rate for β-catenin and Dvl1 in the membrane was 15.4%  was significantly upregulated . After adding siRNA-β-catenin, Dvl1-induced biological behaviours, including invasion, proliferation, and migration, were significantly inhibited  or β-catenin-3′-UTR (WT). The luciferase activity in the cells transfected with β-catenin-3′-UTR and miR-214 mimic was significantly decreased compared with that in the control; however, cells transfected with the β-catenin/mut-3′-UTR and miR-214 mimic showed no significant changes in activity relative to control cells , axin, casein kinase Iα, and adenomatous polyposis coli miR-214 is a negative factor for canonical Wnt signalling, whereas Dvl1 is a positive factor. This indicates potential antagonistic effects between them. We found that Dvl1 reverses miR-214-mediated inhibition of Wnt/β-catenin signalling in NSCLC cells. Our results indicate that the interaction of miR-214 with β-catenin inhibits the Dvl1-induced activation of canonical Wnt signalling. In recent years, many databases have been developed, such as TargetScan and PicTar, facilitating bioinformatics analyses. Bioinformatics-based prediction indicates that miR-214 targets β-catenin at the 3′-UTR and regulates its expression. Our luciferase reporter assay confirmed this prediction in NSCLC cells.let-7 miRNA was associated with poor prognosis in NSCLC miRNAs detected in tissues, either down- or upregulated, are associated with various diseases. Takamizawa et al. discovered that the reduced expression of In summary, we confirmed that miR-214 binds to the 3′-UTR of β-catenin. Dvl1 activates canonical Wnt signalling by inhibiting the interaction of miR-214 with β-catenin and promoting stable β-catenin nuclear translocation in NSCLC cells, which contributes to pulmonary tumorigenesis. We showed that miR-214 is not only sufficient but also required for Dvl1-induced activation of the Wnt/β-catenin pathway, and the interaction between miR-214 and β-catenin is critical for this process. Notably, the inhibition of miR-214 and β-catenin interaction is critical for promoting Dvl1-induced β-catenin nuclear translocation and canonical Wnt signalling activation. Therefore, our results suggest that Dvl1 is a potential therapeutic target for NSCLC and that miR-214 plays an inhibitory role in Dvl1-mediated activation of Wnt/β-catenin signalling in NSCLC cells, which may affect the progression of NSCLC.Supplementary figures and table.Click here for additional data file.


The drug content was 96.69 ± 1.18% and 97.24 ± 1.27% for naproxen and eugenol, respectively. The maximum release of naproxen after 12 h was 85.14 ± 1.11%, whereas eugenol was 86.67 ± 1.23% from emulgel following anomalous non-Fickian mechanism. The maximum % permeation of naproxen across skin was 78.5 ± 1.30, whereas maximum % permeation of eugenol was 83.7 ± 1.33 after 12 h. The skin retention of eugenol and naproxen was 8.52 ± 0.22% and 6.98 ± 0.24%, respectively. The optimized emulgel inhibited the carrageenan induced paw edema. The pain reaction times of optimized emulgel and standard marketed product  were 11.16 ± 0.17 and 10.36 ± 0.47, respectively, with no statistically significant difference (p > 0.05). This study concluded that transdermal delivery of naproxen-eugenol emulgel synergized the anti-inflammatory and analgesic effects of naproxen and eugenol.The aim of this study was to fabricate and characterize a pharmaceutical emulgel co-loaded with naproxen/eugenol for transdermal delivery to improve the analgesic and anti-inflammatory effects and to eliminate GIT adverse reactions. Emulgel was prepared using a slow emulsification method and evaluated for physical appearance, thermodynamic stability, viscosity, pH, spreadability, extrudability, in-vitro drug release, drug content, ex-vivo permeation, drug retention studies and in-vivo studies. The emulgel exhibited good physical attributes, being thermodynamically stable with no phase separation, having excellent homogeneity, and pH 5.5 to 6.5. Slight changes in viscosity, spreadability and extrudability with respect to high temperature were observed ( Acute pain is an unacceptable and vigorous psychophysiological process, which may result in response to any injury in tissues or organs and involves many inflammatory progresses . Acute pNSAIDs  are the class of most widely used drugs for their proved efficacy against pain and inflammation. NSAIDs constitute 5 to 10% of all medication prescribed every year ,8,9. NapCinnamomum zeylanicum, Ocimum basilicum, Myristica fragrans, Houtt gratissimum, but its main source is Eugenia caryophyllata (Clove), which contains about 45 to 90% of eugenol [Eugenol is an aromatic hydroxyl phenolic compound and obtained from  eugenol . Eugenol eugenol . Eugenol eugenol .Drug products delivered through skin varies with respect to formulation ranging from liquid to powders in terms of consistency, but, among them, semisolid preparations gain more importance ,16. TopiIn this research, presenting a unique work rather than an old idea has been tried. Naproxen, a non-steroidal anti-inflammatory drug, belongs to the Biopharmaceutics classification system (BCS) class II drug where the bioavailability is rate-limited by its dissolution i.e., having low aqueous solubility. In this study, the aforementioned research gap is attempted to be filled by formulating it as emulgel. Emulgel can load a high quantity of drugs with slow release and high solubility. Moreover, co-loading of drugs in a single topically applied transdermal dosage form can provide better compliance as taking two separate drugs is not easy and patients get exhausted. It avoids the GIT side effects related especially to naproxen. It will reduce costs because formulating two drugs separately required separate excipients, human resources, productions, and marketing costs. This study was conducted on an R&D basis, so it provides basic insights to conduct large scale commercial studies. Therefore, this study was designed to fabricate and characterize a pharmaceutical emulgel of naproxen and eugenol for analgesic and anti-inflammatory effects using an animal model.The stability of formulation is an important feature that determines the product consistency, therapeutic efficacy, and quality of drug product under different storage conditions of temperature, humidity, and pH . The optp > 0.05).The pH could be considered as an essential parameter for effective delivery, stability, and compatibility of topical formulations for human skin ,19. The p < 0.05) difference in viscosity with respect to temperature and duration of time was observed at 5% level of significance. The decrease in viscosity at higher temperature was also noted by Burki et al., and they proposed that the decrease in viscosity may be due to migration of water molecules from a dispersed oil phase to a continuous aqueous phase, and because of overflowing of numerous droplets due to osmotic pressure [The viscosity plays a major role in the delivery of topically formulated drugs as it controls the release of drug at absorption site and thus ultimately influences the therapeutic efficiency of formulation . The ingpressure ,23.t-test p < 0.05).Spreadability is the property expressed to represent the extent of area to which formulation spread easily over the skin upon topical application . Al-Suwat-test considering a 5% level of significance (p < 0.05), the change in extrudability values of optimized formulation at different temperatures was significant (p < 0.05).Extrudability test is an important factor to be considered in the evaluation of semisolid dosage forms and is mainly dependent on the viscosity and consistency of formulation . The extThe drug content uniformity is an essential parameter which directly affects the therapeutic efficacy of any pharmaceutical dosage form and can be determined by the percentage assay of drug or active pharmaceutical ingredient . The perThe therapeutic efficacy of any drug depends upon the drug release from pharmaceutical dosage forms . The per2) closer to (1) [2 value of naproxen (R2 = 0.994) and eugenol (R2 = 0.992) are the highest in the Korsmeyer–Peppas model among all applicable models, so they obeyed the Korsmeyer–Peppas model. The value of N is greater than 0.5 for both drugs, i.e., Naproxen (N = 0.784) and Eugenol (N = 0.719), which indicates that the drug followed the anomalous non-Fickian, zero order release mechanism [The results obtained from the release kinetics of naproxen and eugenol by applying different drug release kinetics models were summarized in r to (1) . From thechanism .Drug permeation across skin is the main contributing factor for efficient delivery of drug via a transdermal route . The perThe drug retention in the skin can be considered as an important parameter for the treatment of local skin disorders . The perIn transdermal drug delivery systems, the active ingredients are sometimes associated with skin irritation if retained for a longer duration of time after application ,19. Thus® emulgel, GSK, Karachi, Pakistan) and test group (treated with optimized naproxen-eugenol emulgel) at different time intervals are summarized in p < 0.05) as compared to the control group, whereas the difference in standard and test group was insignificant (p > 0.05).The efficacy of emulgel in enhancing transdermal therapeutic effects of naproxen and eugenol from emulgel was evaluated using a hot plate method for determining in-vivo analgesic activity. The hot plate method is considered a central anti-nociceptive test for in-vivo studies . The mea®) and optimized naproxen-eugenol emulgel continued to decrease. The same phenomenon was observed by the Cong et al. when they evaluated the therapeutic intensity and duration of NSAIDs by assessing anti-inflammatory activities of naproxen and indomethacin; after 5 h, no anti-inflammatory activity was observed, and they concluded that NSAIDs inhibit the early signs of inflammation more effectively, and it is well known that NSAIDs had limited outcomes in chronic conditions [p < 0.05) inhibited and reduced edema in the hind paws of rats. However, the difference in percentage inhibition of standard group and test group was insignificant (p > 0.05). The dual (penetration enhancement and anti-inflammatory) properties of eugenol had not only improved the permeation of naproxen across skin but also synergized the in-vivo anti-inflammatory effects of optimized emulgel. Eventually, transdermal permeation of drugs stabilized in rationally designed and optimally characterized drug carriers, such as naproxen-eugenol emulgel, is a critical parameter to consider enhancing the effects of drugs  while minimizing their side-effects [For the assessment of in-vivo anti-inflammatory activity of experimental drugs, carrageenan induced edema could be considered a standard experimental model, which shows highly predictive results . Figure nditions ,39. Stat-effects ,41,42.From the obtained results, it can be concluded that optimized emulgel formulation with Carbopol and sepineo-p600 possessed desired thermodynamic and other physicochemical stability. The release pattern of the drug from optimized formulation followed the Korsmeyer–Peppas model. The present study has affirmed that the developed optimized formulation is a promising alternative for better pain management.® .All chemicals used were of analytical grade which include Naproxen Sodium , Eugenol and Tween-80 , Propylene glycol , Ethanol , Liquid paraffin , Carbopol 934 , Sepineo-P600 (Alternative Chemica (Pvt.) Ltd., Punjab, Pakistan), Methanol , Carrageenan  and VoltralA digital pH meter , Weighing balance , Magnetic stirrer  CO., LTD, Jintan, China), Water bath 1042 , Viscometer NDJ-8S, , Sonicator Bath , Franz diffusion cell , Vernier caliper , Refrigerator PEL PRLP-2350 , Stability chamber WS-150 , UV-visible spectrophotometer Shimadzu-1700  and HPLC Shimadzu-SPD-20AT  were used in the experiments.The naproxen-eugenol emulsion was prepared by the method adapted by Burki et al. with minor modifications . AqueousAccurately weighed 2 g of Carbopol 934 was dispersed in 100 mL distilled water with constant stirring at moderate speed and kept overnight to form a thick gel network. pH was adjusted to 5–6 by adding a few drops of triethanolamine.The emulsion was incorporated into a gel base in a 1:1 ratio until a viscous gel network of NEE was formed and pH was adjusted.Six different batches (Pre-formulation data not shown) of naproxen-eugenol emulgel were incubated at 25 °C for 28 days and evaluated for color change, homogeneity, creaming/phase separation and grittiness. Based on results of physical parameters, the optimized formulation was selected and further subjected to in-vitro, in-vivo and ex-vivo studies.The pH of optimized naproxen-eugenol emulgel formulation was determined by a digital pH meter which was stored at different temperatures , and, at the intervals of 0, 1, 2, 7, 14 and 28 days, the pH values were noted. The test was performed in triplicate, and average values were calculated .The viscosity of optimized formulations was determined according to a slightly modified method previously published . The visThe chemical assay of naproxen and eugenol in emulgel was determined by using a U/V spectrophotometer and HPLC respectively, as reported in literature . It has The standard stock solution of naproxen was prepared by taking 25 mg of naproxen in a 25 mL volumetric flask. It was dissolved in 10 to 15 mL of methanol and sonicated for 2–3 min. The remaining volume was made with methanol up to the mark. A required concentration, i.e., 30 µg/mL, was prepared by transferring 1.5 mL from stock solution into a 50 mL volumetric flask, and the final volume was made with methanol up to the mark. The standard solution was then filtered through 0.45-micron filter paper.max 331 nm:The sample stock solution of naproxen was prepared by taking 500 mg of optimized naproxen/eugenol emulgel equivalent to 25 mg of naproxen in a 25 mL volumetric flask. It was dissolved in 10 to 15 mL of methanol and sonicated for 10 min. The remaining volume was made with methanol up to the mark, filtered through a 0.45-micron filter. The required concentration, i.e., 30 µg/mL, was prepared by transferring 1.5 mL of this solution into a 50 mL volumetric flask, the final volume was made with methanol and maximum absorbance was noted at λThe mobile phase was prepared by mixing methanol and distilled water in a ratio of 60:40, which was then passed through a filter having a pore size of 0.45 microns . After filtration, the mobile phase was degassed by placing it in the sonicator for 10 min.The standard stock solution of eugenol was prepared by taking 25 mg of eugenol in a 25 mL volumetric flask. It was dissolved in 10 to 15 mL of methanol and sonicated for 2–3 min. The remaining volume was made with methanol up to the mark. The required concentration, i.e., 40 µg/mL, was prepared by transferring 1 mL from stock solution into a 25 mL volumetric flask, and the final volume was made with methanol up to the mark. The standard solution was filtered through 0.45-micron filter paper.The sample stock solution of eugenol was prepared by taking 1 g of optimized naproxen/eugenol emulgel equivalent to 10 mg of eugenol in a 50 mL volumetric flask. It was dissolved in 20 to 25 mL of methanol and sonicated for 10 min. The remaining volume was made with methanol up to the mark, filtered through a 0.45-micron filter. The required concentration, i.e., 40 µg/mL, was prepared by transferring 5 mL of this solution into a 50 mL volumetric flask, and final volume was made with methanol up to the mark.A C-18 column  was used for this procedure. Mobile phase consisted of methanol and water in a 60:40 ratio, and the flow rate was 0.8 mL/min (Isocratic flow). The injection volume was 20 μL, and the effluent was observed at 280 nm. The temperature was set at 30 °C and retention time was 5 min. A specified volume (20 µL) of standard and sample solutions was separately injected into the chromatograph. The chromatograms were recorded, and the responses of the individual components peaks in standard and sample eugenol solution were measured. The results were taken in triplicate, and average values were calculated. The percent assay of eugenol was determined by using the following formula:Spreadability of emulgel was determined by a drag and slip apparatus according to the method adapted by Akram et al. . The appM = weight tied to upper glass slide,L = length of glass slides,T = time taken to separate the glass slides from each other.The extrudability test is performed to measure the force that is required to extrude the emulgel from a collapsible tube. The evaluation of formulation extrudability was based upon the quantity of emulgel extruded from aluminum collapsible tubes by applying weight in grams required to extrude at least a 0.5-cm ribbon of emulgel from tube in 10 s. This test was performed in triplicate, and average values were calculated .The extrudability of emulgel was then calculated by using the following formula:The in-vitro drug release studies of optimized formulation were carried out using a Franz diffusion cell. The cellulose acetate membrane  having a pore size of 0.45 µm was clamped between the donor and receptor compartment. Prior to the loading of formulation, the receptor compartment was filled with a 5 mL phosphate buffer pH 5.5, and temperature was maintained at 37 ± 1 °C under constant stirring at 300 rpm. About 1 gram of optimized emulgel equivalent to (naproxen = 100 mg and eugenol = 20 mg) was placed on the donor compartment. The samples were drawn at specific time intervals, i.e., at time 0, 1, 2, 4, 8 and 12 h, and were replaced with 2 mL fresh phosphate buffer to maintain the sink condition. All the samples were then analyzed by a UV visible spectrophotometer at 282 and 331 nm for eugenol and naproxen, respectively.The data obtained from in-vitro drug release studies were evaluated for kinetics of drug release from the naproxen-eugenol emulgel by employing the different mathematical kinetic models such as zero order kinetic, first order kinetic, the Korsmeyer–Peppas model, the Hixson–Crowell model, the Higuchi model, and the model having a high correlation co-efficient was considered the best fitting model.For ex-vivo studies, permission for animal use was granted from the institutional ethical review board (ERB) of Gomal University Dera Ismail Khan, Pakistan under reference No. 851/QEC/GU. All the protocols were followed according to the NIH guidelines for lab animals.The ex-vivo permeation study was carried out using a Franz diffusion cell. The excised rat skin, previously refrigerated, was first hydrated with normal saline at least 1.5 h before experiment and then clamped between the donor and receptor compartment of the Franz diffusion cell in such a way that the epidermis layer of skin interacts with the donor compartment. Prior to the loading of formulation, the receptor compartment was filled with phosphate buffer pH 7.4. About 1 g of emulgel was placed on the donor compartment, and a 2 mL sample was drawn at specific time intervals, i.e., at time 0, 1, 2, 4, 8 and 12 h, and was replaced with 2 mL of fresh phosphate buffer to maintain the sink condition. All the samples were then analyzed by a UV visible spectrophotometer at 282 and 331 nm for eugenol and naproxen, respectively.The skin was removed from a Franz diffusion cell and washed with phosphate buffer (7.4) to remove the drug on the surface of skin. The skin was then cut into small pieces and dropped into the beaker containing methanol to extract the drugs from skin and stirred for 2 h. The solution was filtered, and samples were analyzed by a UV visible spectrophotometer at 282 and 331 nm for eugenol and naproxen retention, respectively.n = 4). Control group was treated with blank emulgel, and the test group was treated with naproxen-eugenol emulgel. The blank emulgel and drugs loaded emulgel were applied over the hairless rat’s skin at intervals of 8 h. After 24 h, the skin was washed with water, the signs of irritation (erythema and edema) were observed for the period of 0, 1, 2 and 3 days, and sensitivity reactions were scored as 0 (no reaction), 1 (slightly erythema), 2 (moderate erythema) and 3 (severe erythema with or without edema). The total score for skin irritation was calculated using the following equation:For the skin irritation study, rats were divided into control and test groups containing 4 rats in each group  as follows:Group 1: Control group, Treated with 0.1 mL of 1% carrageenan injection only,Group 2: Test group, Treated with optimized naproxen-eugenol emulgel + 0.1 mL of 1% carrageenan injection.Group 3: Standard group, Treated with marketed emulgel  + 0.1 mL of 1% carrageenan injection.For in-vivo anti-inflammatory activity, animals were divided into 3 groups containing 6 rats in each group  as follows:Group 1: Control group, no treatment was given,Group 2: Test group, Treated with optimized naproxen-eugenol emulgel,Group 3: Standard group, Treated with marketed diclofenac emulgel .For in-vivo analgesic activity, animals were divided into 3 groups containing 6 rats in each group (t-test considering p < 0.05 as statistically significant.All the experiments were performed in triplicate, and the results were expressed as average ± sd. The statistical analysis was carried out by using ANOVA and the Student’s


To investigate the catalytic mechanism of IKKβ, we performed pre–steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32 s−1 and the binding affinity of ATP for the IKKβ•IκBα complex (Kd) was 12 μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1–54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event.Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser Dysrpoptosis , 4, 5. TThe mammalian NF-κB family consists of RelA p65), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2), which form 15 separate homodimeric or heterodimeric complexes 5, RelB, ).The IKK complex consists of the nonenzymatic protein NEMO  and a homodimer or heterodimer of the catalytic subunits IKKα and IKKβ , 11, 12.A) consisting of an N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (A). This activation loop is essential for the activity of IKKβ as mutational analysis indicates that changing these Ser residues to Ala prevents IKKβ activation, whereas substitution of these critical Ser residues with phosphomimetic Glu residues renders the kinase constitutively active  during a single binding event. Our study also suggests that IKKβ preferentially phosphorylates IκBα sequentially at Ser32, followed by Ser36. We conclude that these aspects of the kinetic mechanism of IKKβ may be shared among the IKK and IKK-related kinases.Although the phosphorylation of IκB proteins by IKKβ is a critical step within the canonical pathway of NF-κB activation, the molecular mechanism of IKKβ is poorly understood. Here, we used pre–steady-state kinetic analysis to investigate the molecular mechanism of IκBα phosphorylation catalyzed by constitutively active IKKβ. Our data indicate that IKKβ phosphorylates full-length IκBα twice within the conserved DSGXXS destruction box motif C during A) is significantly increased upon phosphorylation of activation loop residues Ser177 and Ser181 within the kinase domain (32 and Ser36 (B and C) to induce the proteolytic degradation of IκBα and the subsequent release of NF-κB dimers. Importantly, IκBα is also phosphorylated at other sites in vivo, particularly within the C-terminal PEST domain (B)  for the IKKβ•IκBα complex and the maximum observed rate (kp) of IKKβ-catalyzed phosphorylation were determined by using pre–steady-state kinetic analysis. To this end, we measured the ATP concentration dependence of the phosphorylation rate of IKKβ under conditions where IKKβ was in a 4-fold molar excess compared to the IκBα substrate to ensure that nearly all of the IκBα substrate was initially bound by the kinase. IKKβ has previously been shown to bind IκBα with an equilibrium dissociation constant (Kd) of 56 nM . The resulting plots indicated that IKKβ  phosphorylated IκBα with an initial, fast exponential phase rate (ke), followed by a significantly slower linear phase rate (kl) and therefore, these data were fit to biphasic Equation Figure 4A when longer reaction time points were included in the plot. We hypothesized that the initial exponential rate corresponded to phosphorylation of IκBα at residues Ser32 and Ser36 within the destruction box motif, and the significantly slower linear rate resulted from either the phosphorylation of IκBα at sites outside of the destruction box motif, such as the PEST domain (B), or phosphorylation of IκBα substrate that was not initially bound in a productive complex by IKKβ, or both. The exponential rates of IKKβ-catalyzed phosphorylation of IκBα were then plotted as a function of ATP concentration and fit to hyperbolic Equation Fig. 3B), yielding a kp of 0.32 ± 0.01 s−1 for the maximum observed rate of IKKβ-catalyzed phosphorylation of full-length IκBα and a Kd of 12 ± 1 μM for ATP binding to the IKKβ•IκBα complex.A preincubated solution of full-length IκBα and IKKβ  was rapidly mixed with a solution containing increasing concentrations of ATP from MP Biomedicals, ATP from Thermo Fisher Scientific, Optikinase from USB corporation, and plasmid pBluescript II KS + from Agilent Technologies.Reagents were purchased from the following companies: ATP  and a linearized, 5′-[32P]-radiolabeled pBlueScript loading control (250 CPM/μl). The reaction mixtures were quenched at various times by the addition of EDTA to a final concentration of 375 mM. Reactions were carried out by using a rapid-chemical quench flow apparatus (KinTek). Reaction products were resolved by using SDS-PAGE (12% polyacrylamide) and quantified by using a Typhoon TRIO . The detected amount of [32P]-radiolabeled IκBα was normalized to the radiolabeled pBlueScript internal standard at each time point, and the normalized data was plotted as a function of time. The time courses of product formation at each nucleotide concentration were fit to biphasic Equation A is the exponential phase amplitude, t is the reaction time, ke is the exponential phase rate, and kl is the linear rate  of IκBα phosphorylation catalyzed by IKKβ  and the binding affinity of ATP for the IKKβ•IκBα complex (Kd)  .(2)ke=kp32P]ATP  and a linearized, 5′-[32P]-radiolabeled pBlueScript loading control (250 CPM/μl). The reaction mixtures were quenched and product formation was quantified as described above. The time courses of full-length IκBα phosphorylation were then fit to biphasic Equation A preincubated solution containing IKKβ (3 μM) and either full-length IκBα wt (0.75 μM) or the indicated IκBα amino acid substitution mutant (0.75 μM) was rapidly mixed with a solution containing [γ-A is the exponential phase amplitude, t is the reaction time, and ke is the observed exponential phase rate.The pre–steady-state kinetic parameters of the GST-IκBα(1–54) substrates were determined as described for the full-length IκBα substrates above. However, the IKKβ phosphorylation of the GST-IκBα(1–54) substrates lacked a clearly defined linear rate. Therefore, the time courses of GST-IκBα(1–54) product formation were fit to single-exponential Equation The article contains all data described within the text.The authors declare that they have no conflicts of interest with the contents of this article.


Abnormal expression of p120 catenin is associated with the malignant phenotype in human lung cancer. Numerous studies have focused on the function of p120 catenin in the juxta-membrane compartment. However, the role of nuclear p120 catenin remains unclear. In this study, the dynamic changes in nuclear p120 catenin localization during cell cycle progression were investigated. Immunofluorescent staining, FACS analysis, and western blotting revealed that nuclear p120 catenin is a major architectural constituent of the chromosome periphery during mitosis. During mitosis, granule-like p120 catenin dispersed into a cloudy-like structure and formed cordon-like structures surrounding the condensed chromosomes to create the peri-chromosomal layer. Interestingly, lumican and p120 catenin colocalized at the spindle fiber where the perichromosomal layer connects to the condensed chromosomes during mitosis. Furthermore, downregulation of p120 catenin using a specific siRNA induced cell cycle stalling in the G2/M phase and promoted aneuploidy. This study validates the role of nuclear p120 catenin in the formation of the chromosome periphery and reveals the p120 catenin-lumican interaction may couple orientation of cell division with the segregation of sister chromatids during mitosis. Our data suggest the protective role of p120 catenin in maintaining the integrity of chromosomes, and also warrants further studies to evaluate the contribution of the loss of p120 catenin to the creation of gene rearrangement in cancer evolution and tumor progression. Lung cancers, the leading cause of cancer-related mortality worldwide, are characterized by extensive genomic alternations within the tumors . AneuploP120 catenin is a member of the Armadillo gene family that has emerged as a major regulator of cadherin stability and an important modulator of the activity of small GTPases . SimilarIn this study, we explored the mitotic role of nuclear p120 catenin in lung cancer cells. We demonstrate that p120 catenin functions at the chromosome periphery that coats mitotic chromosomes during mitosis. Furthermore, we show nuclear p120 catenin interacts with lumican, a tubulin-binding protein, to coordinate the separation of sister chromatids during the mitotic phase. Depletion of p120 catenin was accompanied by reduced expression of lumican and led to aneuploidy associated with unpredictable chromosome instability. Thus, our data indicate an emergent role for p120 catenin in the modulation of the segregation of sister chromatids during mitosis.TM) and A549 (CCl-185TM), were purchased from the American Type Culture Collection, USA.Both non-small lung cancer cell lines used in this study, H460 (HTB-177CTNND1 (siRNA 5′-GCUAUGAUGACCUGGAUUA-3′ and 5′-CUAUGAUGACCUGGAUUAU-3′) [Unless otherwise noted, all chemical reagents were purchased from Sigma-Aldrich. The antibodies used in the present study were: lumican , p120 catenin , and α-tubulin . The sequences of the p120 catenin siRNAs target the coding sequence of the gene encoding p120 catenin, UUAU-3′) .We adopted the double thymidine synchronization approach to perform cell cycle analysis . BrieflyTo perform confocal microscopy, lung cancer cells were cultivated in chamber slides for 24 h, fixed in 4% formaldehyde at room temperature, rinsed with PBS, then incubated with a combination of anti-lumican/anti-p120 catenin/anti-α-tubulin antibodies at 4 °C overnight. Following 4’,6-diamidino-2-phenylindole  staining for 1 min, the samples were imaged by confocal microscopy  with the pinhole set at 1 Airy unit under 488, 546, and/or 633 nm excitation using a 63–100/1.4 oil objective lens. The samples were imaged by confocal microscopy  with the pinhole set at 1 Airy unit under 488, 546, and/or 633 nm excitation using a 63–100/1.4 oil objective lens. To avoid saturation of the fluorescence intensity in the scanned images, we optimized the detector settings using the feature Range Indicator provided by the Zeiss software .g for 10 min at 4 °C, and 0.3 mL of the sonicated cell lysates were collected. The Bio-Rad Protein Assay Kit was used to determine the protein concentrations and the cell lysates were resolved by SDS–PAGE and blotted by appropriate antibodies following standard protocols.Cells were lysed in cold lysis buffer  containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific), centrifuged at 12,000×t-test to evaluate the significance of the differences between groups. A p-value < 0.05 was considered to indicate a significant difference.All data are presented as mean and standard deviation. We adopted the unpaired Student’s P120 catenin is enriched in lung cancer cells and associated with the stability of cadherin, adhesion-induced signaling, and cell proliferation . To explOur previous study has shown the subcellular distribution of p120 catenin investigated by western blot . In addiThe dynamic changes in nuclear p120 catenin observed in mitotic cells in different phases of the cell cycle were investigated by immunostaining with anti-p120 catenin and anti-α-tubulin antibodies Fig. . At inteLumican, a tubulin-binding protein, is crucial for the formation of spindle fibers and the assembly of midbodies during cell division . Our preTo further investigate the role of p120 catenin in the perichromosomal layer, we depleted p120 catenin using specific siRNAs (p120si) and performed immunostaining 24 h later using anti-p120 catenin and anti-lumican antibodies. As shown in Fig. To investigate whether depletion of p120 catenin induced mitotic defects, we visualized the nuclei of the NCi- and p120si-treated cells using DAPI staining. Various sizes of rounded micronuclei were observed in the p120si-transfected cells Fig. . The numP120 catenin engages multifaceted functions associated with its various subcellular distribution . NuclearLung cancer is a spectrum of diseases caused by numerous alterations in expression patterns resulting from alterations in various genetic and epigenetic mechanisms . DependiTargeting of p120 catenin to the perichromosomal layer occurred during prophase to telophase in the processes of mitosis Figs. . Our p12Lumican is overexpressed in lung cancer cells and implicated in tumorigenesis and regulation of cancer cell invasion . Our priOur previous works showed that depleting lumican by its siRNA/sgRNA decreased tubulin’s expression level, which led to aneuploidy/cell apoptosis . Here weOur previous works and others showed that p120 catenin is associated with lumican and microtubules to regulate microtubule stability , 9. TherLike p120 catenin, other scaffold proteins, such as CUB-domain containing protein 1 (CDCP1), function as a protein–protein interaction hub that interfaces with the signaling proteins and structural elements that also control cell–cell/cell–substratum adhesion, but not the nucleus event of mitosis , 20. InsMost solid cancer cells are aneuploid, as they display chromosome number alterations including the loss and gain of complete chromosomes . ChromosOur previous study has confirmed the physical interaction between p120 catenin and lumican and their co-localizations in the juxtamembrane compartment by co-immunoprecipitation and co-immunofluorescence, respectively . The preMost importantly, we verified that lumican also plays an essential role in facilitating the faithful sister chromatid’s separation through the physical interaction with the cordon-like p120 catenin. This finding offers a mechanical function of the p120 catenin-mediated cordon-like structure with a mitotic spindle. Consistently, downregulation of p120 catenin decreased the formation of both the perichromosomal layer and spindle fibers due to reduced expression of lumican  Fig. 4)4). FurthLumican belongs to the leucine-rich repeats (LRRs)-containing protein (LRP) . LeucineMicronuclei arose from lagging chromosomes or chromosome fragments caused by mitotic errors or DNA damage . DelayedThe consequences of micronuclei formation on tumor biology are dependent on the frequent and irreversible rupture of their nuclear envelopes, which results in the exposure of their DNA contents to the cytoplasm . NotablyChromosomal instability may facilitate tumor plasticity and adaptation to versatile environments through Darwinian selection processes, and positively correlates with poorer patient prognosis . Loss ofTogether, this study verified a novel role for p120 catenin in the mitotic chromosome periphery and sister chromatid segregation. P120 catenin may function in the perichromosomal layer as a sheath to promote the efficient separation of condensed chromosomes. Moreover, we demonstrate that the interaction of p120 catenin with lumican regulates the microtubule dynamics of the mitotic spindle during mitosis. These activities affect the segregation of sister chromosomes during mitosis, independently of the cadherin adhesion system. Importantly, this study provides new insight into the role of p120 catenin as a guardian of the segregation of sister chromatids during mitosis. Further studies regarding p120 catenin expression, subcellular localization, and chromosome instability in various cancer cell types are currently under investigation.Original Data FileSupplementary Figure legendSupplement Figureaj-checklist


Accumulating evidence supports the existence of glioma stem cells (GSCs) and their critical role in the resistance to conventional treatments for glioblastoma multiforme (GBM). Differentiation therapy represents a promising alternative strategy against GBM by forcing GSCs to exit the cell cycle and reach terminal differentiation. In this study, we demonstrated that cAMP triggered neuronal differentiation and compromised the self-renewal capacity in GSCs. In addition, cAMP induced negative feedback to antagonize the differentiation process by activating β-catenin pathway. Suppression of β-catenin signaling synergized with cAMP activators to eliminate GSCs in vitro and extended the survival of animals in vivo. The cAMP/PKA pathway stabilized β-catenin through direct phosphorylation of the molecule and inhibition of GSK-3β. The activated β-catenin translocated into the nucleus and promoted the transcription of APELA and CARD16, which were found to be responsible for the repression of cAMP-induced differentiation in GSCs. Overall, our findings identified a negative feedback mechanism for cAMP-induced differentiation in GSCs and provided potential targets for the reinforcement of differentiation therapy for GBM. As one of the most prevalent primary intrinsic brain tumors in adults, glioblastoma multiforme (GBM) is a universally lethal cancer with a median survival of less than 2 years . GBMs coDifferentiation therapy with cytokines or chemical agents is a promising alternative strategy to treat cancers by forcing self-renewed tumor cells to a mature post mitotic state . The not5 cells/well) [GSC1 and GSC11 cell lines were derived from human GBM tumor samples and were characterized as reported previously  . The fol3 cells per well. GSCs were treated with designated agents. After 72 h incubation at 37 °C in a humidified 5% CO2 atmosphere, three pictures were taken per condition at 40× magnification and at least 10 newly formed neurospheres diameter were measured per condition under microscopy . Single cells were not included into quantification.Dissociated GSCs were seeded in 48-well flat-bottomed plates at a density of 4 × 10statmod package in R software.Limiting dilution assay was performed as described previously , 22. GSC2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 10 μM pepstatin, 10 μg/mL aprotinin, 5 mM iodoacetic acid, and 2 μg/mL leupeptin. Equal amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes . The membranes were then probed with the following primary antibodies: CD133 , SOX2 , β-catenin , Ser675-phosphorylated β-catenin , Ser9-phosphorylated GSK3β , GSK3β , PKA , GAPDH , and β-actin . Immunoreactivity was visualized by probing with the HRP-conjugated secondary antibody  and detected using the ECL kit . Western blot band intensities were quantified through densitometric analysis using ImageJ and normalized the band intensities with the loading control band. Original uncropped WB are given in Supplementary Material.Soluble proteins from treated cells were harvested and lysed in solution containing 50 mM HEPES (pH 7), 150 mM NaCl, 1.5 mM MgClFlow cytometry (FCM) analysis was performed by using a Gallios flow cytometer (Beckman Coulter). For cell proliferation assessment, the cells were incubated with 10 mM EdU (Sigma-Aldrich) for 2 h, and EdU was chemically conjugated to 50 mM Auto 488 (Sigma-Aldrich) for 1 h and incubated with 1 mg/mL DAPI for another 30 min. For neuronal markers analyses, cells were collected after treatment with vehicle or cAMP activators, and then incubated with 1:100 dilution of MAP2  and 1:100 dilution of TUJ1  for 1 h.Cells cultured as monolayers were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde/PBS for 20 min at room temperature, and then permeabilized in 0.2% Triton/PBS for 10 min. After blocking in 2% bovine serum albumin/PBS at room temperature for 30 min, cells were incubated with the following primary antibodies: β-catenin , MAP2 , and TUJ1  for 1 h at room temperature or overnight at 4 °C. Then, cells were washed three times with PBS for 10 min each and incubated with secondary antibody  for 1 h at room temperature. After washing with PBS, cells were incubated with DAPI for 5 min and then washed three times with PBS and rinsed with ddH2O once. Immunofluorescent imaging was performed on a Nikon A1 confocal microscope. ImageJ software was used to assess immunofluorescence, with thresholds set according to signal intensity. The fluorescence intensity of TUJ1 and MAP2 in approximately 30 GSC cells from random fields was quantified and the mean fluorescence intensity was calculated as Brain tissue from mice were quickly dissected and fixed in 4% paraformaldehyde overnight, then dehydrated with ascending grades of ethyl alcohol, and cleaned in xylene and embedded in paraffin, and sectioned at a thickness of 4 μm. Immunohistochemical (IHC) staining was performed as follows. According to the manufacturer’s instructions of the IHC staining kit . Slices were placed with antigen retrieval for 30 min, and in 3% hydrogen peroxide for 15 min after the gradients of deparaffinization and hydration. Cooling to room temperature, slices were incubated with indicated primary antibodies against Ki-67  and MAP2  overnight at 4 °C, and then incubated with an HRP-conjugated secondary antibody  at room temperature. Washing with PBS three times, slices were stained with a Diaminobenzidine (DAB) substrate–chromogen mixture in turn. Images were captured using an inverted microscope . For quantification, the integrated optical density (IOD) of the IHC results were analyzed using ImageJ software and the relative IOD values between groups were compared.Specific siRNAs targeting CTNNB1, PKA, CARD16, APELA, and MSLN were purchased from RiboBio . siRNAs were transfected using Lipofectamine RNAiMAX  with opti-MEM (Life Technologies) following the manufacturer’s instructions.−ΔΔCt method.Total RNA was extracted using TRIzol  reagent and the concentration of RNA was detected by a NanoDrop2000 spectrophotometer (Thermo Fisher). RNA was reverse transcribed using a PrimeScrpt RT reagent kit. Specific gene expression was quantified with SuperReal PreMix SYBR Green (Tiangen) using an Applied Biosystems 7500 fast real-time PCR system (Life Technologies). All reactions were performed in triplicate, and human actin was served as the endogenous control. The results were analyzed via the 2http://genome.ucsc.edu/) using TOPHAT (v2.0.11) [http://tophat.cbcb.umd.edu/). Default Tophat settings were used, except for the redefined parameter ‘–mate-inner-dist200’. The inner distance between pair ends was estimated by the PICARD program (http://picard.sourceforge.net). The HTSeq program was used to count mapped reads with the parameters ‘-s no –a 20’ [Raw reads were trimmed to 90 nucleotides in length and were mapped to the UCSC Genome Browser Database hg19 reference genome  (http://v2.0.11)  (http://o –a 20’ . Genes wo –a 20’ .http://geneontology.org/), Kyoto Encyclopedia of Genes and Genomes (KEGG), and the Ingenuity Pathways database  [p values from the GSEA were corrected for multiple testing by false discovery rate (FDR). Pathways with corrected p values of less than 0.05 were considered significant.The Piano package  was usedity.com) , 31. RawMetascape was applied to analyze the functional enrichment .5 GSCs. After 5 days, palpable tumors developed (50 mm3), and the mice were randomly divided into six groups (n = 8). These six groups were injected intraperitoneally once per day with vehicle, 20 mg/kg dbcAMP, 20 mg/kg.Mice were housed in a pathogen-free animal facility. The hind flanks of 4-week-old female BALB/c-nu/nu mice were subcutaneously inoculated with 1 × 103) = width2 × length/2. Tumors were dissected and fixed in formalin for immunohistochemical analysis. Data are means ± SD of 8 mice/group.Luteolin, 5 mg/kg FH535, dbcAMP+FH535, or Luteolin+FH535 respectively for 14 days. Tumor diameters were measured every other day with a caliper, and tumor volumes were estimated using the following formula: V , 5 mg/kg FH535, or dL+FH535, once per day for 14 days. The mice were monitored daily and sacrificed when neurological symptoms were observed. Their brains were then dissected and fixed in formalin for H&E staining. A Kaplan–Meier survival curve was generated using the GraphPad Prism 6.0 software package . The points on the curves indicate glioma-related deaths .The orthotopic implantation of glioma cells was performed using 5 × 10t test  or ANOVA followed by LSD post hoc test using the SPSS 20 statistical software package . Statistical significance was defined as p values ≤ 0.05.Statistical differences were evaluated with Student’s Previous studies have reported that the activation of cAMP induces differentiation in the established glioma cell lines , 34. To To determine whether the cAMP-induced differentiation is accompanied by the impairment of stemness in GSCs, we initially evaluated the effect of cAMP activation on the neuroshpere formation of GSCs. Quantification analysis demonstrated that the size of neurospheres was reduced by more than 50% when GSCs were treated with dbcAMP or forskolin Fig. . We nextThe mechanisms underlying cAMP-induced neuronal differentiation in GSCs have not been fully elucidated. To investigate pathways involved in the differentiation process, we explored the signaling signatures in GSCs incubated with dbcAMP. We identified that genes closely related to the Wnt/β-catenin pathway and those enhancing nuclear translocation of β-catenin were enriched in dbcAMP-treated GSCs Fig. . The proPKA as a key downstream effector of cAMP has been found to inhibit the ubiquitination of β-catenin by phosphorylating the molecule at Ser675 . To deteβ-catenin has been found to promote differentiation in stem cells, although conflicting reports also exist , 39. To To explore whether the cooperative effect of β-catenin deactivation and cAMP agonists occurs in vivo, we implanted GSCs subcutaneously in immunocompromised mice. After the establishment of tumors, mice were treated with vehicle, cAMP activators (dbcAMP or Luteolin), FH535, and combined cAMP activators with FH535, respectively. Monotherapy with the agents mentioned above mildly retarded the glioma growth whereas a more notable decrease in tumor size was observed in mice administered with cAMP activators and FH535 in combination Fig. . The bodTo understand the biological function of genes downstream of β-catenin in cAMP-treated GSCs, we firstly analyzed the gene expression profiles of GSCs treated with dbcAMP in the presence of silencing RNA against β-catenin. A total of 749 differential expression genes  were found after β-catenin knock-down in GSCs treated with dbcAMP, compared to those incubated with dbcAMP alone Fig. . The genGBMs are the most common malignant primary brain cancers in adults with devastating outcomes. Most of the current treatments mainly focus on the glioma bulk mass and fail to be curative . High ceThe role of cAMP in regulating differentiation in gliomas has been established for more than four decades. The early observation that cAMP promotes morphological and biochemical changes of glioma cells towards mature glia elicited the interest in the feasible application of differentiation therapy in gliomas , 44. SubWnt/β-catenin signaling is a critical mediator that regulates embryonic development and tissue homeostasis. β-catenin has been found to be essential for cell fate determination and differentiation. Knockout of β-catenin is lethal to mouse embryonic stem cells (mESC) due to the failure of mESC to develop the endodermal and mesodermal germ layers . In addiWe demonstrated that APELA and CARD16 are potential downstream genes of β-catenin, which account for the inhibition of cAMP-induced differentiation in GSCs. Apelin early ligand A (APELA), also called Toddler or ELABELA, is a peptide hormone that binds the G protein-coupled receptor apelin receptor (APLNR) and acts as a critical regulator of gastrulation and cardiovascular development in zebrafish , 53. In The caspase recruitment domain (CARD) is a member of death domain (DD) superfamily that modulates protein-protein interaction. CARD has been found in a diversity of adaptor proteins and regulates crucial signaling pathways involved in caspase activation, inflammasome assembly and innate immune response . Among mIn conclusion, we identify a negative feedback mechanism in differentiation therapy for GSCs. cAMP agonists induce neuronal differentiation and simultaneously inhibit the degradation of β-catenin through PKA and GSK3β. The activated β-catenin translocates into the nucleus and promotes the transcription of APELA and CARD16, facilitating the maintenance of the stemness and compromise differentiation in GSCs. Suppression of β-catenin signaling dramatically improves the efficacy of differentiation therapy both in vitro and in vivo. Our findings enhance the understanding of the interaction of signaling pathways in cAMP-induced differentiation and provide attractive targets for the reinforcement of differentiation therapy for GBM.Figure S1Figure S2Figure S3Figure S4Figure S5Supplementary figure legendsReproducibility checklistOriginal Data of WB


Ubiquilin-2 (UBQLN2) mutations lead to familial amyotrophic lateral sclerosis (FALS)/and frontotemporal dementia (FTLD) through unknown mechanisms. The combination of iPSC technology and CRISPR-mediated genome editing technology can generate an iPSC-derived motor neuron (iPSC-MN) model with disease-relevant mutations, which results in increased opportunities for disease mechanism research and drug screening. In this study, we introduced a UBQLN2-P497H mutation into a healthy control iPSC line using CRISPR/Cas9, and differentiated into MNs to study the pathology of UBQLN2-related ALS. Our in vitro MN model faithfully recapitulated specific aspects of the disease, including MN apoptosis. Under sodium arsenite (SA) treatment, we found differences in the number and the size of UBQLN2+ inclusions in UBQLN2P497H MNs and wild-type (WT) MNs. We also observed cytoplasmic TAR DNA-binding protein  aggregates in UBQLN2P497H MNs, but not in WT MNs, as well as the recruitment of TDP-43 into stress granules (SGs) upon SA treatment. We noted that UBQLN2-P497H mutation induced MNs DNA damage, which is an early event in UBQLN2-ALS. Additionally, DNA damage led to an increase in compensation for FUS, whereas UBQLN2-P497H mutation impaired this function. Therefore, FUS may be involved in DNA damage repair signaling. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by the selective loss of upper and lower motoneurons ,2. PatieALS-related UBQLN2 mutations were identified in 2011, including P497H, P497S, P506T, P509S, and P525S .To date, many findings have been obtained using the iPSC-MN model, including exploring pathogenic mechanisms, drug screening, and the search for new therapeutic targets ,10,11,12The mechanism of protein aggregation and its clearance has been widely considered ,14. ReceP497H iPSC-MNs were recruited into SG and the UBQLN2 formed aggregates that were more difficult to degrade than WT MNs under oxidative stress. Thus, the P497H-mutant MNs recapitulated the hallmark features of the human disease, including neuron apoptosis and protein aggregation, which may be exploited to study UBQLN2 pathology. In addition, increased DNA damage was already visible in MNs prior to UBQLN2 and TDP-43 protein aggregates and neuron apoptosis, suggesting that DNA damage is an early event in the pathophysiology of UBQLN2-ALS. The level of FUS increased as DNA damage increased, which supports the findings of previous studies that FUS was involved in DNA damage repair [Here, we introduced a UBQLN2-P497H mutation in the genome of a healthy control iPSC line using CRISPR/Cas9. UBQLN2-P497H mutation caused age-dependent axon damage and MN death through an apoptotic-like pathway involving capsase3. The TDP-43 of UBQLN2e repair . We noteWe introduced a UBQLN2-P497H mutation in a healthy control iPSC line using CRISPR/Cas9. Homologous sgRNA and a single-stranded oligo donor (ssODN) are shown in P497H iPSC line expressed high levels of pluripotency markers OCT4, SOX2, SSEA4, NANOG, and TRA-1-60 , mesoderm (cartilage), as well as endoderm   G.P497H iPSC lines into MNs using a small-molecule cocktail [+/OTX2+-induced neuroepithelial progenitors (iNEPs) at day 6 (+-induced motor neuron progenitors (MNPs) at day 12  in all analyzed MN cultures   B. Howevet day 30 B, support day 30 C,D, recaactivity E. Aging P497H MNs, we focused on axonal changes in the MNs during in vitro maturation and aging. We observed that the increased apoptosis of UBQLN2P497H MNs was also accompanied by morphological changes, which were consistent with the observation of postmortem spinal cord tissue in ALS patients [P497H MNs exhibited damaged morphologies of fragile axons (SMI32) with more thin branchings (indicated as “damaged”)  both in controls and UBQLN2-P497H under SA treatment (10 μM for 24 h)  C. IntereMNs only C,D. AfteMNs only C. OveralWe next sought to determine whether ALS iPSC-MNs endogenously develop UBQLN2 protein aggregates, as this is an important phenotypic hallmark of UBQLN2-related ALS pathology . We obseP497H MNs in terms of the stress response, MNs were exposed to oxidative stress through treatment for 0.5 h with SA (0.5 mM). We found differences in the number and size of UBQLN2 aggregates (>0.1 μm2)  A. Our ime stress B.P497H MNs, whereas UBQLN2P497H MNs still showed more and larger UBQLN2 aggregates in comparison with WT MNs , ATP-binding cassette-ATPase (RAD50), and phosphopeptide-binding Nijmegen breakage syndrome protein 1 (NBS1), checkpoint kinase 2 (Chk2), etc.) will be recruited to the DNA damage site to play their repair role . Here, wiPSC-MNs F–H. TherCurrently, the pathogenic molecular mechanism of ALS is not entirely known. Although significant progress has been made in research using transgenic mice, the relevance of these findings to human ALS has been questioned due to genetic differences between rodents and humans and the fact that most rodent models overexpress the mutant proteins ,33. ThusBy analyzing the expression of SMI32 and ChAT, we confirmed the maturity of our iPSC-MNs. The challenge of the in vitro iPSC-MN model is that it is considered fetal-like , so it iP497H MNs began to be damaged and tended toward axon regression during longer aging in vitro, accompanied by the loss of MNs through an apoptotic-like pathway involving capsase3  was co-transfected into Neuro-2a cells, and some UBQLN2-positive inclusions were TDP-43-negative . Consistreatment .P497H MNs . Here, we found that acute oxidative stress (0.5 mM SA 0.5 h) was able to induce more cytoplasmic TDP-43 in all groups A, while 497H MNs C, which WT or mutated hUBQLN2P497H in Neuro2a cells can promote cytoplasmic mislocalization of TDP-43 [UBQLN2 inclusions significantly accumulated in the brain and spinal cord tissues of UBQLN2-linked patients with ALS/FTD ,40. We af TDP-43 ,42,44. TP497H MNs.This study showed that DNA damage is an early event in the pathophysiology of UBQLN2-ALS. Previous studies revealed that DNA damage leads to distal axon degeneration and finally MN death . AlthougP497HiPSC-MNs , then the sgRNA oligonucleotides were annealed and ligated into lentiCRISPR v2 plasmids , and sequenced via Sanger sequencing to confirm that no errors were introduced. After TrypLETM Select  was used to detach WT iPSCs into a single cell, 1 × 106 cells, 1 μL of 40 μM ssODN, and 5 μg of leti-UBQLN2-sgRNA-cas9 plasmid were added to 18 μL Supplement 1 and 82 μL Solution 2 of Human Stem Cell Nucleofector Kit 2 (Lonza). The transfer procedure was B-016. The cells were seeded on Matrigel (BD Biosciences)-coated 6-well plates in mTeSR1 , containing 10 μm Y27632 for 24 h, then selected using mTeSR1 with a final concentration of 0.3 μg/mL puromycin. When the confluence of cells reached 70%, the cells were detached with TrypLETM Select. One thousand cells were seeded on a Matrigel-coated 6 cm dish in mTeSR1 containing 10% clone R (Stem cell) for four days and then replaced with mTesR1 without clone R every day until clones were picked.The sgRNA for UBQLN2 was designed using the CRISPR design tool  was coated with Matrigel.Wild-type iPSCs were previously generated by our group . All iPSTM Select and incubated at 37 °C with 0.075 M KCl for 10 min, then fixed with 500 μL methanol and 10 mL glacial acetic acid. The metaphase chromosome spreads were prepared via air-drying. The chromosomes were baked at 75 °C, digested with trypsin, and then G-banded via Giemsa staining .UBQLN2-P497H iPSCs were detached with trypLEThe use and care of animals complied with the guidelines of the Ethics Committee of the Center for Medical Genetics of Central South University.6 cells were dissociated with 0.05% trypsin/EDTA (Gibco), then resuspended in 70 μL Matrigel and 140 μL mTesR1. After injecting the mixture into the groins of nude mice for about 8 weeks, the teratoma was fixed in 4% paraformaldehyde, embedded in paraffin, sliced, and stained with HE. All procedures for animal care and use were carried out in accordance with institutional guidelines. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Center for Medical Genetics of Central South University.First, 5 × 10The differentiation of motoneurons derived from iPSCs was performed as previously described . BrieflyTotal RNA was extracted using the Trizol method (Sigma-Aldrich). RNA was purified and reverse-transcribed to cDNA with Q RT SuperMix . qPCR analysis was performed with a ChamQ Universal SYBR qPCR Master Mix (Vazyme#Q711) according to the manufacturer’s instructions in a Bio-Rad CFX96 Touch q-PCR system. The GAPDH gene was used as an endogenous control. All the primers are shown in TM overnight. After 48 to 72 h post-plating, the cells were fixed with 4% paraformaldehyde for 20 min. After washing with DPBS, the cells were permeabilized for 15 min with 0.1% PBST (DPBS added 0.1% Triton X-100) and blocked for 30 min with 5% bovine serum albumin (BSA) dissolved in DPBS. Then, diluted with 5% BSA, the primary antibodies were added to the cells and incubated overnight at 4 °C. Next, the cells were washed with 0.1% PBST, and the secondary antibodies were diluted with 5% BSA and incubated at room temperature for 1 h. Finally, the nucleus was stained with DAPI for 5 min and washed with DPBS. Images were acquired with a LAS X SP-5 confocal microscope .Cells were seeded on 24-well slides coated with MatrigelCell fractionation was performed as previously described . Total pt-test was used to compare data between two groups. Two-way analysis of variance (ANOVA) was performed to compare data among two or three groups, followed by Tukey’s and Sidak’s multiple comparison test. Statistical significance was determined as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.Data were analyzed with GraphPad Prism 8.3. Student’s