Scleraxis (Scx) and Mohawk Homeobox (Mkx) gene and maintain the tissue homeostasis, while its role in the periodontal ligament is unclear. The aim of this study was to investigate the effects of Wnt/β-catenin signaling induced by Wnt-3a stimulation on the inhibition of osteogenic differentiation of human periodontal ligament fibroblasts (HPLFs). During osteogenic differentiation of HPLFs, they formed bone nodules independently of alkaline phosphatase (ALP) activity. After stimulation of Wnt-3a, the expression of β-catenin increased, and nuclear translocation of β-catenin was observed. These data indicate that Wnt-3a activated Wnt/β-catenin signaling. Furthermore, the stimulation of Wnt-3a inhibited the bone nodule formation and suppressed the expression of osteogenic differentiation-related genes such as Runx2, Osteopontin and Osteocalcin, and upregulated the gene expression of Type-I collagen and Periostin (Postn). Scx may be involved in the suppression of osteogenic differentiation in HPLFs. In conclusion, Wnt/β-catenin signaling may be an important signaling pathway that inhibits the osteogenic differentiation in HPLFs by the upregulation of Scx gene expression and downregulation of osteogenic differentiation-related genes.The periodontal ligament is a collagenous tissue that is important for maintaining the homeostasis of cementum and alveolar bone. In tendon cells, Wnt/β-catenin signaling has been reported to regulate the expression level of  The periodontal ligament is a tissue that plays a variety of roles, including buffering mechanical stress during chewing, providing a sensory organ, and providing an immune response to periodontal disease ,2. Once Runx2, Osteocalcin and Osteopontin [The periodontal ligament also plays a dual role in maintaining the soft tissue periodontal ligament while acting as a source of osteoblasts for the surrounding bone tissue. The periodontal ligament contains a multicellular population of undifferentiated mesenchymal stem cells that can differentiate into cementoblasts, osteoblasts and fibroblasts to supply the surrounding tissues, but the mechanism of differentiation is unknown ,6,7. Humeopontin ,9. ThereMohawk Homeobox (Mkx) [Scleraxis (Scx) [Scx regulates the expression of periostin (Postn) in periodontal ligament-derived cells, suggesting that these tendon-specific genes are important for the maintenance of the periodontal ligament [Recently, it has been reported that tendon-specific genes, ox (Mkx)  and Scleis (Scx) , are expligament ,12. Periligament , and oveligament . Wnt/β-cligament ,16. Wnt-ligament ,18, but Scx is involved in the suppression of osteogenic differentiation. These results indicate that Wnt/β-catenin signaling in the periodontal ligament may be involved in the suppression of osteogenic differentiation through the expression of Scx.The aim of this study was to investigate the effect of Wnt/β-catenin signaling on the osteogenic differentiation of HPLFs. HPLFs cultured in DMEM and osteogenic medium supplemented with Wnt-3a were subjected to immunofluorescence staining, Western blotting, ALP activity staining and measurement, alizarin red S staining and real time quantitative PCR. HPLFs cultured in osteogenic medium without Wnt-3a showed osteogenic differentiation independent of ALP activity and increased the expression of osteogenic differentiation related genes. The addition of Wnt-3a to the osteogenic medium activated Wnt/β-catenin signaling and suppressed osteogenic differentiation in a signal-dependent manner, suggesting that the tendon-specific gene Chemicals and reagents were purchased from the following manufacturers.TM cell lysis reagent, and Ascorbic acid were purchased from Sigma-Aldrich . Fetal bovine serum (FBS), penicillin, streptomycin, phosphate-buffered saline , dimethyl sulfoxide (DMSO), Triton X-100, bovine serum albumin (BSA), Disodium Glycerophosphate 5.5 Hydrate, Dexamethasone, Alizarin Red S, Cetyl pyridinium Chloride (CPC), ImmunoStar LD, 4% Paraformaldehyde Phosphate Buffer Solution, Sirius red, LabAssayTM ALP, TRAP/ALP Stain Kit and protein assay Bradford reagent were purchased from FUJIFILM Wako Pure Chemical . Alexa Flour® 488-labeled phalloidin and Alexa Flour® 594-labeled goat anti-rabbit IgG1 were purchased from Invitrogen . Anti-β-Catenin antibody (ab16051), Goat anti-rabbit IgG (H+L) antibody, HRP conjugate (SA00001-2) and α-Tubulin antibody (11224-1-AP) were purchased from Proteintech . A sample of 4′,6-diamino-2-phenylindole (DAPI) was purchased from Dojindo . Human Wnt-3a protein (#5036-WN) was purchased from R&D Systems . RNeasy Mini kit was purchased from QIAGEN . TaKaRa Ex Taq was purchased from Takara bio-Inc. . Thermanox® Coverslips (13 mm dia.) were purchased from Thermo Fisher Scientific . LightCycler® 480 SYBR® Green I Master and Transcriptor First Strand cDNA Synthesis Kit were purchased from Roche . Human periodontal ligament fibroblasts (HPLFs) were purchased from ScienCell research laboratories .Dulbecco’s modified Eagle’s medium–high glucose (DMEM), trypsin-EDTA, CelLytic2 at 37 °C. In this study, all experiments were conducted using the HPLFs at passage 3–5 and cultured in 24-well plates. The osteogenic differentiation medium was DMEM with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 µL/mL streptomycin, plus three osteogenic differentiation factors: 10 nM dexamethasone, 50 mg/mL ascorbic acid, and 10 mM β-glycerophosphate. The types of media are shown in HPLFs were cultured in DMEM supplemented with 10% heat inactivated FBS, 100 U/mL penicillin, and 100 µL/mL streptomycin. HPLFs were grown in a humidified atmosphere containing 5% CO4 cells/cm2 and cultured in DMEM. After two days, the medium was replaced by four different media and HPLFs were cultured for 14 days. The cells were lysed in CelLyticTM M and homogenized by sonication. The cell lysates were separated on SDS-PAGE and a nitrocellulose membrane followed by immunoblotting with antibodies against β-catenin , α-tubulin . Band intensities were quantified in four independent experiments for each group and were normalized by α-tubulin.HPLFs were seeded at a density of 5 × 10® Coverslips (ϕ13 mm) were placed on the bottom using sterile tweezers. HPLFs were seeded at a density of 1.25 × 104 cells/cm2 and cultured in DMEM. After one day, the medium was replaced by four different media and HPLFs were incubated for 15 min. The cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature. After rinsing with PBS twice, the cells were blocked with 3% BSA in PBS for 1 h at room temperature and incubated with a primary antibody, β-catenin  which was diluted in PBS overnight at 4 °C. The cells were washed with PBS twice and then stained with Alexa Flour® 488-labeled Phalloidin (1:250) for F-actin, Alexa Flour® 594-labeled goat anti-mouse IgG (1:250) for β-catenin and DAPI (1:500) for the nucleus diluted in PBS for 1 h at room temperature in the dark. The cells were washed with PBS and then examined by fluorescence microscopy .In a 24-well plate, Thermanox4 cells/cm2 and cultured in DMEM. After two days, the medium was replaced by four different media and HPLFs were cultured for 14 days. The cells were stained with alkaline phosphatase (ALP) stain according to the manufacture’s protocol.HPLFs were seeded at a density of 5 × 104 cells/cm2 and cultured in DMEM. After two days, the medium was replaced by four different media and HPLFs were cultured for 14 days. HPLFs were collected and stored at −80 °C. The cells were lysed in CelLyticTM M and homogenized by sonication. This experiment was conducted using LabAssayTM ALP and following the manufacturer’s protocol. Total protein concentrations were determined by the Bradford standard method.HPLFs were seeded at a density of 5 × 104 cells/cm2 and cultured in DMEM. After two days, the medium was replaced by four different media and HPLFs were cultured for 21 days. The cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were then washed twice with dH2O and stained with 0.1% alizarin red S solution (pH 4.1) for 1 h at room temperature in the dark. The cells were washed four times with dH2O and then examined by fluorescence microscopy . Ten percent (wt/vol) cetyl pyridinium chloride was added to the 24-well plate. The optical density values were read at 570 nm.HPLFs were seeded at a density of 5 × 10Runx2, Osteocalcin, Osteopontin, Type-I collagen, Periostin, Mohawk, and Scleraxis HPLFs were seeded at a density of 5 × 104 cells/cm2 and cultured in DMEM. After two days, the medium was replaced by four different media and HPLFs were cultured for 14 days. Total RNA was extracted using the RNeasy Mini kit and complementary DNA was synthesized using the Transcriptor First Strand cDNA synthesis kit. The primer sequences used in the experiment are listed in ® 480 Real Time PCR System  using a LightCycler® 480 SYBR® Green I Master. The amounts of mRNA were calculated as relative quantities in comparison to 18S mRNA and analyzed with the 2ΔΔCt method.The expression levels of p < 0.05 and p < 0.01.The data were statistically analyzed for determination of the mean and the standard deviation (SD) of the mean. The Student’s t-test was carried out with a significance level of To investigate whether Wnt-3a activates Wnt/β-catenin signaling and induces the transfer of β-catenin to the cell nucleus, cells were observed using fluorescence microscopy  15 min after the treatment of HPLFs with Wnt-3a.When HPLFs were cultured in (−/+) and (+/+) medium supplemented with Wnt-3a for 15 min, the nuclear translocation of β-catenin was observed as indicated by yellow arrows . In contWnt-3a activates Wnt/β-catenin signaling by binding to the Frizzled receptor on the plasma membrane and inhibiting the Axin complex, which degrades β-catenin, thereby stabilizing β-catenin accumulation in the cytoplasm and inducing nuclear translocation of β-catenin. To further investigate whether stimulation with Wnt-3a contributes to the activation of Wnt/β-catenin signaling in HPLFs, Western blotting was performed, and the amount of β-catenin expression accumulated in the cells was measured. The expression of β-catenin in HPLFs cultured in (−/+) and (+/+) medium was significantly increased compared to those cultured in (−/−) and (+/−) medium . The expIn contrast, there was no change in β-catenin levels between (−/−) and (+/−) medium. These results indicate that HPLFs activate Wnt/β-catenin signaling by treatment with Wnt-3a. Osteogenic differentiation in periodontal ligament-derived cells is thought to involve a variety of signals, including MAPK, JNK and Erk1/2 signaling . It has ALP staining and ALP activity were conducted on HPLFs after 14 days of culture in each medium to investigate the production of ALP, a marker associated with early bone differentiation. ALP is a hydrolytic enzyme that plays an important role in the progression of osteogenic differentiation, removing pyrophosphate, an inhibitor of the synthesis of hydroxyapatite, the main component of bone, by hydrolysis, and providing phosphate ions .Osteoblasts are known to exhibit high ALP activity during osteogenic differentiation and high ALP activity is one of the indicators of osteogenic differentiation ,25,26. HAfter 21 days in culture, HPLFs were stained with alizarin red S, a chelate complex with calcium. As a result, HPLFs stained red when cultured in (+/−) medium, indicating osteogenic differentiation with the formation of calcified nodules .The results of ALP activity  indicateRunx2  medium, the expression of Type-I collagen was significantly increased compared to (+/+) medium and was at the same level compared to (−/−) medium, suggesting that the suppression of Type-I collagen expression by osteogenic induction was abrogated by Wnt-3a. Therefore, Wnt-3a was considered to be an important factor for the maintenance of periodontal ligament tissue, because Wnt-3a abolished the suppression of Type-I collagen expression by (+/−) medium. The gene expression level of Periostin (Postn) also displayed similar behavior to that of Type-I collagen (Postn) is a protein that is expressed at sites of mechanical stress, and the periodontal ligament is constantly exposed to the mechanical stress of mastication [Postn gene expression was increased in osteoblasts in which Osterix (Osx), a gene essential for osteoblast osteogenesis, was knocked down [Postn is highly expressed in cells within the periodontal ligament and in osteoblasts on the surface of the alveolar bone, the site of insertion of Sharpey fibers, and is thought to be important in periodontal ligament remodeling as it is highly expressed during tooth movement [Postn in (+/+) medium compared to (+/−) medium suggests that Postn plays an important role in suppressing osteogenic differentiation and maintaining the multiple differentiation potential of the periodontal ligament. The gene expression levels of Mkx and Scx, which are tendon-specific genes, were investigated by real time quantitative PCR  medium was significantly decreased compared to that in (−/−) medium (Type-I collagen gene was significantly increased in the culture with (+/+) medium compared with that with (+/−) medium and was recovered to the same level as that in the culture with (−/−) medium. This suggests that matrix formation by collagen other than Type-I collagen is preferentially induced by culture in (+/−) medium. The collagen composition of the human mandible differs from that of other bone tissues, including the fact that it is more flexible than long bones due to looser collagen cross-linking [The most abundant matrix protein in bone tissue is collagen, of which Type-I collagen accounts for 95%. Lysyl oxidase accelerates the intermolecular covalent bonding of collagen and contributes to the maintenance of bone strength . The per) medium D. The ex-linking  to withsRunx2, Osterix, Osteopontin and Osteocalcin [Runx2, Osteopontin and Osteocalcin compared to culture in (−/−) medium  and Cementum attachment protein (CAP), proteins extracted from cementum, are cementum-related genes [CEMP1 may be preferentially expressed in ALP-positive cells among periodontal ligament-derived cells [CEMP1 gene expression in periodontal ligament-derived cells is suppressed by the induction of osteogenic differentiation [CEMP1 and CAP gene expression levels do not correlate with ALP expression levels in different human individuals from whom periodontal ligament-derived stem cells are extracted [CAP gene expression was not significantly different between the four mediums  medium A–C. Runxer birth . Osteopoem cells  and osteem cells . In addiormation . In the ormation  and alteeocalcin . It has eopontin  in stem ed genes . In addied genes ,54. It hed cells . It has ntiation . However mediums . Thus, tormation , and thePostn [Postn genes was significantly decreased in HPLFs cultured in (+/−) medium compared to (−/−) medium, and the expression levels of these genes were significantly increased in (+/+) medium compared to (+/−) medium  medium D,E. ThusMkx and Scx genes is highly expressed in tendon cells and is important in the maintenance of tendons [Mkx and Scx were investigated. The Mkx affects the maintenance of adequate periodontal ligament width and cell morphology in the periodontal ligament [Mkx knockout mice. The present study suggests that the expression of the Mkx gene may not be involved in the osteogenic differentiation of HPLFs or in the suppression of osteogenic differentiation  medium compared to other media (Scx gene suppresses osteogenic differentiation in periodontal ligament-derived cells by inhibiting the expression of Postn, Osteopontin and Osteocalcin [Scx induced by Wnt-3a.The expression of  tendons ,61. Becaligament . It is antiation A. The Scligament ,62 and ier media B. It haseocalcin . TherefoScx and Postn genes. This is the first report to investigate the relationship of the tendon-specific gene Scx via Wnt/β-catenin signaling in HPLFs.It can be concluded from this study that the activation of Wnt/β-catenin signaling induced by Wnt-3a in HPLFs is an important signal responsible for the maintenance of periodontal ligament homeostasis by inhibiting osteogenic differentiation through the upregulation of the expression of


Its excellent adsorption-photocatalytic activity and durability were ascribed to the sufficient vacant sites of octahedral structure for TCH adsorption and the feasible band-gap structure for visible-light response. In addition, the band gap structure (1.37 eV) of MnS with a conduction band value of −0.58 eV and a valence band value of 0.79 eV was favorable for the generation of O2−, while unsuitable for the formation of OH. Hence, octahedral MnS was a potential material for the removal of antibiotics from wastewater.To disclose the effect of crystal plane on the adsorption-photocatalytic activity of MnS, octahedral MnS was prepared via the hydrothermal route to enhance the adsorption and photocatalytic efficiencies of tetracycline hydrochloride (TCH) in visible light region. The optimal MnS treated at 433 K for 16 h could remove 94.83% TCH solution of 260 mg L PL s2− and OH. The high pH is not favorable for the formation of OH in photocatalytic system, leading to the inferior photocatalytic activity of photocatalysts [−1 , while the corresponding ECB value (0.79 eV) was less positive than the redox potential value of OH/OH− (1.99 eV). It is indicated that photo-induced e− was favorable for the conversion of dissolved O2 into O2−, but the separated h+ was unsuitable for the reaction of OH− into OH, which was confirmed by the stronger ESR intensity of O2− in comparison to OH  as solvent. Typically, 3.0 mmol manganous nitrate (Mn(NO3)·4H2O), 4.0 mmol thiourea (CH4N2S), and 1.5 g polyvinylpyrrolidone  were dispersed into 40 mL above ethylenediamine solution under intensive stirring at room temperature for 40 min, and then poured into a 50 mL Teflon-lined autoclave for the heating treatment at 433 K for 16 h. After it was cooled down to room temperature, the above suspension was centrifuged, washed, and dried at 333 K to obtain MnS. MnS treated at 423, 443, and 453 K for 16 h as well as treated at 433 K for 14, 18, and 20 h were also prepared through the above process for comparison. The obtained samples were characterized by various technologies of MnS was synthesized via a solvothermal route using ethylenediamine solution . Effects of hydrothermal temperature, hydrothermal time, inorganic salts , and quenchers  on the adsorption-photocatalytic activity of MnS were also performed.The adsorption-photocatalytic performance of MnS was performed in a 200 mL Pyrex reactor with a 300 W Xe lamp with the detected power intensity of 500 mW cm−1 within 180 min, and decreased to 89.68% after five cycles, exhibiting its superior adsorption-photocatalytic activity. This is attributed to the abundant vacant sites of octahedral structure for TCH removal and the suitable band-gap structure for radicals’ formation. The photo-induced e− and O2− played avital role in the MnS-assisted photocatalytic reaction.The adsorption-photocatalytic of octahedral MnS for visible-light driven removal of TCH was intensively affected by the hydrothermal temperature, hydrothermal time, and inorganic salts. The optimal MnS treated at 433 K for 16 h could remove 94.83% of TCH solution of 260 mg L


Wnt/β-catenin signaling is a delicate and complex signal transduction pathway mediated by multiple signaling molecules, which plays a significant role in regulating human physiology and pathology. Abnormally activated Wnt/β-catenin signaling pathway plays a crucial role in promoting malignant tumor occurrence, development, recurrence, and metastasis, particularly in cancer stem cells. Studies have shown that the Wnt/β-catenin signaling pathway controls cell fate and function through the transcriptional and post-translational regulation of omics networks. Therefore, precise regulation of Wnt/β-catenin signaling as a cancer-targeting strategy may contribute to the treatment of some malignancies. SUMOylation is a post-translational modification of proteins that has been found to play a major role in the Wnt/β-catenin signaling pathway. Here, we review the complex regulation of Wnt/β-catenin signaling by SUMOylation and discuss the potential targets of SUMOylation therapy. The wingless and Int-1 (Wnt)/β-catenin signaling pathway is a key cascade in embryonic development, self-renewal of organisms, cell proliferation, differentiation , and usuFollowing the first member of the Wnt family identified over the last four decades , severalvia ATP hydrolysis. Consequently, upon interaction of the charged E1 enzyme with E2, SUMO is transferred from E1 to E2, forming an E2-SUMO thioester. Finally, SUMO is linked to the lysine residues of target proteins via an isopeptide bond, with or without stimulation by the ligase enzyme (E3). SUMOylation of proteins provide a platform for enhancing protein-protein interactions  proteins bind to substrates covalently or non-covalently under the catalysis of SUMO proteases . SUMO prractions , 52.DeSUMOylation is the process of removing the SUMO from SUMOylated substrates , 54. TheSUMOylation and deSUMOylation remain in a dynamic equilibrium, allowing cells to respond rapidly to changing external and internal pressures and stimuli , 62. SUMβ-catenin is an evolutionarily conserved molecule that plays crucial roles in a variety of developmental and homeostatic processes . AberranRanBP2 is part of the nuclear pore complex (NPC) and acts as a SUMO E3 enzyme, which helps macromolecular proteins that lack nuclear localization signals to enter the nucleus . It promJiang et al. demonstrated that the deSUMOylation activity of SENP2 was important for β-catenin stability. SENP2, a deSUMOylation enzyme, was downregulated in hepatocellular carcinoma (HCC) tissues . The stuThese studies suggest that the dynamic balance between SUMOylation and deSUMOylation can maintain the stability and transcriptional activity of β-catenin. The TCF-4-SUMO1-RanGAP1-Ubc9-RanBP2 complex can be used as a target to prevent the SUMO modification of β-catenin. β-catenin without SUMO modification cannot enter into the nuclear and play the role in the initiation and development of cancer.GSK-3β is a ubiquitously expressed, broadly specific, serine/threonine kinase. Therefore, GSK-3β regulates a number of intracellular signaling pathways and is involved in a variety of biological events, including autophagy, cell apoptosis, and cancer progression –77. Thisin vitro or in vivo and is critical for GSK-3β function  belongs to the ubiquitin-like protease 1 (Ulp1) cysteine protease family, which possesses hydrolase and isopeptidase activities of the SUMO-specific proteases . Axam us of Axam . Usuallyin vitro and in vivo  bind to various transcription factors and play a bidirectional regulatory role as a gene-specific activator and repressor of Wnt target gene transcription , 101. InWnt/β-catenin and Smad3/4 can crosstalk with each other . In the in vivo and in vitro , and Foundation of committee on science and technology of Tianjin . The funders had no roles in the design of the study, data collection, analysis and interpretation, or decision to write and publish the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PRMT5 underwent alternative splicing (AS) and generated a spliced isoform PRMT5-ISO5 in hepatocellular carcinoma (HCC) patients after radiotherapy. However, the regulatory mechanism and the clinical implications of IR-induced PRMT5 AS are unclear. This work revealed that serine and arginine rich splicing factor 3 (SRSF3) silencing increased PRMT5-ISO5 level, whereas heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1) silencing reduced it. Then, we found that SRSF3 and HNRNPH1 competitively combined with PRMT5 pre-mRNA located at the region around the 3′- splicing site on intron 2 and the alternative 3′- splicing site on exon 4. IR-induced SRSF3 downregulation led to an elevated level of PRMT5-ISO5, and exogenous expression of PRMT5-ISO5 enhanced cell radiosensitivity. Finally, we confirmed in vivo that IR induced the increased level of PRMT5-ISO5 which in turn enhanced tumor killing and regression, and liver-specific Prmt5 depletion reduced hepatic steatosis and delayed tumor progression of spontaneous HCC. In conclusion, our data uncover the competitive antagonistic interaction of SRSF3 and HNRNPH1 in regulating PRMT5 splicing induced by IR, providing potentially effective radiotherapy by modulating PRMT5 splicing against HCC.Protein arginine methyltransferase 5 (PRMT5) is an epigenetic regulator which has been proven to be a potential target for cancer therapy. We observed that  Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide and represents three-fourths of primary hepatic malignancies. Unfortunately, more than 70% of patients harboring a high tumor burden or liver dysfunction remain unsuitable for definitive resection . StereotPRMT5 isoform a  translates into full-length protein, whereas another transcript PRMT5 isoform e  generates truncated protein by skipping exon 3 and part of exon 4. When compared to full-length PRMT5, the truncated protein can exhibit distinct localization and preferential control of critical genes for cell cycle arrest [PRMT5 splicing may be an effective radiosensitization strategy for cancer radiotherapy.Alternative splicing (AS) of RNA is a posttranscriptional process found in the majority of human multiexon genes. Differential AS events were predicted to be risk factors for HCC patient survival and were significantly enriched in metabolism-related pathways . As a prPRMT5 splicing. In vitro cell experiments mainly explored that (1) IR induced PRMT5 AS by changing the levels of serine/arginine-rich splicing factor 3 (SRSF3) and heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1); (2) the interaction of SRSF3 and HNRNPH1 involved in the process of PRMT5 splicing; (3) the elevated level of PRMT5-ISO5 sensitized HCC cells to radiation. The clinical and preclinical studies mainly involved the analysis of PRMT5-ISO5 levels responded to radiation, the effects of increased PRMT-ISO5 and PRMT5 deficiency on xenograft tumor and primary HCC, by establishing xenograft tumor models, and liver-specific Prmt5 knockout spontaneous HCC mice. Considering the increased incidence and death rates of HCC, we hope modulating PRMT5 splicing will be an effective choice to enhance the radiotherapeutic effect on HCC.Based on the above consideration, we focus on the molecular mechanism and preclinical evaluation of IR-induced PRMT5 precursor mRNA (pre-mRNA) can be alternatively spliced into different splicing isoforms. The splice variant PRMT5-ISO5 lacks exon 3 and part of exon 4  were treated with IR   and prediceAid 2   [PRMT5 depletion at the protein level, but the transcriptional levels of PRMT5-minigene achieved dozens of times the residual expression of endogenous PRMT5 mRNA  , we geneMT5 mRNA . Subsequilencing E. Meanwhsplicing F. These PRMT5 splicing, a mixture of the SRSF3 construct and HNRNPH1 construct was co-transfected into PRMT5 KO clones accompanied by PRMT5-minigene plasmid. We achieved a decrease in exogenous HNRNPH1 and a simultaneous increase in exogenous SRSF3 in the assay to investigate the level change in PRMT5-ISO5. In the results, we observed that exogenous HNRNPH1 downregulation and exogenous SRSF3 upregulation  on intron 2 and the alternative 3′ss on exon 4  probably increases cellular radiosensitivity and influences the methyltransferase activity of PRMT5.To investigate the effects of PRMT5 protein or the truncated isoform on radiosensitivity, PRMT5 KO and isoform rescue assays were used in HCC cells. We observed that PRMT5 deficiency sensitized Huh7 cells to IR treatment by suppressing cell proliferation A. AlthouPRMT5-ISO5 transcript, the HCC xenograft model was constructed via subcutaneous injection of Huh7 cells . The single-cell RNA sequencing (scRNA-seq) dataset (GSE149614) was derived from the Gene Expression Omnibus database .The liver hepatocellular carcinoma (LIHC) patient datasets were derived from the TSVdb TCGA Splicing Variants Database  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum  and penicillin/streptomycin at 37 °C with 5% COThe X-Rad 320 Biological Irradiator  was used at a dose rate of 300 cGy/min.PRMT5-minigene, a segment of PRMT5 including the region from exon 1 to exon 4, was amplified by polymerase chain reaction (PCR) from genomic DNA. The human HNRNPH1 and SRSF3 open reading frames (ORFs) were amplified by RT-PCR. The primers are listed in PRMT5-ISO5) plasmids were generous gifts from Prof. Jiuyong Xie  as described [The escribed .The normal siRNA and In vivo siRNA (2′ O-methyl + 5′ cholesterol-modified) against HNRNPH1 or SRSF3 and nonsense siRNA  were dissolved in rNase-free water for cell transfection or phosphate buffered saline (5 nmol siRNA with 50 μL PBS) for intratumoral injection. The sequences of si-HNRNPH1 and si-SRSF3 are shown in PRMT5-minigene plasmid was co-transfected with 4 μL siRNA  or 2 μg plasmid in PRMT5 KO cells cultivated in 6-well plates. To investigate the HNRNPH1 and SRSF3 interaction, 4 μg PRMT5-minigene plasmid was co-transfected with a total of 4 μg plasmid mixture (pcDNA3.1-FLAG-HNRNPH1 plus pcDNA3.1-FLAG-SRSF3) in PRMT5 KO cells grown in 60 mm Petri dishes. The cells were harvested 48 h post-transfection. The levels of PRMT5-ISO5 were determined by RT-qPCR, and the expression of HNRNPH1 and SRSF3 was detected by western blotting.RNA interference and construct overexpression were carried out in 6-well plates using Lipofectamine 2000  with 5 μL siRNA  or 2.5 μg plasmid. For the cotransfection assay, 2 μg PRMT5-sgRNA lentivirus were designed and generated in HEK 293T cell lines by cotransfection of psPAX2, pMD2.G, and Cas9 or PRMT5-sgRNA plasmids at a 4:3:1 ratio. A total of 2.5 μg plasmids were mixed with Lipofectamine 2000 reagent  in a well of 6-well plates when HEK 293T cells were 60–70% confluent. Huh7 and MHCC-97H cell lines were infected with Cas9 lentivirus for 24 h and then treated with hygromycin for 7–10 d. Subsequently, Cas9-positive cells were infected with PRMT5-sgRNA lentivirus for another 24 h and then treated with puromycin for 5–7 d. Furthermore, serial dilution was performed to generate single clones of stable PRMT5 knockout (KO) cell lines. Finally, all clones were evaluated for PRMT5 KO efficiency by western blotting.Cas9 lentivirus and 3, 2 × 103, 3 × 103, 4 × 103, or 6 × 103 cells per well in 6-well plates for another 24 h. Subsequently, the cells with different starting cell numbers were untreated (0 Gy) or treated with IR of 2 Gy, 4 Gy, 6 Gy, and 8 Gy and then incubated with DMEM for 10 days at 37 °C. Finally, paraformaldehyde  was used for fixation, and crystal violet  was employed to stain the forming colonies.PRMT5 KO Huh7 clones were rescued with either Myc-PRMT5L or Myc-PRMT5S plasmids separately. The transfected cells (48 h post-transfection), Cas9-positive Huh7 cells, and PRMT5 KO Huh7 clones were seeded at a density of 1 × 10PRMT5-ISO5 was carried out with 10 μL TB Green™ Premix Ex Taq™ II  by the QuantStudio 3 Real-Time PCR System . The primers for RT-qPCR are shown in Total RNA was extracted using RNAiso Plus  according to the manufacturer’s protocol. Quantitative PCR analysis of Proteins extracted from cells and tissues were measured and heat denatured. Equal amounts of proteins were separated using 10% or 12% SDS-PAGE and transferred to PVDF membranes . After saturating, the membranes were incubated with anti-PRMT5 , anti-HNRNPH1 , anti-SRSF3 , anti-AKT1 , anti-phospho-AKT (Ser473) , anti-SDMA  or anti-GAPDH , and subsequently incubated with HRP-conjugated secondary antibodies . Finally, the membranes were detected using Pierce™ ECL Western Blotting Substrate  and visualized with ChemiScope 6100 .Cells were transfected with FLAG-tagged SRSF3 or HNRNPH1 using Lipofectamine 2000 reagent . Forty-eight hours post-transfection, the cells were washed and collected in lysis buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 0.5% NP-40, 1% Triton X-100) containing EDTA-free Protease Inhibitor Cocktail , 1 mM DTT  and 200 units/mL RNase OUT . Then, 10% of the cell lysate was taken as the “input”, and the remains were divided into equal amounts and incubated overnight at 4 °C with anti-FLAG magnetic beads  or preprepared IgG-protein A/G magnetic beads . Subsequently, all the mixtures were washed 6 times with 1×NT-2 buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40) using the DynaMagTM-2 Magnet . Next, the washed samples were digested at 55 °C for 30 min with proteinase K digestion buffer (1 × NT-2 buffer containing 1% SDS and 1.2 mg/mL proteinase K). Finally, immunoprecipitated RNA was extracted using RNAiso Plus . Fold enrichment of the target region was determined after normalization to the input and compared with the IgG control. The primers for RT-qPCR are shown in 7 Huh7 cells were mixed with Matrigel Basement Membrane Matrix  at 4 °C and then injected into the right flanks of nude mice. When the tumors were visible (160 mm3 in volume), the mice were divided into 4 groups and treated with (1) 15 Gy; (2) 15 Gy and intratumoral injection of si-HNRNPH1 (5 nmol for each); (3) 15 Gy and intratumoral injection of si-SRSF3 (5 nmol for each) [Male BALB/c nude mice  were kept under pathogen-free conditions with a 12 h light/dark cycle and had free access to water and food. After acclimation for a week, 1 × 10or each) ; and (4)flox/flox-Alb-CreERT2 mice with a C57BL/6J background (4–5 weeks old) were generated and purchased from Cyagen Biosciences. The mice were housed under pathogen-free conditions with a 12 h light/dark cycle and allowed free access to water and food. pCMV(CAT)T7-SB100 and pT3-myr-AKT-HA were purchased from Fenghui Biotechnology (Addgene plasmids #34879 and #31789), and pT3-EF1α-NRasV12 (RPT-ZL 2101C2) was purchased from RiboBio. After acclimation for a week, hydrodynamic tail-vein injection (HTVi) of a volume equivalent to 10% body weight of endotoxin-free plasmids (10 μg of pCMV(CAT)T7-SB100, 10 μg of pT3-myr-AKT-HA, and 10 μg of pT3-EF1α-NRasV12 for each mouse [Male Prmt5ch mouse ,50,51) dch mouse ,38. Regi2/2. At the end of the experiment, mice were sacrificed by euthanasia, tumor tissues were excised and weighed, images were captured, and immunohistochemistry was performed.The xenograft tumor volume was measured every 2 days and calculated as follows: volume = longest tumor diameter × (shortest tumor diameter)Liver (tumor) tissue sections were paraffin-embedded, deparaffinized, and rehydrated, followed by hematoxylin and eosin (H&E) staining. For detection of lipid droplets, liver tissue sections were incubated with oil red O dye and washed with 60% isopropanol. Finally, the images were captured and evaluated.The LIHC patient datasets were analyzed using the Kruskal–Wallis test and Kaplan–Meier curves (log-rank tests) in R software. The scRNA-seq data were preprocessed by using the Seurat package. The harmony package and t-distributed stochastic neighbor embedding (t-SNE) algorithm were used to perform normalization, dimensionality reduction, and cluster classification. Gene Ontology (GO) analysis was conducted with clusterProfiler packages. GEPIA2  was usedt-test, as appropriate. All data are presented as the mean ± S.D. Differences were considered statistically significant if p < 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.Each experiment was performed in triplicate and the representative data from one experiment are shown. Statistical analysis for biological assays was performed by GraphPad Prism 7 software using one-way or two-way ANOVA and a two-tailed unpaired PRMT5-ISO5 might be a putative enhancer of radiosensitivity and a potential therapeutic approach for HCC patients. Briefly, IR induces an increase in PRMT5-ISO5 which improves poor prognosis. SRSF3 and HNRNPH1 antagonistically regulate PRMT5 AS by virtue of their competitive binding to PRMT5 pre-mRNA and promoting the choice of alternative 3′-ss on exon 4 rather than 3′-ss on intron 2. Notably, IR decreases SRSF3 expression and results in an increase in PRMT5-ISO5 levels, which enhances cell radiosensitivity and xenograft tumor regression (In conclusion, we provide preclinical evidence that gression . Finally


Median nerve stimulation (MNS) diminishes regional myocardial ischemia and ventricular arrhythmia; however, the underlying mechanism has not been elucidated.via bilateral vagotomy. Venous blood and ventricular tissue were collected to detect molecular indicators related to inflammation and cholinergic pathways by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and Western blot (WB).In this study, we randomly categorized 22 adult mongrel dogs into a control group, MNS group 1, and MNS group 2. After a 4-week experimental myocardial infarction (MI), ventricular electrophysiology was measured in the MNS group 1 before and after 30 min of MNS. The same measurements were performed in the MNS group 2 dogs P < 0.01). The levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nuclear transcription factor-κB (NF-κB) were lower (P < 0.01), whereas the levels of Ach were higher in the peri-infarct zone tissues in the MNS group 1 dogs than those in the MNS group 2 dogs (P < 0.01).No significant changes were reported in the ventricular effective refractory period (ERP) in the MNS group 1 and MNS group 2 dogs before and after MNS. The ventricular fibrillation threshold (VFT) in the MNS group 1 was significantly higher than that in the MNS group 2 (20.3 ± 3.7 V vs. 8.7 ± 2.9 V, This study demonstrated that MNS increases VFT in a canine model with MI. The effects of MNS on VFT are potentially associated with the cholinergic anti-inflammatory pathway. In appry and VA –10. Stimy and VA . In a suy and VA .The median nerve is paired and arises from some of the spinal nerves of the cervical and thoracic regions. In our previous study, we found that stimulation of the median nerve causes a cardiac sympatho-vagal balance, decreasing stellate ganglion nerve activity, and enhancing cardiac vagal nerve activity . Median This experiment was approved by the animal studies subcommittee of our institutional review board and complied with the guidelines of the National Institutes of Health for the care and use of laboratory animals. A total of 22 adult mongrel dogs (weighing 15–18 kg) were used in the experiment. Before pentobarbital sodium premedication, we subjected the dogs to an intramuscular injection of 25 mg/kg ketamine sulfate. All dogs were premedicated with sodium pentobarbital , intubated, and ventilated in a room atmosphere supplemented with oxygen from a respirator . Continuous electrocardiogram (ECG) monitoring was performed.The MI model was established using catheter intervention. After administering stable anesthesia to all dogs, we injected 1000 U of heparin; then, we inserted hemostatic sheaths into their right femoral artery. Using X-ray fluoroscopy, a 5 F catheter was inserted into the left coronary artery to determine a coronary angiogram. Keeping the 5 F catheter positioned at the left anterior descending artery, a Radifocus SP catheter was delivered to the distal anterior descending artery through the 5 F catheter. A gelatin sponge with normal saline was injected into the distal anterior descending artery in 15 dogs. Notably, if the ST segment had no significant changes, the gelatin sponge with normal saline was reinjected. When the chest lead ST-segment was elevated, acute MI was achieved . The conTransthoracic 2D and Doppler echocardiography were performed in all animals with Vivid 7  at baseline and after 4 weeks. Standard 2-D short- and long-parasternal views and 4-, 2-, and 3-chamber apical views were obtained. The left ventricular diastolic dimension (LVDD) and right ventricular diastolic dimension (RVDD) were measured using Simpson’s biplane formula. We took the measurements for all volumes in triplicate and reported the averages. An independent echocardiography expert reviewed the images and the parameters.After 4 weeks, the dogs with MI were randomly categorized into two groups, i.e., MNS group 1 and MNS group 2. The heart was exposed in a pericardial cradle by a median sternotomy performed under anesthesia for the two dog groups. In the MNS group 1, the median nerve in the left forelimbs was exposed, and a pair of bipolar hook electrodes were attached to the nerve. We identified the stimulated nerve as the median nerve by anatomical localization and functional localization of the stimulation effect, and any paw twitches resulting from stimulation were identified as an MNS effect. The electrodes were then connected to a constant current stimulator  with a stimulus isolation unit  generated 5 Hz, 0.5 ms duration pulses. MNS was performed for 30 min as shown in 1), a premature extra stimulus (S2) was introduced. As the S1–S2 intervals approached the ERP, decrements were reduced to 2 ms. ERP was the most prolonged S1–S2 interval at which S2 failed to capture. ERP dispersion was the maximum difference among all sites tested.The electrophysiological testing was performed before and after MNS. Effective refractory period (ERP) in the right ventricular epicardium at the apex (RVA), free wall (RVFW), and base (RVB) and left ventricular epicardium at the apex (LVA), free wall (LVFW), and base (LVB) were evaluated during ventricular pacing at 300 ms cycle lengths (CLs) using stimuli at twice threshold. 2) and double (S3) extra stimuli were delivered after eight beats of the ventricular drive (S1) at 300 ms . To reduce the influence of repeated defibrillation on the results, we measured the ventricular fibrillation threshold (VFT) at the end of the study. After all electrophysiological tests were completed, the VFT was obtained with right ventricular pacing using a train of 30 stimuli , which was performed at the end of a 20-beat drive train at 300 ms; this ensured that all measurements were taken at the same heart rate. To determine the VFT, we progressively increased the pacing voltage in 1 V steps, with a 30 s rest period before the next pacing train if no VF was induced. The VFT was defined as the minimal voltage required to induce sustained VF.The inducibility of VA was conducted before and after MNS and was assessed with programmed ventricular stimulation from the right free wall. Single  vacutainers from the MNS group 1 and MNS group 2 dogs before acute MI, before MNS, and after MNS. At the end of the protocol, the animals were euthanized, and the hearts were immediately excised. The control group was assigned to measure the concentration of inflammation cytokines in the tissues. Tissue specimens were obtained from the peri-infarct zone of LV in the MNS group 1, MNS group 2, and the same areas in the control group dogs. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and acetylcholine (Ach) were examined through enzyme-linked immunosorbent assay (ELISA).via Western blot (WB). We incubated the membranes with the primary antibody NF-κB , then blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) for 1 h, and incubated with the primary antibody overnight at 4°C. The membranes were then washed in TBST thrice, incubated with the secondary antibody for 1 h at 37°C, and imaged using Immun-Star horseradish peroxidase substrate. The relative expression levels of the proteins were evaluated using image analyzer software .The expression of nuclear transcription factor-κB (NF-κB) and signal transducers in the peri-infarct zone of LV in the MNS group 1, MNS group 2, and the same areas in the control group dogs were measured Tissues were obtained from the peri-infarct zone of LV for immunohistochemical analysis. We used CD68  and CD163  antibodies for immunohistochemical staining. The tissues were stained in the same session. We determined staining densities by a computer-assisted image analysis system .t-tests were used to compare means for two groups. ANOVA followed by Newman-Keuls tests were applied to compare the mean values of continuous variables among multiple groups; any significant difference was further analyzed using the Tukey–Kramer test. All of the statistical tests were two-sided, and a probability value of <0.05 depicted statistical significance.Data were expressed as the mean ± SD. Two-sample independent Student’s P > 0.05) in the MNS group 1. The heart rate and blood pressure in the baseline, acute MI, and before and after 30 min of MNS are shown in We performed successful experiments on 13 of 15 dogs with acute MI. Two dogs died during the rearing period. After 4 weeks, six dogs and seven dogs were assigned to MNS group 1 and MNS group 2, respectively. During MNS, the heart rate exhibited a decreasing trend in the MNS group 1 and MNS group 2, but this did not contribute to the significant difference. For instance, the heart rate was 125 ± 12 beats/min before MNS and 121 ± 11 beats/min after 30 min of MNS  and reduced LVEF  were noted. However, there was no significant change in the RVDD between baseline and after 4 weeks in the MNS group 1 and the MNS group 2 dogs. Moreover, we reported no significant differences in the LVDD and RVDD between baseline and after 4 weeks in the control group   . DispersP < 0.01). These results demonstrated that it was more difficult to induce VF in the MNS group 1 than in the MNS group 2.The VFT in the MNS group 1 was significantly higher than that in the MNS group 2  and the MNS group 2 dogs  than in the control group dogs. However, compared to the MNS group 2, the concentrations of TNF-α  and IL-6  were lower in the MNS group 1 dogs, whereas the levels of Ach were higher in the MNS group 1 dogs than that in the control  and MNS group 2 dogs .As highlighted in P < 0.01) and in the MNS group 1 . Compared to the control group, NF-κB protein expression was higher in the MNS group 1 .We compared Western blot results of ventricular tissues from the three groups . All immP < 0.01; CD163: 4.83 ± 0.26 vs. 1.67 ± 0.22, P < 0.01) and in the MNS group 1 . Compared to the control group, CD68 and CD163 protein expressions were higher in the MNS group 1 .Immunohistochemical staining showed that macrophage markers of CD68 and CD163 were elevated to varying degrees in different groups . The levBy exploring the influence of MNS on ventricular electrical remodeling and VA vulnerability in a canine model with experimental MI, this study provides evidence for the following: (1) MNS elevates post-infarction VFT and reduces the post-infarction inflammatory response and macrophage infiltration; (2) dissociation of the vagus nerve reverses the above effects of MNS; and (3) cholinergic anti-inflammatory pathways may play an important role in the effects of MNS on VFT.Several compelling bodies of evidence have shown that the risk of SCD after MI is highest during the first 30 days , 16, 17.In this study, we observed the effects of MNS on VA vulnerability in a canine model with MI for 4 weeks. Of note, it was revealed that the ventricular ERP and VT vulnerability had not changed obviously. However, the VFT increased significantly after 30 min of MNS. Furthermore, TNF-α, IL-6, and NF-κB levels in the peri-infarct zone of LV decreased after MNS. Interestingly, the effects of MNS on the VFT and ventricular inflammation factors were suppressed by bilateral vagotomy. In our previous study, we found that MNS not only attenuated left stellate ganglion nerve activity but also enhanced cardiac vagal nerve activity . CollectPrevious studies demonstrated that the levels of inflammation factors increased after MI, and vagal nerve stimulation could inhibit the inflammatory reaction and relieve myocardial ischemia/reperfusion injury by activating the cholinergic anti-inflammatory pathway , 23. TheIn clinical practice, the stimulation of the Neiguan spot has been utilized to treat MI . The NeiThis study has some limitations. First, we only explored the effect of 30 min of MNS on ventricular electrophysiology and VT vulnerability in a canine model with MI. No significant changes were found in ventricular ERP. Therefore, the effects of long-term intermittent or stronger MNS on ventricular electrophysiology and VT vulnerability are unclear. Second, previous studies revealed that local heterogeneity of refractoriness is more significant in the peri-infarct zone than remote zone, and widely different monophasic action potential among the epicardial, mid-myocardial, and endocardial layers increased transmural heterogeneity of repolarization after MI. Moreover, we did not investigate the changes in transmural action potential during MNS. Whether MNS influences the transmural action potential after MI is unknown. Third, our previous study demonstrated that MNS attenuated stellate ganglion nerve activity and enhanced vagal nerve activity, whether MNS affects the tissue of vagus nerve and ganglion plexus-related changes is unknown, and harvesting cardiac ganglion plexus tissue for relevant assays will help further analysis. Fourth, monitoring imaging for the cardiac mapping and the hypo voltage area can better understand the mechanism of MNS in cardiac electrophysiology.This study revealed that MNS increases VFT induced by a train of rapid stimulation post-MI. Additionally, bilateral vagotomy suppresses the effects of MNS on VFT; these findings validate the hypothesis on whether the effects of MNS on VFT may be associated with the cholinergic anti-inflammatory pathway.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the Renmin Hospital of Wuhan University. Written informed consent was obtained from the owners for the participation of their animals in this study.QZ performed the conception or design of the work. XW, YQ, YY, YW, YZ, and SZ contributed to the acquisition, analysis, and interpretation of data for the work. XW and YQ drafted the manuscript. QZ and YY critically revised the manuscript. All authors gave final approval and agreed to be accountable for all aspects of the work ensuring integrity and accuracy.


Targeting of glucagon‐like peptide 1 receptor (GLP‐1R), expressed on the surface of pancreatic β‐cells, is of great interest for the development of advanced therapies for diabetes and diagnostics for insulinoma. We report the conjugation of exendin‐4 (Ex‐4), an approved drug to treat type 2 diabetes, to poly‐γ‐glutamic acid (γ‐PGA) to obtain more stable and effective GLP‐1R ligands. Exendin‐4 modified at Lysine‐27 with PEG4‐maleimide was conjugated to γ‐PGA functionalized with furan, in different molar ratios, exploiting a chemoselective Diels‐Alder cycloaddition. The γ‐PGA presenting the highest number of conjugated Ex‐4 molecules (average 120 per polymeric chain) showed a double affinity towards GLP‐1R with respect to exendin per se, paving the way to improved therapeutic and diagnostic applications. Exendin‐4‐γ‐polyglutamic acid conjugates have been generated by efficient Diels‐Alder click reaction. The highly functionalized conjugate (average 106 Exendin‐4 molecules/γ‐Polyglutamic acid) showed affinity towards GLP1 receptor higher with respect to Enendin‐4 a labile peptide approved for type 2 diabetes treatment. Type 2 diabetes is an unresolved pathology, related to genetic and environmental factors, which gradually decline the pancreatic islet functionality, finally culminating in a pathological phenomenon defined as hyperglycemia.The conjugation of Ex‐4 with biodegradable FDA approved polymers in a multivalent fashion can represent an innovative, affordable and effective therapeutic solution. Therefore, the simultaneous exhibition of multiple GLP‐1R agonists may lead to a cluster effect, enhancing the relative affinity of each of them towards the target receptor.40, 41Herein we present an efficient and chemoselective protocol for the conjugation of Ex‐4 to γ‐PGA. γ‐PGA exhibits multiple attractive properties, including absence of toxicity, biodegradability, edibility, and water solubility.A γ‐PGA‐Ex‐4 conjugate may represent the starting point for the development of novel therapeutic polymeric nanotools targeting β‐cells with high specificity. γ‐PGA, and therefore γ‐PGA‐Ex‐4 conjugate, is stable in aqueous solutions, requiring high temperatures and low pH valuesThe generation of polymer conjugates for therapeutic purposes requires efficient, chemoselective and clean protocols usually defined click reactions. Their attractive features include very high yields (quantitative), modularity, absence of toxic byproducts.60, 61We developed an efficient click chemistry protocol based on Diels‐Alder cycloaddition to conjugate Ex‐4 to γ‐PGA and evaluated the affinity of the γ‐PGA‐Ex4 conjugate towards GLP‐1 receptors of β‐TC6 cells  as condensing agents.Diels‐Alder cycloaddition reaction was selected to conjugate of Ex‐4 to γ‐PGA Scheme . To thisDifferent degrees of functionalization (%DoF), 2.1 %, 8.5 % and 16.8 %, were obtained adjusting the amount of FA and EDC/NHS .The assignment of the peaks in NMR spectra was first carried out for γ‐PGA‐FA‐1H‐NMR peak integral at 6.2, 6.3 and 7.4 ppm of furan‐H  vs. the γ‐PGA CHNH2 peak (H‐2) at 4.1 ppm and/or CH2−C−CN peak at 2.3 ppm (H‐3) , C=C bend and C−O−C stretch of furan ring are detectable respectively at 745, 884 and 1016 cm−1. Furthermore, the peak of amide N−H bend and C=C stretch are visible respectively at 1533 and 1641 cm−1.FT‐IR analysis confirmed the functionalization with furan, by comparison between the derivatized and the untreated γ‐PGA Figure C. The sp1, γ‐PGA‐FA‐2 and γ‐PGA‐FA‐3 were employed to generate the γ‐PGA‐Exendin‐4 conjugates  by Diels‐Alder reaction with Ex‐4 functionalized with PEG4‐maleimide at Lys‐27 (Ex4‐PEG‐MA). The reactions were performed in PBS buffer 0.01 м at pH 7.5. The purified products were analyzed by 1H‐NMR and SEC‐HPLC , at 2.4–2.3 ppm, and the signals of the aromatic residues of conjugated Ex‐4  at 7.4–6.9 ppm.The derivatization of γ‐PGA with Ex‐4 was evaluated by In SEC‐HPLC analyses, the elution of γ‐PGA‐Ex‐4 compounds, as well as γ‐PGA and Ex4‐PEG‐MA as controls, was monitored at 214 and 280 nm. The derivatized polymers experience a delay in the elution with respect to γ‐PGA, presumably due to the conformation change caused by the peptide conjugation. The protein content was determined from the ratio between the area of the peak at 280 nm, absent in the starting γ‐PGA‐FA, and the peak at 214 nm  for the fluorescent anti‐Ex‐4 antibodies. The quantification of antibodies bound to the polymers was determined in a competitive flow cytometric assay. The calibration using known amounts of free Ex‐4 allowed to determine the average number of Ex‐4 molecules/γ‐PGA molecules. The results are in good correlation the data obtained by SEC‐HPLC and MR Table .The binding ability of γ‐PGA‐Ex‐4 compounds to GLP‐1R expressed on β‐cells surface was examined in a competitive flow cytometric assay. This receptor binding assay was developed based on similar, receptor targeting studies, of Ex‐4 targetingThe results are plotted on Figure Conjugation of Ex‐4, an approved drug for diabetes type‐2 with γ‐PGA, a biocompatible and biodegradable FDA approved polymer, resulted in multivalent adducts with high affinity for GLP‐1R. The conjugation of Ex‐4 to γ‐PGA assures two key progresses: an increased affinity for GLP‐1R for the more intensively functionalized γ‐PGA‐Ex‐4 conjugate, and an increased stability due to the polymeric component. The γ‐PGA‐Ex‐4 conjugate presenting an average of 120 conjugated Ex‐4 molecules showed a double affinity towards GLP‐1R with respect to Ex4‐PEG‐MA. These results open the way to new improved therapeutic for diabetes and pancreas tumors.Materials: Ex‐4‐PEG4‐Maleimide with the sequence HGEGTFTSDLSKQMEEE‐AVRLFIEWLysKNGGPSSGAPPP S‐NH2 was purchased from Caslo ApS (Denmark) as freeze‐dried. Poly‐γ‐glutamic acid (γ‐PGA), with a molecular weight of 100 kDa, was gently granted by BBS Nanotechnology Ltd. (Hungary). N‐(3‐dimethylaminopropyl)‐N’‐ethylcarbodiimide hydrochloride , 2‐(N‐morpholino) ethanesulfonic acid hydrate , 2‐(aminomethyl)furan (FA), phosphate buffered saline  and deuterium oxide were purchased by Sigma‐Aldrich (Germany). Polymers purification was performed using cellulose membranes  or Vivaspin tubes with a molecular weight cut‐off of 30 kDa, MilliQ water and a solution 0.1 м NaCl in MilliQ. The polymers have been freeze‐dried using a ALPHA 1–2 LO freeze‐dryer.γ‐PGA functionalization with furan : γ‐PGA  were solubilized in MES buffer solution 0.1 м pH=5.5 (10 mL). EDC/NHS  were added as powder and the solution stirred at room temperature for 30 minutes. Following, FA was added dropwise  and left reacting for 24 hours. The solution was dialyzed in a cellulose membrane dialyzing tube with a molecular weight cut‐off of 14 kDa, against NaCl 0.1 м and then MilliQ water, for 48 hours. Finally, the solution was freeze‐dried, giving the products γ‐PGA‐FA‐1, γ‐PGA‐FA‐2 and γ‐PGA‐FA‐3 respectively as powder.Conjugation of γ‐PGA‐FA with Ex‐4. : γ‐PGA‐FA‐1 , solubilized in PBS 0.01 м pH=7.5 (1 mL) was treated with 1 eq. of Ex4‐PEG‐MA solubilized in PBS pH=7.5 . After stirred overnight at room temperature, the product was isolated with PBS in Amicon® Ultra‐15 Centrifugal Filter Units (MWCO 30 kDa) at 5,000 g for 10 minutes 15 times, and finally freeze‐dried. γ‐PGA‐FA‐2  and γ‐PGA‐FA‐3  were separately treated with the same protocol, with 1 eq of Ex4‐PEG‐MA solubilized respectively 2 and 4 mL of PBS pH=7.5.NMR analysis: All the analyzed samples were dissolved in D2O‐d2 . 1H‐NMR spectra were acquired using a Bruker BioSpin FT‐NMR AvanceTMI 600 (1H frequency=600 MHz) equipped with a superconducting ultrashield magnet of 14,1 Tesla, with pulse field gradient module (Z axis) and a tunable 5 mm reverse broadband BBI probe NMR, with high sensitivity on 1H nucleus and on a Bruker BioSpin FT‐NMR AvanceTM 500 (1H frequency=500 MHz) equipped with a superconducting ultrashield magnet of 11.7 Tesla, with pulse field gradient module (Z axis) and a tunable 5 mm direct QNP probe , to increase sensitivity on 13C nucleus., setting the pulse angle at 90° and the relaxation delay at 5 seconds, while the number of scans was varied between 160 and 320, depending on the signal‐to‐noise ratio.FI‐IR analysis: FT‐IR spectra have been performed with a spectrometer PerkinElmer Spectrum 100 equipped with a Universal ATR (UATR). The range of acquisition is 4000–650 cm−1 with a resolution of 2 cm−1, while the absorbances of the samples and backgrounds were measured using 30 scans each.HPLC‐SEC analysis: Size exclusion chromatography analysis was carried out with a Waters e2695 Separations Module  HPLC system using an Ultrahydrogel 1000 column  equipped with a PDA detector (Waters 2998 PDA detector). The flow rate was set to 0.60 mL/min using gradient elution and column was maintained at 303 K. The eluent was 100 % PBS buffer which pH was adjusted to 7.4 and changed to 90 % PBS: 10 % Methanol from the 35th minute.Determination of average number of Ex4 molecules conjugated to γ‐PGA molecules (antibody‐based assay): γ‐PGA‐Ex‐4 compounds were analyzed based on their recognition by Alexa Fluor 488 conjugated polyclonal anti‐Ex4 antibody (Bioss Inc.). The number of antibodies bound to the polymers was determined in a competitive flow cytometric assay, Ex4‐conjugated polymers competed with Ex4‐coated beads for the A488‐anti‐Ex‐4.Preparation of Ex‐4 coated beads: avidin‐coated beads  were mixed to six times excess of Ex4‐biotin (AnaSpec Inc) in PBS‐0.1 % BSA at RT, after 0.5 h the beads were washed two times with 1 mL PBS‐0.1 % BSA .6 Ex‐4 coated beads in presence of 5 μg/mL A488 conjugated anti‐Ex‐4 in 60 μl PBS‐0.1 %BSA. After 1 h incubation at 310 K the beads were washed with 1 mL PBS‐0.1 %BSA  and the fluorescence of the beads was measured by flow cytometer . Flowing Software was used for data evaluation. The calibration of this assay with known amounts of free Ex‐4 allowed the determination of the average number of Ex‐4 molecules/γ‐PGA molecules.The competitive test: the Ex‐4‐containing samples in different concentration were incubated with 3×10Affinity test (cell‐based assay): GLP‐1R positive Beta‐TC‐6 cells (ATCC) were dissociated using TrypLE™ Express Enzyme (Gibco).5 cells were incubated with 10 nм fluorescein‐labelled Ex‐4 (Eurogentec) alone or together with the tested compounds at 310 K in RPMI supplemented with 10 % FBS. After 0.5 h the cells were washed with ice cold PBS and fluorescence of the cells was measured by flow cytometer . Flowing Software was used for data evaluation. The binding ability of γ‐PGA‐Ex‐4 conjugates and peptides to the GLP‐1R positive cells was calculated based on the inhibition of the fluorescent Ex‐4 derived signal. The relative affinities were defined as the ratio of molar concentrations of Ex4‐PEG‐MA and the conjugated sample required to displace 50 % of fluorescent Ex‐4 bound to the GLP‐1R of beta cells:5×10The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Hair loss or alopecia is an unpleasant symptom that exacerbates an individual's self-esteem and requires appropriate treatment. The Wnt/β-catenin signaling is a central pathway that promotes dermal papilla induction and keratinocyte proliferation during hair follicle renewal. GSK-3β inactivated by its upstream Akt and ubiquitin-specific protease 47 (USP47) has been shown to inhibit β-catenin degradation. The cold atmospheric microwave plasma (CAMP) is microwave energy enriched with mixtures of radicals. CAMP has been reported to have antibacterial and antifungal activities with wound healing activity against skin infection; however, the effect of CAMP on hair loss treatment has not been reported. We aimed to investigate the effect of CAMP on promoting hair renewal in vitro and to elucidate the molecular mechanism, targeting β-catenin signaling and YAP/TAZ, the co-activators in the Hippo pathway, in human dermal papilla cells (hDPCs). We also evaluated plasma effects on the interaction between hDPCs and HaCaT keratinocytes. The hDPCs were treated with plasma-activating media (PAM) or gas-activating media (GAM). The biological outcomes were determined by MTT assay, qRT-PCR, western blot analysis, immunoprecipitation, and immunofluorescence. We found that β-catenin signaling and YAP/TAZ were significantly increased in PAM-treated hDPCs. PAM treatment also induced β-catenin translocation and inhibited β-catenin ubiquitination by activating Akt/GSK-3β signaling and upregulating USP47 expression. In addition, hDPCs were more aggregated with keratinocytes in PAM-treated cells compared with control. HaCaT cells cultured in a conditioned medium derived from PAM-treated hDPCs exhibited an enhancing effect on activating YAP/TAZ and β-catenin signaling. These findings suggested that CAMP may be a new therapeutic alternative for alopecic treatment. Therefore, finding effective and alternative hair loss therapy is a key topic that needs further research.Hair is served as an important structure that fulfills an individual's self-confidence. Although hair loss is not considered a severe disease, it can impair an overall appearance, lowering self-esteem and quality of life. However, the FDA-approved drugs, minoxidil and finasteride, require repeated use for effective treatment, which has been shown to induce adverse effects3. The CAP has further developed into a cold atmospheric microwave plasma (CAMP) to generate a high density of radicals in the atmospheric condition. Compared to other conventional CAP, CAMP has several benefits in biomedical fields. This CAMP generates mild heat, probably providing additional benefits for hair growth, and plasma density or reactive species concentration can reach further distance from the device.The cold atmospheric plasma (CAP) is an ionized gas that contains mixtures of reactive oxygen species, reactive nitrogen species, ultraviolet (UV), and photons. CAP is ignited from a high-voltage electrode, and plasma is delivered to the target sites through the carrier gas at a low temperature. The plasma level can be controlled by adjusting the power consumption and gas velocity4, antibacterial activity5, anticancer activity6, and promotes stem cell proliferation7. Although CAP activates dermal papilla cell proliferation via Wnt/β-catenin signaling8, the effect of CAMP on stimulating hair growth has not been elucidated. In this research, we used the CAMP, a 2.45 GHz microwave energy containing a higher density of radicals than other types of CAP.Recent reports suggested that CAMP affects antifungal activity9. The β-catenin is usually located in the cytoplasm and translocated into the nucleus after phosphorylation at the serine 552 position10. However, the phosphorylation at the serine 33, 37, or threonine 41 sites of β-catenin recruits ubiquitin to bind to itself, resulting in the degradation of β-catenin11. The activated β-catenin is a transcriptional activator of cell proliferation-related genes, cyclin D1, and c-Myc. During the anagen phase, GSK-3β is also phosphorylated to an inactive form by its upstream suppressor Akt. Since GSK-3β regulates β-catenin ubiquitination, the inactivation of GSK-3β results in β-catenin stabilization, which induces DPCs and keratinocytes proliferation12. A study also found that USP47, a deubiquitination protein, significantly inhibits β-catenin ubiquitination and promotes cell proliferation13.The hair follicle cycle consists of three phases; anagen, catagen, and telogen. Among these processes, the β-catenin signaling pathway is vital in promoting dermal papilla cell (DPCs) induction and keratinocyte division, which is responsible for maintaining the hair follicular compartment during hair regeneration15. Therefore, we aimed to study the effect of CAMP on enhancing Akt/β-catenin and USP47 signaling pathways in hDPCs. We hypothesized that CAMP promotes hDPCs aggregation in co-culture conditions between hDPCs and HaCaT keratinocytes. Since the epithelial-mesenchymal interactions are the main mechanism for initiating hair follicle formation, we also determined the effect of conditioned media (CM) derived from hDPCs on inducing CAMP-treated HaCaT keratinocyte proliferation.In the bald area, hair follicles decrease in size, indicating a defective DPCs induction which contributes to an impairment of hair follicle renewal; however, restoring the β-catenin signaling pathway significantly induces hair follicle development16. To investigate the cell viability of PAM-treated cells, hDPCs were treated with different doses of PAM for 15 min and further incubated for 18 h after replacing media. Cell viability was evaluated by MTT assay. The results showed that PAM treatment significantly increases cell viability in hDPCs, compared with GAM control. In contrast, the other doses showed no significant difference  at 15 min and 1 h after treatment . Hence, we selected these genes to further investigate the expression levels in hDPCs by conducting qRT-PCR. Similarly, PAM significantly induced FER and USP47 expression, compared to GAM Fig. A. Based 20. To determine the effect of PAM on inducing the PI3K/Akt signaling pathway, hDPCs were treated with PAM or GAM, and mRNA expression levels were measured using TaqMan™ Array Human PI3K Signaling. As shown in the heat map -β, and notch signaling, have been implicated in the hair regeneration process. Among those, Wnt/β-catenin signaling has been well investigated.Alopecia, abnormal hair loss or baldness, is a disorder characterized by hair loss due to an interrupted hair production cycle. When a hair follicle is damaged, or this cycle is disrupted, hair may begin to fall out more quickly than regenerate, leading to symptoms such as hair falling out in patches or thinning. Some dogs, like humans, also exhibit hair loss when they have thickened skin, scaling, excessive shedding, genetic factors, and itching27. In addition, studies indicated that the Wnt-dependent signaling pathway in the epidermis is the principle for folliculogenesis and increases the number of regenerated hair follicles29. Efforts are made to target these signaling pathways to design novel therapies for alopecia. Our results demonstrated that CAMP induces phospho-β-catenin (S552) protein expression and nuclear translocation to activate its target genes, thereby enhancing the effects on hair growth.The concept of the involvement of Wnt/β-catenin signals in follicular development stemmed from the β-catenin activation for cell proliferation, and the secreted Wnt proteins bind with the Frizzled receptor, thereby inactivating glycogen synthase kinase-3β (GSK-3β), which is an enzyme responsible for phosphorylation and ubiquitination-mediated degradation of β-catenin. Subsequent studies have reported that genetic ablation of the β-catenin gene in the epidermis leads to the failure of placode morphogenesis in mice30. The Akt/PI3K signaling assessed by the gene expression array demonstrated that INPP5D (SHIP1), a protein that is a main regulator on PI3K/Akt signaling, is the potential gene upregulated by CAMP treatment. The western blot data confirmed that CAMP upregulated p-Akt and p-GSK3β protein expression in hDPCs. The downstream protein of Akt/PI3K and GSK-3β plays a role in the Wnt/β-catenin signaling pathway as β-catenin does not enter the degradation pathway. CAMP activates β-catenin activation via several mechanisms, including USP47 and Akt/PI3K signaling proteins.Moreover, CAMP also reduced the interaction between ubiquitin and phospho-β-catenin (Ser33/37/Thr41), diminishing β-catenin degradation. CAMP also increases mRNA and protein expression of USP47, a ubiquitin-specific proteinase that causes protein deubiquitination and then augments the protein level of β-catenin in the cells. Akt/PI3K signaling pathway is another important mechanism for hair maintenance and follicle regeneration18. YAP and TAZ also have been involved in hair growth, and YAP/TAZ activation increased cell growth and proliferation in dermal papillary cells18. This study demonstrated the induction of YAP and TAZ protein expression in hDPCs by CAMP treatment.Besides Wnt/β-catenin and Akt/PI3K signaling, YAP and TAZ are the well-known crucial downstream proteins and transcription activators in the Hippo pathway, which regulates cell proliferation, renewal, and differentiation. The studies reported that the inhibition of transcription factor TEAD in human/mouse keratinocytes contributes to reducing cell proliferation and activating cell differentiation. The expression of YAP and TAZ in the basal stem/progenitor cell layer of the epidermis is required for skin homeostasis and cell proliferation in the mouse model31. Our study suggests that CAMP affected many beneficial signaling pathways that increase cell proliferation in hDPCs.Moreover, the cell-cycle regulating markers, including c-Myc and cyclin D1, are also increased upon treating the cell with CAMP. The study reported that c-Myc was significantly expressed in the proliferative germinative epithelial cells, terminally differentiating matrix cells at the human follicle bulb, leading to hair fiber induction. c-Myc affects the follicles and plays a role in stem-transit amplifying cell switching, which is probably assumed as the site of human follicles stem cells32. Hence, we investigated the effect of CM-derived from hDPCs and the enhancing effect of PAM and CM on increasing protein expression of β-catenin signaling in keratinocytes (HaCaT cells). The results showed that CM-GAM increases the protein expression of β-catenin signaling compared to GAM, while CM-PAM indicates the enhancing effect on stimulation of β-catenin signaling  derived from DPCs contain growth factors that induces keratinocytes proliferationing Fig. D,E. This33. In this study, we found that PAM treatment can induce DPC aggregation, when co-cultured with keratinocytes. To elucidate the mechanism, we determine the mRNA expression of versican which plays a key role in maintaining DPCs induction during the anagen phase and promotes DPCs aggregation34. The result showed that PAM significantly upregulates versican expression, compared to GAM. Therefore, we concluded that PAM induces versican expression which subsequently promotes DPCs aggregation surrounded by keratinocytes.For DPC aggregation analysis, the studies showed that DPCs become inductive and aggregate into the center of the keratinocytes to enhance the interaction between epithelium and mesenchyme during the anagen phase35, surface decontamination of fruits36, or deposition of thin layers37. Our finding suggested that CAMP has a therapeutic effect on stimulating hair regrowth through Akt/β-catenin signaling, YAP/TAZ Hippo pathway, and USP47-associated β-catenin deubiquitination in hDPCs , Fetal bovine serum (FBS), phosphate-buffered saline (PBS), Penicillin–Streptomycin, SuperSignal™ West Femto Maximum Sensitivity Substrate, and Pierce™ BCA Protein Assay Kit were purchased from Thermo Scientific . 3--2,5-diphenyltetrazolium bromide (MTT) and Akt1/2 kinase inhibitor (Cat. #A6730) was purchased from Sigma-Aldrich . RIPA lysis buffer was purchased from Biomax, Seoul, Korea. The fluorescence mounting medium (S3023) was purchased from Agilent, CA, USA. Mouse anti-β-catenin  was purchased from BD Biosciences, Franklin Lakes, USA. Rabbit anti-cyclin D1 (Cat. #92G2), rabbit anti-c-Myc (Cat. #5605P), rabbit anti-YAP/TAZ (D24F4) (Cat. #8418S), rabbit anti-p-β-catenin (S552) (Cat. #9566P), rabbit anti-p-β-catenin (S33/37/T41) (Cat. #9561P), rabbit anti-p-Akt (S473) (Cat. #9271), rabbit anti-Akt (Cat. #9272), p-GSK-3α/β (Ser21/9) (Cat. #9331) were purchased from Cell Signaling Technology . Mouse anti-USP47 (Cat. #SC-100633), mouse anti-HA tag (F-7) (Cat. #SC-7392), mouse anti-β-actin (C4) HRP (Cat. #SC47778), HRP-conjugated anti-GAPDH antibody (G-9) (Cat. #SC-365062 HRP) were purchased from Santa Cruz Biotechnology . Goat anti-rabbit IgG (H + L) Secondary antibody HRP conjugated  and Rabbit anti-Mouse IgG (H + L) Secondary antibody HRP conjugated  were purchased from Invitrogen .2 humidified incubator at 37 °C.Human hair DPCs (hDPCs) were purchased from ScienCell . Cells were cultured in mesenchymal stem cell medium  containing 10% FBS, 1% mesenchymal stem cell growth supplement , and 1% penicillin–streptomycin. The immortalized human keratinocyte (HaCaT) cells were purchased from Cell Line Service . Cells were cultured in high glucose DMEM containing 10% FBS and 1% penicillin–streptomycin. Both cells were cultured and maintained in a 5% COA CAMP device  was used for producing plasma in this research  and were incubated with the same condition as above  for 40 s or PAM  for 10, 20, or 40 s. After 18 h incubation, cell viability was investigated by adding an MTT reagent and further incubated for 3 h. The purple formazan precipitate was dissolved by adding 10% SDS and incubated overnight at 37 °C. The absorbance was measured by a microplate reader at 570 nm .The hDPCs were seeded in 96-well plates at a density of 3 × 105 cells/well, and then the cells were treated with GAM or PAM at 30W for 10 s. After incubation for 18 h, RNA was extracted using Trizol reagent  and was converted to cDNA using Verso cDNA Synthesis Kit . The quantitative RT-PCR was performed using Applied Biosystems™ SYBR Green Master Mixes  and analyzed by QuantStudio 1 Real-Time PCR System . The lists of primers are indicated below.hDPCs were seeded in 6-well plates at a density of 1 × 10The hDPCs were seeded and treated with the same conditions as quantitative RT-PCR. After RNA extraction, cDNA synthesis and reaction preparation were conducted, followed by the protocol from TaqMan™ Array Human PI3K Signaling . Briefly, the cDNA sample (50 ng per 20 µL of reaction) was mixed with 2 × TaqMan® Fast Advanced Master Mix  to obtain the final volume of 2,160 µL. The cDNA‑Master Mix was added to the 96‑well Standard (0.2 mL) TaqMan® Array Plates at 20 µL per well. The plates were sealed with microplate sealing film . The experiments were set up following the protocol and analyzed by QuantStudio 1 Real-Time PCR System.5 cells/well, and then the cells were treated with GAM or PAM at 30W for 10 s. For p-β-catenin and USP47 analysis, cells were further incubated in the replaced media for 15 min and 1 and 2 h after GAM or PAM removal, respectively. After incubation, cell proteins were extracted with RIPA buffer, including a protease inhibitor. The proteins were sonicated and centrifuged at 14,000 rpm, 20 min, and 4 °C. The supernatants were collected, and the proteins were measured by Pierce™ BCA Protein Assay Kit . The proteins were equalized with 5 × SDS-PAGE diluted in RIPA buffer and heated at 98 °C for 5 min. Then, 5% stacking and 8% or 10% separating acrylamide gel were used for electrophoresis, and polyvinylidene fluoride (PVDF) membranes (0.2 μm) were used for protein transfer. The membranes were blocked with 5% non-fat dry milk in TBST (0.1% Tween 20) for 30 min before being incubated with primary antibodies overnight on the shaker at 4 °C. Goat anti-Mouse/Rabbit IgG (H + L) antibodies   were used as secondary antibodies. Supersignal West Femto maximum sensitivity substrate  was used for visualization. The membranes were detected by Alliance Q9 advanced chemiluminescence imager , and the band intensity was calculated by Image J software (Version 1.53).The hDPCs were seeded in 60-mm dishes at a density of 2 × 105 cells/dish. Cells were then transfected with HA tag-ubiquitin (Ub-HA) plasmids using PolyJet reagent according to the following protocol . Cells were treated with GAM or PAM at a low dose for 10 s and incubated for 15 min before harvesting protein. The proteins (0.5 mg) were incubated with HA-Tag mouse mAb or normal mouse IgG by rotating overnight at 4 °C. Then, the complexes were pulled down by adding Pierce™ Protein A/G Magnetic Beads . After incubation, the bead complexes were collected and washed with 0.05% TBST. The protein complexes were eluted by adding 1 × SDS-PAGE reducing buffer and rotated for 20 min at RT. The expression levels were determined by western blot analysis, and the membrane was probed with rabbit anti-p-β-catenin (S33/37/T41) for IP conditions and mouse anti-β-catenin for input conditions.The hDPCs were seeded in 10-cm cell culture dishes at a density of 8 × 105 cells/well. The cells were then treated with GAM or PAM for 10 s and further incubated for 15 min after replacing with fresh culture media. Cells were washed and fixed with cold PBS and 4% PFA, respectively. Cells were permeabilized and blocked using 0.3% Triton X-100 and 5% BSA, respectively. Cells were stained with rabbit anti-β-catenin (1:200) and mouse anti-USP47 (1:200) antibodies overnight at 4 °C. After washing, cells were stained with fluorescein goat anti-rabbit (H + L)  and Alexa Fluor 568 goat anti-mouse (H + L)  for 1 h at RT. Hoechst 33,342 (10 µg/mL) was used for nuclei staining. Cells were mounted on slides with anti-fade and sealed with a mounting medium. Cells were visualized under a confocal microscope, and the data were analyzed by Image J software.The hDPCs were seeded in PDL-coated cover slip placed on 6-well plates at a density of 1 × 105 cells/well density and seeded in 35-mm dishes. The cells were treated with GAM or PAM for 10 s. The cells were visualized on day 0 and day 8 by Nikon Eclipse Ti-U inverted microscope. For monitoring cell location, hDPCs were transfected with pmEGFP-C1  using PolyJet™ reagent. The cells were trypsinized and co-cultured with HaCaT cells. The co-cultured cells were treated as above. After incubation, both cells were stained with Hoechst 33342 (10 µg/mL) for 10 min at 37 °C. The cells were mounted on slides and visualized under a confocal microscope.For co-culture preparation, HaCaT and hDPCs were mixed in the same tube at 1 × 106 cells/well, and then the cells were treated with GAM or PAM at 30W for 10 s. After incubation, cells were replaced with CM derived from hDPCs and further incubated for 18 h before harvesting.For CM preparation, hDPCs and HaCaT cells were separately seeded in 60-mm dishes at a density of 1 × 10The results were indicated as means ± standard error (SE) with three independent experiments. The data were analyzed by Student's t-test and one-way analysis of variance followed by post hoc Dunnett test using GraphPad Prism 9 software. The p-values less than 0.05 were indicated as statistically significant.


Vasculogenic Mimicry (VM) refers to the capacity to form a blood network from aggressive cancer cells in an independent way of endothelial cells, to provide nutrients and oxygen leading to enhanced microenvironment complexity and treatment failure. In a previous study, we demonstrated that VE-Cadherin and its phosphorylation at Y658 modulated kaiso-dependent gene expression (CCND1 and Wnt 11) through a pathway involving Focal Adhesion kinase (FAK). In the present research, using a proteomic approach, we have found that β-catenin/TCF-4 is associated with nuclear VE-cadherin and enhances the capacity of malignant melanoma cells to undergo VM in cooperation with VE-Cadherin; in addition, preventing the phosphorylation of Y658 of VE-cadherin upon FAK disabling resulted in VE-Cadherin/β-catenin complex dissociation, increased β-catenin degradation while reducing TCF-4-dependent genes transcription (C-Myc and Twist-1). Uveal melanoma cells knockout for VE-Cadherin loses β-catenin expression while the rescue of VE-Cadherin (but not of the phosphorylation defective VE-Cadherin Y658F mutant) permits stabilization of β-catenin and tumor growth reduction in vivo experiments. In vivo, the concomitant treatment with the FAK inhibitor PF-271 and the anti-angiogenic agent bevacizumab leads to a strong reduction in tumor growth concerning the single treatment. In conclusion, the anomalous expression of VE-Cadherin in metastatic melanoma cells , together with its permanent phosphorylation at Y658, favors the induction of the aggressive VM phenotype through the cooperation of β-catenin with VE-Cadherin and by enhancing TCF-4 genes-dependent transcription. Vasculogenic Mimicry or Vascular Mimicry (VM) characterizes the capacity of establishing perfusion gateway in a variety of tumors by highly invasive, genetically deregulated tumors cells: vasculogenic because they diffuse plasma and red blood cells that flow around tumor cells and mimicry by considering that vessels are not authentic blood vessels and exclusively mimic the vascular function in an angiogenesis dependent manner but not excluding both pathways. Although VM formation is an indicator of highly invasive can cell phenotype, the mechanisms by which this particular configuration may participate to negatively affect the tumor outcome are not well understood. It has been suggested that VM formation may facilitate possible tumor perfusion and the physical connection between VM and blood vessels may also help the hematogenous propagation of tumor cells. There is a robust correlation between the histological presence of VM patterns in primary uveal and cutaneous melanoma and the resulting death by metastasis progression –3 constaβ-catenin signaling pathway is crucial to confer homeostasis of the cells and embryonic progress; it was significantly proven to be associated with tumor cell proliferation, apoptosis, invasion, self-renewal, and chemotherapy resistance in distinct tumor models, particularly in colon cancer [The Wnt/n cancer , 9. Wnt n cancer . It actsn cancer , 12. Oncn cancer . In the Non-endothelial VE-Cadherin expression has been examined in many different cancer types associated with VM. Although the expression of VE-Cadherin CD144 or CDH5) has been correlated with an endothelial context, its role in the transformation of tumor cells has received increasing attention in the last decades. We recently reported that the VM-prone cells express Y658 of VE-Cad through the intrinsically active FAK 4 or CDH5.Since the first description of β-catenin/VE-cadherin in 1995 , a vast To get more specific information about the consequences of the anomalous expression of VE-cadherin in the nucleus in VM prone cells we performed a proteomic analysis to identify VE-cadherin-interacting proteins. Proteomic analysis of proteins complexes to VE-cadherin using Liquid Chromatographic-Mass Spectrometry (LC-MS) in a cytosol-nucleus subfractionation Tables , S2 showTo confirm the association between β-catenin/FAK/VE-Cadherin identified by LC/MS, we performed a group of experiments aiming to analyze the status of these proteins in VM prone cells. Total expression of β-catenin or non-phospho β-catenin (active β-catenin), focal adhesion kinase (FAK) or the active form phospho FAK (Y397) were measured in different melanoma cell lines from either uveal melanoma cells: MUM 2B  and forms a complex with p120-catenin and the transcriptional repressor Kaiso or ZBTB33 in the nucleus of the cells. Immunoprecipitation experiments of VE-Cadherin and β-catenin were performed in the absence/presence of the FAK inhibitor PF-271 Fig.  or afterN = 79, TCGA) and cutaneous melanoma . Some of the alterations identified are drivers of different tumors and for VM formation: S1PR1 , PDGFRBt cancer ), LOXL2 t cancer ) and intWnt pathway-dependent gene expression is transduced by the nuclear effector β-catenin. Due to the lack of a DNA-binding domain, β-catenin binds indirectly Wnt-responsive genes by interacting with TCF/LEF family members .After the decoupling of the β-catenin/VE-Cadherin complex following VE-Cadherin silencing Fig. , the absWe further examined the effect of preventing VE-Cadherin phosphorylation in the recruitment of TCF-4 to target promoters by performing chromatin IP. FAKi treatment robustly decreased TCF-4 binding to the CCND1 and C-myc promoters and induced a moderate decrease of WNT-11 Fig.  and S1PRBecause of the elevated expression of FAK/Y397 in uveal melanoma samples with predominantly high levels of pVEC (Y658) by malignant melanoma cells, we investigated the cell’s capacity to generate de novo vasculogenic-like network in a 3D matrix (Matrigel) after the alteration of the upstream of TCF-4 target genes (Twist-1) and directly attack to β-catenin; results in Fig. To further substantiate the impact of the phosphorylation of Y658 of VE-Cadherin in the capacity to form VM and β-catenin stability, we performed a cytosol-nucleus subfractionation experiment in MUM 2B cells permanently knock-out for VE-Cad after gene editing with CRISPR/Cas9, which were rescued with either VE-Cadherin WT or the mutant/non-phosphorylable VE-Cadherin (Y658F) Fig. . The re-N = 6 per group), the combination of Bevacizumab plus PF-271 reduced global tumor progression, as well as the final tumor growth , . AlthougAberrant microcirculation, including VM, is a major driving factor for the formation of hypoxic areas and treatment failure in solid tumors by the aggressiveness and complex tumor microenvironment is mainly determined by an aberrant tumor microcirculation. VM is the predominant blood supply in early malignant melanoma tumor growth; prevailing endothelial-based vessels are assisted by VM in the later stages of tumor expansion . In addiFAKi prevents nuclear relocation of the complex and allows, in the context of VM formation during tumor development, to enhance TCF-4 activity by boosting the expression of TCF-4-dependent genes. For instance, silencing of β-catenin produces an important reduction of VE-Cadherin, suggesting that β-catenin is involved in its degradation probably by autophagy; about this degradation process, we have shown in a previous study  that theThe following reagents were used: Avastin/Bevacizumab (Roche) 5 mg/Kg I.P two days per week, PF-562271 (Selleckchem) 1 μM during 24 h in vitro assays and 30 mg/Kg oral gavage every two days in tumor xenograft assay, PND-1186 (Selleckchem) 1 μM during 24 h in vitro assays, XAV-939 2.5-5 μM during 24 h and G007-LK 5 μM during 24 h. Corning Matrigel Basement Membrane Matrix for in vitro angiogenesis experiments. Antibodies used were: Y658 VEC rabbit , VEC C-ter mouse , β-catenin mouse , non-phopho β-catenin mouse , phopho Y397 FAK rabbit , FAK rabiit , TCF-4/TCF7L2 rabbit , TNKS mouse , phopho-YAP S127 rabbit , YAP rabbit , α-tubulin mouse , lamin B1 rabbit  and PARP-1 mouse , Twist-1 rabbit , c-Myc mouse , TIE-1 rabbit .2 in incubator cells. cDNA of human VEC fused in-frame with GFP at the COOH-terminus (VEC-GFP), VEC Y658F was a kind of gift from Dr Masahiro Murakami. These constructs were subcloned into pcDNA3.1 (Invitrogen), and previously validate [Human melanoma cells MUM 2B (GNAQ mutant) , 55, C81validate .The effect of PF-271, XAV-939, and G007-LK after 24 h and siβ-catenin, siTWIST-1 after 48 h on the formation of tube-like structures in Matrigel (BD Biosciences) was resolved according to the manufacturer’s instructions and previously describe . After 4Total RNA was isolated by RNeasy Mini Kit (Qiagen) according to the manufacturer’s recommendations and previously described . Each reMUM2B knockout (ko) cells for the VE-Cad gene were composed using the CRISPR-Cas9 technology. Five different sgRNAs were designed using the Zhang Lab Optimized CRISPR design tool and cloned into the pL-CRISPR.EFS.GFP which was purchased from the Addgene public repository (#57818) and previously describe .Cultured cells were transiently transfected with an irrelevant siRNA: 5′- CCUACAUCCCGAUCGAUGAUG-3′, siVE-Cad: 5′- AGAUGCAGAGGCUCAUGAUTT -3′ 50 nM, siβ-catenin: 5′-CAGGGGGUUGUGGUUAAGCUCUU-3′ 50 nM, siTwist1: 5′- AAGCTGAGCAAGATTCAGACC-3′ 50 nM and siFAK: 5′-GGUUCAAGCUGGAUUAUUU-3′ 50 nM were transfected for 48 h using JetPrime (Polyplus transfection) according to the recommendations.6 cells per IP) confluence with PF-271 1 µM for 24 h with the respective controls. The culture medium was aspirated and cells were washed twice with cold PBS. ChIP was performed following SimpleChIP Enzymatic Chromatin IP Kit (Magnetics beads) . qPCR promoter specific primers used: c-Myc promoter, Forward 5′-GCTCAGTCTTTGCCCCTTTGTGG-3′, Reverse 5′- TAACACCTTCCCGATTCCCAAGTG-3′; CCND1 promoter [Cells were grown to approximately 80–90%  for 30 min at 4 °C and previously describe [Simple coimmunoprecipitation, cells were lysed in lysis buffer  for 10 min at 4 °C.Accordingly with the article of , for subTube-like 3D structures in Matrigel (BD Biosciences) were calculated according to the protocol from . Images q value ≤ 0.01.Cells were resuspended in 60 μl after IP-CE, NE subfractionation, were precipitated with acetone and resuspended in 50 mM ammonium bicarbonate solution containing 8 M urea. After reduction and alkylation, samples were digested with trypsin and desalted with Clean-Up. Enriched samples were analyzed on nano-LC-MS in Easy-nLC 1000 (Proxeon) coupled to an ionic source with nano electrospray (ThermoScientific). Raw files were analyzed in the Uniprot database using Sequest in Proteome Discoverer (ThermoScientific) (Proteomic department from UCO). Peptide identification was validated by Percolator using q < 0.25). Non-specific adjustment IP-IgG was measured as a control in LC-MS experiments  Fig. S. The liss Tables , S5.6 MUM 2B cells, MUM 2B KO, MUM 2B KO WT, and MUM 2B KO Y658F in 100 µL PBS were subcutaneously injected in the flank of 7-week-old mice. Animals (n = 6 per group) were monitored every two days after cell injection until the final time point when they were sacrificed and tumors were dissected for further analyses. Tumor volume was calculated as: in progress tumor volume = (π × length × width2)/6 and final tumor volume = (π × length × width × height)/6).Male Swiss Nude (SwN) mice were purchased from Charles River Laboratories and housed at IPBLN-CSIC and IBIS animal facility according to institutional guidelines . For xenograft generation, 2 × 10Immunofluorescence was performed on cells plated onto coverslips and grown for 24 h before experimental treatment. The cultured media was removed and washed two times with PBS 1× and the cells were fixed  for 15 min at room temperature. Permeabilization was handled using 0.25% Triton-X100 in PBS for 10 min and previously describe . Immunof2O2 aqueous solution for 30 min to exhaust endogenous peroxidase activity, then covered with 1% blocking reagent  in 0.05% Tween 20-PBS, to block nonspecific binding sites. Sections were incubated with primary antibodies for 1 h for anti-Y658 VE-Cadherin (Red) (Thermofisher) (1/50) and previously reported [Four-micrometer-thick tissue sections from paraffin blocks were dewaxed in xylene and rehydrated in a series of graded alcohols. Sections were immersed in 3% Hreported . The perFigS1FigS2FigS3FigS4Table S1Table S2Table S3Table S4Table S5Table S6Figure legends SuppOriginal Data Filechecklist