ABSTRACTXenopus oocytes leads to the inhibition of GSK-3β and cellular accumulation of β-catenin. This study investigated whether Gαq-mediated cellular accumulation of β-catenin could affect the transcriptional activity of this protein.  The canonical Wnt signal transduction or the Wnt/β-catenin pathway plays a crucial role in both carcinogenesis and development of animals. Activation of the Gαq class of Gα proteins positively regulates Wnt/β-catenin pathway, and expression of Gαq in HEK293T cells or HEK-293T and HT-29 cells were used for cell culture and transfection. Protein localization and quantification were assessed by using immunofluorescence microscopy, cell fractionation assay, and Western blotting analysis. Gene expression at the transcription level was examined by quantitative reverse transcriptase/real-time PCR method.c-MYC and CCND1) and the β-catenin/TCF reporter luciferase gene (TopFlash plasmid) significantly increased by Gαq activation. The Gαq-mediated increase in the expression level of the β-catenin-target genes was sensitive to the expression of a minigene encoding a specific Gαq blocking peptide. The results of cell fractionation and Western blotting experiments showed that activation of Gαq signaling increased the intracellular β-catenin protein level, but it blocked its membrane localization. Transcription of two cellular β-catenin target genes ( Our results reveal that the Gαq-dependent cellular accumulation of β-catenin can enhance β-catenin transcriptional activity.  Specificity in signal transduction pathway is mediated through many known and unknown mechanisms. One of these mechanisms is the cross-talk between the components of two or more signaling pathways. Both the heterotrimeric G protein and canonical Wnt/β-catenin signaling pathways play essential roles in various cellular processes and deregulation of these pathways leads to abnormalities in animal development, as well as many human diseases, including cancers. Translocation of β-catenin into the nucleus and its interaction with the TCF/LEF transcription factors leads to the transcriptional regulation of the genes involving in diverse cellular processes. In addition, β-catenin has an important role in maintaining epithelial tissues by interacting with the cadherin cell-cell adhesion proteins[4-5]. Activation of the canonical Wnt signaling pathway initiates by interacting the Wnt glycoproteins with the Frizzle/LRP5 receptors. While the mechanism of this pathway is still not well understood, its activation results in the inhibition of GSK activity and stabilization of cellular β-catenin protein[5], the possibility of regulating Wnt signaling by heterotrimeric G-proteins has been investigated by several researchers who demonstrated that heterotrimeric G-proteins regulate both canonical and non-canonical Wnt signaling pathways[6-11]. Formerly, we have also shown that activation of the Gαq class of G proteins in two different cell systems (Xenopus oocytes and HEK293T cells) leads to the inhibition of GSK activity and accumulation of β-catenin in the cell membrane. Given the structural similarity between GPCRs and Frizzled receptors in terms of possessing a heptahelical transmembrane domainIn this study, we have examined whether the Gαq-mediated increase in cellular β-catenin protein level affects the β-catenin-dependent gene transcription. Consistent with our previous results, we herein showed that Gαq signaling induces the transcription of the β-catenin target genes. Moreover, we provided preliminary results supporting that Gαq signaling increases the intracellular β-catenin level, at least partially, by inhibiting the membrane localization of this protein. Cell culture and transfection 2 at 37 °C. HEK293T cells are responsive to the canonical Wnt signaling and have therefore been used extensively by the researchers in the field of Wnt signaling pathways. At 60% confluency, the medium of HEK-293T cells was replaced with the fresh medium, and then two hours later, the cells were transfected with the expression plasmids. The standard calcium phosphate protocol was used for transfection[13]. Six hours post transfection, the medium was changed, and 48 hours later, the cells were harvested. The cell pellets were used directly or stored at -70 °C until use. Treatment of cells with carbachol (100 µM) or thrombin (0.5 U/ml) was performed 3 and 10 hours before the cell harvest, respectively. HEK293T and HT-29 cells were respectively grown in DMEM and RPMI 1640 medium supplemented with 10% FBS and antibiotics (100 µg/ml of streptomycin and 100 U/ml of penicillin) in 5% COCell fractionation and Western blotting analysis [13]. Briefly, the cells were suspended in a buffer containing 50 mM of Tris-HCl (pH 7.8), 100 mM of NaCl, 2 mM of EDTA, 1 mM of DTT, 0.5 mM of PMSF, 1.5 µM of pepstatin A and lysed by passing them 25 times through a 27-gauge needle. The lysates were centrifuged at maximum speed in a refrigerated microfuge (15.339 ×g) for 15 min, and the supernatants containing the cytoplasmic proteins were transferred to a new tube and stored at -70 °C until use. The pellet containing membrane proteins was rinsed using the above-mentioned buffer and centrifuged, accordingly. The washed pellet was dissolved in 120 µl of 50 mM of Tris-HCl (pH 7.8), 100 mM of NaCl, 1 mM of EDTA, 1 mM of DTT, 2% SDS, 100 mM of β-mecaptoethanol, 10% glycerol, 0.5 mM of PMSF, and 1.5 µM of pepstatin A. The suspension was heated in a 95 °C water bath for 4 min, vortexed and heated again at 95 °C for 3 min. The suspension was centrifuged (15.339 ×g) at RT for 30 min, and the supernatant was preserved as membrane proteins. Protein concentration was measured using a previously described method[14]. To this end, 20 µg of proteins from each fraction was separated by SDS-PAGE (8%) and transferred to a nitrocellulose membrane. Following electrical transfer, the gel was stained with Coomassie blue overnight to verify transfer efficiency. The membrane was washed using TBS  for 15 min and incubated in fat-free milk 5% (in TBS) at RT for 1 hour to block the nonspecific binding. The blot was then incubated with a polyclonal primary antibody against β-catenin at a 1:1000 dilution at RT for 1 hour. The blot was again washed four times  with TBS and incubated for another one hour with horseradish peroxidase-labelled goat anti-rabbit IgG (1:1000 dilution) at RT. The membrane was finally washed in TBS for 15 min, and the antigen-antibody complex was visualized using an ECL detection kit . Isolation of cytoplasmic and membrane proteins from HEK293T cells, as well as measurement of protein concentration, were performed as described beforeIndirect immunofluorescence [13]. In summary, HEK293T cells grown on glass coverslips were washed in PBS, fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 in PBS for three minutes. The coverslips were washed with PBS and then incubated in blocking buffer (3% BSA) for 30 min before being incubated in a 1:500 dilution of β-catenin antibody at RT for 1 h. The coverslips were again rinsed three times (10 min each) in PBS and then incubated with FITC-conjugated secondary antibody (1:250) at RT for 1 h. The coverslips were finally washed and visualized by a fluorescence microscope . Indirect immunofluorescence experiments were performed as described previouslyQuantitative reverse transcriptase and real-time PCR experiments GAPDH and c-MYC at 59 °C, CCND1 at 57 °C, and luciferase at 62 °C) for 60 s, and extension at 72 °C for 60 s. The 30 cycles of PCR were followed by a final extension at 72 °C for 10 min. PCR products were separated on a 1% agarose gel and visualized by ethidium bromide. The results of RT-PCR experiments were quantified by the Image J software. Real time-PCR experiments were performed using the QuantiFast™ SYBR Green PCR Master Mix kit  under the following programs: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 25 s, and extension at 72 °C for 25 s. For each 10 µl reaction, 1 µl of complementary DNA (100 ng) was used with 1 µl of each primer (1.0 µM) plus 5 µl of SYBR Green I Master Mix on a real-time thermo cycler Rotor Gene 6000 (Qiagen). Analysis was performed using Corbett rotor-gene 6000 software based on the comparative Ct method. The relative amount of the target gene expression was quantified relative to the reference gene (GAPDH) expression. The primers are listed in  HEK293T cells were harvested 48 hours post transfection with Trypsin/EDTA (0.53 mM of EDTA and 0.05% [w/v] Trypsin in PBS) and washed twice with cold PBS. Total RNA was extracted from the cell pellets using the RNX-Plus kit  as described by the supplier. Next, 2 µg of RNA was treated with 1 U of DNase I in a total volume of 10 µl at 37 °C for 30 min. The DNase enzyme was inactivated in 2.5 mM of EDTA at 65 °C for 10 min, and then the reaction was used for reverse transcription by adding 200 U of reverse transcriptase enzyme , 1× RT buffer, 20 U of RiboLock RNase Inhibitor, 1 mM of dNTP mix, and 0.2 µg of random hexamer primer in a total volume of 25 µl. PCR amplification was performed on 1 µl of the reverse transcription reaction using 30 pmol of each primer and 2.5 U of Taq polymerase in a total volume of 25 µl. The amplification protocol included denaturating at 95 °C for 60 s, annealing  and c-MYC plus the reporter luciferase gene cloned into the TopFlash plasmid[15].To evaluate β-catenin transcriptional activities, we selected two native cellular genes, [15-18], we measured their transcription in the presence and absence of exogenous β-catenin to further verify that the expression of the chosen genes can be induced by β-catenin  by about two-fold. This increase was blocked by the expression of a Gαq minigene, which encodes a short peptide corresponding to the C-terminal 11 amino acids of Gαq. The specificity of this peptide in blocking Gαq has previously been verified. We then examined if endogenous Gαq activation could induce β-catenin target gene expression. In this regard, we treated HEK293T cells with thrombin or carbachol. Thrombin is a known agonist for PAR1, a GPCR that preferentially couples to Gq [21]. Carbachol is a cholinergic agonist that activates M1/3 muscarinic acetyl choline receptors, the GPCRs that also couple to Gq[22]. We have previously shown that treatment of HEK293T cells with these two agonists leads to more than two-fold increase in the cellular protein level of β-catenin[13]. Treatment of HEK293T cells with these agonists (0.5 U/ml thrombin or 100 µM carbachol) did not significantly change the expression of the β-catenin target genes . Although there are reports that HEK293T cells endogenously express both PAR1 and muscarinic receptors, the expression levels may not be sufficient to induce Gαq-mediated β-catenin transcriptional activity. To verify this assumption, we used HT-29 colon cancer cells, which express higher levels of several Gq-coupled GPCRs including PAR1, as compared to the corresponding normal cells[25]. As shown in CCND1 and c-MYC gene transcription, the thrombin treatment led to about 2- to 2.5-fold increase in the expression of these two genes  plus the reporter luciferase gene  were selected[15]. As we intended to assess β-catenin transcriptional activity, RT-PCR  experiments were used to measure the luciferase gene transcription. Altogether, our results clearly showed that the Gαq signaling activation induced β-catenin transcriptional activity.We have previously reported that activation of the Gαq class of Gα proteins inhibits GSK activity, which in turn increases the intracellular level of β-catenin protein[7]. Furthermore, it has been reported that treatment of F9 teratocarcinoma cells with Wnt-3a activates a Gαq-mediated phosphatidylinositol signaling, which results in generating inositol polyphosphates such as IP5[10]. Evidence has suggested that transient accumulation of IP5 inhibits GSK-3β and, therefore, leads to the accumulation of β-catenin and TCF/LEF-dependent transcription[10]. Moreover, Gαq signaling is required for the Wnt-mediated disruption of Axin/GSK-3β interaction, which may result in β-catenin cellular stabilization[8]. β-catenin is a known proto-oncoprotein, which its upregulation is involved in tumorigenesis of several human cancers including colorectal cancer. It is believed that this protein is a highly potential target for cancer prevention and therapy[26-28]. In addition to the Gq class of heterotrimeric G-proteins, the involvement of other classes of G-proteins in the regulation of β-catenin expression and function has been reported[29]. GPCRs are the most diverse class of proteins in mammals involved in many critical cellular processes. Around 900 different GPCRs are encoded by human genome, which are the target of more than 30% of the approved therapeutic drugs. Regulation of β-catenin by heterotrimeric G-proteins raises an interesting question: would it be possible to target G-protein signaling pathways in order to control the expression and function of β-catenin in human cancers? The answer to this question definitely needs much further investigation. Fortunately, the data on the deregulation of G protein signaling pathways in human cancers is growing, and several excellent review articles have already been published, supporting that these important classes of proteins can be considered as potential targets for cancer therapeutics.In this study, we also found that the Gαq activation could inhibit β-catenin membrane localization . This isc-MYC and CCND1) and a β-catenin-responsive reporter gene. Since β-catenin deregulation is associated with many human cancers, our results further support this hypothesis that trimeric G-proteins and their receptors are among the potential targets for cancer therapy.In conclusion, the activation of Gαq signaling not only increases the intracellular β-catenin protein level but also results in the regulation of β-catenin nuclear activity. The present study clearly shows that the expression of the wild-type Gαq or activation of the endogenous Gαq causes transcriptional upregulation of two known β-catenin cellular target genes  to S Mahmoud A Najafi and also by the University of Tehran Research Department, Tehran, Iran.


Activation of the Wnt pathway has been linked to colorectal cancer (CRC). Previous reports suggest that Wnt3a can activate p38. Besides, p38α feeds into the canonical Wnt/β-catenin pathway by inhibiting GSK3β through phosphorylation. Recently, we identified p38α as a new druggable member of β-catenin chromatin-associated kinase complexes in CRC.Min/+ mice). The role of p38α in β-catenin transcriptional activity was assessed by pharmacological inhibition with ralimetinib.The functional relationship between p38α and β-catenin was characterized in CRC cells, patient-derived CRC stem cells, patient-derived tumor intestinal organoids, and in vivo models (C57BL/6-APCWe used the GSK3β inhibitor TWS-119, which promotes the activation of Wnt signaling, to uncouple p38α nuclear/cytoplasmatic functions in the Wnt pathway. Upon GSK3β inhibition, nuclear p38α phosphorylates β-catenin at residues S111 and T112, allowing its binding to promoter regions of Wnt target genes and the activation of a transcriptional program implicated in cancer progression. If p38α is pharmacologically inhibited in addition to GSK3β, β-catenin is prevented from promoting target gene transcription, which is expected to impair carcinogenesis.p38α seems to play a dual role as a member of the β-catenin destruction complex and as a β-catenin chromatin-associated kinase in CRC. This finding may help elucidate mechanisms contributing to human colon tumor pathogenesis and devise new strategies for personalized CRC treatment.The online version contains supplementary material available at 10.1186/s13578-023-01175-4. APC) gene on chromosome 5q (90%) or by CTNNB1 activating mutations on chromosome 3p (5%). One of the hyperproliferating cells may then give rise to a small adenoma by clonal expansion. Subsequently, the occurrence of mutations in the RAS pathway  is the main malignant tumor affecting the gastrointestinal tract and the third most common cancer in both men and women , 2. CRC se death .o and Gαq as well as the downstream phosphoprotein Dishevelled, thereby inhibiting GSK3β activity. Stabilized β-catenin then translocates into the nucleus and forms complexes with members of the T cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors, exerting its oncogenic role by activating the transcription of many regulatory genes, such as c-Myc [Activation of the Wnt pathway has been linked to CRC; indeed, abnormalities of chromosome 5q have been recognized as early events in the carcinogenic process leading to sporadic and hereditary CRCs . In the as c-Myc .Recent advances suggest the existence of a functional relationship between the Wnt and p38 MAPK signaling pathways.Currently, p38 is believed to act as a tumor suppressor in normal cells at the onset of cellular transformation, while it seems to function as an oncogene after the acquisition of a malignant phenotype , 12. OurVarious studies have suggested that p38α plays a role in chemoresistance by supporting survival or delaying ongoing cell death processes –18. Indep38α is relatively inactive in its non-phosphorylated form and becomes rapidly activated upon phosphorylation of two Thr-Gly-Tyr motifs , 20. Phoq and Dishevelled, which ultimately promotes transient activation of p38 [Recent findings identified crosstalk between the p38 and Wnt pathways. Indeed, Wnt ligands have been reported to be able to activate p38. In totipotent mouse F9 teratocarcinoma cells, Wnt3a binds to Frizzled and activates Gαn of p38 . Moreoven of p38 . On the n of p38 . Recentln of p38 .Despite this evidence, the exact role played by p38α in the Wnt signaling pathway remains unclear. Elucidating the underlying molecular mechanisms might be beneficial for designing new therapeutic strategies to counter chemoresistance and improve treatment response in CRC patients.HCT-116 and HT-29 cells were purchased from ATCC and cultured in DMEM  with 10% FBS  and 100 IU/mL penicillin–streptomycin .l-glutamine , 10 ng/mL bFGF , 20 ng/mL EGF , B27 supplement , and N-2 supplement . Patient-derived CRC organoids were generated using IntestiCult™-SF Organoid Growth Medium (Human)  according to the manufacturer’s instructions.CRC-stem cells (CRC-SCs) were isolated from CRC patients and propagated as tumorspheres. Briefly, human colon tissue fragments were obtained in accordance with the ethical standards of the Institutional Committee on Human Experimentation after informed consent. Tumor samples were subjected to mechanical and enzymatic dissociation using the human Tumor Dissociation Kit  according to the manufacturer’s instructions. For magnetic separation, cells were labeled with CD44 microbeads  24–48 h after dissociation using the Miltenyi Biotec cell isolation kit . CRC-SCs were maintained in DMEM/F12 Advanced  supplemented with 6 mg/mL glucose , 2 mM 2 incubator.All cell lines were tested to be mycoplasma-free . All cell cultures were performed in a 37 °C and 5% COHCT-116 cells were transfected with 50 nM validated siRNAs (Ambion) directed against (p38α) MAPK14 using HiPerfect reagent  according to the manufacturer’s instructions.Min/+ mice (n = 24) treated with azoxymethane (AOM) , respectively. Four-month-old APCMin/+ male mice were administered with AOM (14 mg/kg body weight) once a week for 5 weeks; one month later, they were subjected to daily intraperitoneal injections of the p38α inhibitor SB202190  (0.05 μmol/kg body weight) or DMSO for 14 days and then sacrificed. Body weight was recorded daily. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that comply with national and international laws and policies.For in vivo studies, normal and adenocarcinoma colon mucosa tissues were obtained from C57BL/6 mice (n = 12) and from APC2O. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 min. Antigen retrieval was conducted in 10 mM sodium citrate buffer (pH 6.0) for 30 min. Sections were incubated overnight with anti-p38α  or anti-β-catenin  primary antibodies. Then, they were incubated with secondary biotinylated antibodies and subsequently with streptavidin–biotin-peroxidase . Samples were developed with DAB and mounted with permanent mounting media. Negative controls were used in each experiment. p38α and β-catenin immunoreactivity was evaluated through a semiquantitative approach by two independent pathologists. Ten fields with an equal area were selected for analysis at 40 × magnification. Protein expression was assessed with ImageJ software and reported as a positivity percentage.Tissue specimens were fixed in 4% buffered formalin, embedded in paraffin, and sectioned at 4 mm thickness. Sections were dewaxed and rehydrated in dHTWS-119 (S1590) and ralimetinib (S1494) were purchased from SelleckChem. Wnt3a (H17001) and SB202190 (S7067) were purchased from Sigma-Aldrich.Nuclear and cytoplasmic fractions were obtained by using the Nuclear Extraction Kit  according to the manufacturer’s instructions. Immunoblots were carried out as previously described [Primary antibodies: anti-p38α MAPK (9228), anti-Lamin B1 (12586S), anti-PDI (2446S), anti-β-catenin (9562S), anti-c-Myc (9402), anti-p-β-catenin (9561), anti-p-GSK3β (35480), anti-GSK3β (9315), and anti-Vinculin (13901) all from Cell Signaling Technologies. Secondary antibodies: Rabbit IgG HRP and Mouse IgG HRP . Densitometric evaluation was performed using ImageJ software.Cells were seeded on glass coverslips, fixed in 4% paraformaldehyde, and permeabilized using 0.01–0.1% Triton X-100. Coverslips were incubated with the indicated primary antibodies and then with Alexa Fluor 488  and 647  secondary antibodies; nuclei were counterstained using DAPI . Slides were sealed using Vectashield Mounting Medium . Images were acquired using a Zeiss fluorescence microscope. Primary antibodies: p38α , β-catenin , c-Myc , and FLAG .Total RNA was isolated using TRIzol reagent  according to the manufacturer’s instructions. To avoid possible DNA contamination, RNA was treated with DNase-1 . RNA purity was confirmed by spectrophotometry and RNA integrity by agarose gel electrophoresis. cDNA was synthesized by retro-transcribing 1 μg of total RNA using the iScript cDNA Synthesis Kit  according to the manufacturer’s instructions. Real-time PCR primers were designed using Primer Express software. PCR assays were performed in 96-well optical reaction plates using a QuantStudio 3 machine (Thermo Fisher Scientific). Each assay was carried out in triplicate wells. Baseline values of amplification plots were set automatically and threshold values were kept constant to obtain normalized cycle times and linear regression data. The following reaction mixture per well was used: 5 μL of SYBR Green PCR Master Mix , 1.2 μL of primers at a final concentration of 100 nM, 2.3 μL of RNAse-free water, 1.5 μL of cDNA. For all experiments, the following PCR conditions were used: denaturation at 95 °C for 30 s, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C. Quantitative normalization of cDNA in each sample was performed using β-actin as an internal control. Relative quantification was done using the ddCT method. Primer sequences are available upon request. Results are representative of at least three independent experiments.SDM_ beta-catenin _S111A_FW 5'-P-GCAGATCCCAGCTACACAGTT,SDM_ beta-catenin _S111A_RV 5'-P-ATGCCCTCATCTAATGTCTCA,SDM_ beta-catenin _T112A_FW 5'-P-GATCCCATCTGCACAGTTTGATG.SDM_ beta-catenin _T112A_ RV 5'-P- TGCATGCCCTCATCTAATG.The plasmids described in this manuscript were generated with specific primers, as previously described . Site-diThe pGEX-4T-3 GST-beta-catenin Wild-Type, pGEX-4T-3 GST-beta-catenin S111A, and pGEX-4T-3 GST-beta-catenin T112A constructs were generated by cloning the relevant β-catenin fragments from human beta-catenin pcDNA3, human beta-catenin pcDNA3-S111A, and human beta-catenin pcDNA3-T112A, respectively, into the pGEX-4T-3 GST Expression Vector , using the BamHI and NotI enzymes.g for 20 min at 4 °C. Recombinant protein expression was confirmed by SDS-PAGE. GST-Fusion proteins were purified using Pierce Glutathione Magnetic Agarose Beads  according to the manufacturer’s instructions. GST-fused proteins were evaluated and quantified by SDS-PAGE.BL21(DE3) Competent Cells  transformed with different constructs were grown in Luria Broth medium with ampicillin  and induced with 200 nM IPTG when they reached the optical density of 0.6 (A600) at 37 °C, overnight. Cells were then collected by centrifugation, and pellets were lysed with B-PER lysis buffer . The lysate was centrifuged at 20,000×Cells were collected and homogenized in lysis buffer  supplemented with protease and phosphatase inhibitors. Coupling was performed between Dynabeads Protein A or G  and antibodies in 100 μL of 0.01% Tween20-1X PBS for 45 min at room temperature on a rocking platform. Cell lysates were immunoprecipitated with antibody-bead complexes. Immunocomplexes were washed extensively and boiled in Laemli sample buffer and subjected to SDS-PAGE and immunoblot analysis. Input corresponds to 10% of the lysate. IgGs were used as negative controls. Primary antibodies: p38α , β-catenin , GSK3β , APC , Axin1 , FLAG-M2 . Rabbit IgG HRP  was used as a secondary antibody and revealed using the ECL-plus chemiluminescence reagent . Results are representative of at least three independent experiments.2 resin for phospho-enrichment. Then, the flow-through was treated with C18 Spin Tips & Columns  for desalting. The samples enriched for phospho-peptides and the desalted flow-through were further purified with SP3 and then analyzed by nLC-ESI–MS/MS on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) with a 32 min gradient. Samples were run in technical duplicate, in a positive mode with electrospray ionization. Data acquisition and processing were performed with Analyst TF . Data were analyzed using the Proteome Discoverer, Mascot, and Scaffold setting software. The parameter settings of data processing were as follows: DataBase = Uniprot_CP_Human_2020_GST_CTNB1 ; Enzyme = Trypsin ; Modifications = Acetyl (Protein N-term), Carbamidomethyl (C), Oxidation (M), Phosphorylation (STY); Peptide Thresholds: 95.0% minimum; Protein Thresholds: 99.0% minimum; 2 peptides/protein minimum.Mass spectrometry analysis was performed by the Cogentech SRL service. Gel bands were subjected to reduction with dithiothreitol (DTT) , alkylation with iodoacetamide (IAA) , and digestion with trypsin . Peptides were loaded onto a TiOChromatin isolated from HT-29 and HCT-116 cells was subjected to chromatin immunoprecipitation using the MAGnify Chromatin Immunoprecipitation System  according to the manufacturer’s instructions. Briefly, cells were cross-linked in 1% formaldehyde  for 10 min. Cross-linking was blocked with 0.125 M glycine  for 5 min; then, cells were washed with 1 × PBS and lysed in lysis buffer. Chromatin was sonicated to a fragment length of about 200–500 bp and immunoprecipitated with 1 µg of anti-p38α , anti-β-catenin , rabbit IgGs, FLAG-M2 , or mouse IgGs. Quantitative real-time PCR was performed using SYBR Green IQ reagent  with the CFX Connect detection system (Bio-Rad Laboratories). Primer sequences are available upon request. Results are representative of at least three independent experiments.The analysis of p38α kinase activity was performed using the ADP-Glo Kinase Assay  according to the manufacturer’s instructions. 10 ng of p38α active protein  were assayed in a kinase reaction buffer with 500 ng of human recombinant β-catenin protein , 150 μM ATP, and varying concentrations  of ralimetinib . A total of 500 ng of p38 peptide substrate was used as a control. The generated luminescence was measured using a SPECTROstar Omega microplate reader (BMG Labtech).ddPCR was performed using 1 ng of RNA extracted from the HCT-116 CRC cell line or 20 ng of RNA extracted from patient-derived CRC-SC tumorspheres using the Purelink RNA Micro Kit  and retro-transcribed to cDNA using the SuperScript VILO cDNA Synthesis Kit  according to the manufacturer’s instructions. Reactions were prepared using the ddPCR Supermix for Probes Kit  according to the manufacturer’s instructions. 20 µL of each reaction mix were converted into droplets with the QX200 Droplet Generator (Bio-Rad Laboratories). The droplets were transferred to a 96-well plate, sealed, and cycled in a C100 Thermocycler (Bio-Rad Laboratories) under the following cycling protocol: 25 °C for 3 min, 95 °C for 10 min followed by 40 cycles of 94 °C for 30 s and 60 °C for 1 min, a post-cycling step of 98 °C for 10 min, and infinite hold at 4 °C. The plate was then transferred into a QX200 Reader (Bio-Rad Laboratories).For patient-derived CRC organoids, 10 ng of RNA were extracted using the Purelink RNA Micro Kit  according to the manufacturer’s instructions. The ddPCR assay was prepared using the One-Step RT-ddPCR Advanced Kit for Probes  according to the manufacturer’s instructions. 20 µL of each reaction mix were converted into droplets with the QX200 Droplet Generator (Bio-Rad Laboratories). The droplets were transferred to a 96-well plate, sealed, and cycled in a C100 Thermocycler (Bio-Rad Laboratories) under the following cycling protocol: 25 °C for 3 min, reverse transcription at 50 °C for 60 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s and 55 °C for 1 min, a post-cycling step of 98 °C for 10 min, and infinite hold at 4 °C. The plate was then transferred into a QX200 Reader (Bio-Rad Laboratories).c-Myc: ddPCR Gene Expression Assay, MYC, Human, Homo sapiens .β-catenin: ddPCR Gene Expression Assay, β-catenin, Human, Homo sapiens .The data were analyzed using Bio-Rad QX Manager 1.2 Standard Edition. Probes were as follows:5 HT-29 cells were co-transfected with 1 µg of p38 and 1 µg of β-catenin plasmids, using the Neon transfection system  according to the manufacturer’s instructions, and plated onto 24-wells. After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector  and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid  or the FOPFlash negative control  using Lipofectamine 3000  and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM). After 24 h in culture, cells were harvested and both firefly and Renilla luciferase activity was measured in triplicate according to the manufacturer’s instructions. Firefly luciferase activity was normalized against Renilla luciferase activity and fold increase in TOPFlash-compared to FOPFlash-transfected cells was determined.To assess the transcriptional activity of β-catenin in HT-29 cells, we took advantage of the TOP/FOP reporter system using a dual-luciferase kit . On the day of the seeding, 6 × 10Activation of p38α and nuclear β-catenin is associated with many cancers, and several genes that are regulated by these signaling pathways are crucial for cancer development and progression. Recently, we demonstrated that p38α acts as a β-catenin chromatin-associated kinase involved in tumor proliferation, metastatic dissemination, and chemoresistance . In ordeIn order to confirm the results of Thornton and colleagues  in our CMin/+ mice. This murine model, identified in 1990 in an ethylnitrosourea mutagenesis screen in C57BL/6 mice, represents a very useful tool to study CRC. The genetic basis for the intestinal phenotype is a T-to-A transversion at nucleotide 2549 of the mouse APC gene resulting in the truncation of the APC protein at amino acid 850. For this reason, the Wnt/β-catenin pathway is overactive in these mice, which develop multiple intestinal neoplasia (Min) after spontaneously losing the heterozygous wild-type APC allele in intestinal epithelial cells [Min/+ mice were administered with a potent carcinogen, AOM (14 mg/kg body weight), once a week for 5 weeks to induce epithelial cell transformation from aberrant crypt foci to adenoma and malignant adenocarcinoma. One month later, animals were subjected to daily intraperitoneal injections of the p38α inhibitor SB202190 (0.05 μmol/kg body weight) or dimethyl sulfoxide (DMSO) for 14 days and then sacrificed , followed by inhibition of p38α with ralimetinib, in HT-29 CRC cell lines. HT-29 cells were serum-starved to retain β-catenin in the cytoplasm and then switched to a serum-supplemented medium or a medium containing Wnt3a and TWS-119. Immunoblot analysis showed that β-catenin and p38α expression was barely detectable in the nucleus under serum starvation, while it increased substantially after serum supplementation  , the addThese data were further confirmed by immunofluorescence staining Fig. : Fig. S3TCF7 and LEF1 themselves  are required for β-catenin and p38α physical interaction. Our results showed that this interaction was not affected in HCT-116 cells overexpressing FLAG-β-catenin-S111A or FLAG-β-catenin-T112A as compared to FLAG-β-catenin-WT-transfected cells Fig. A. Subseqion Fig. C. Moreovc-Myc expression in patients-derived CRC-SCs and patient-derived tumor intestinal organoids  decreased significantly upon treatment with ralimetinib  and elicit transcriptional activation. Like other transcription factors, β-catenin functions are tightly regulated by protein phosphorylation and dephosphorylation [Emerging evidence indicates the existence of a functional relationship between p38 MAPK and the Wnt signaling pathway. Specifically, Thornton and colleagues investigated the cytoplasmic activity of p38α, revealing its ability to inhibit GSK3β by phosphorylation . Moreovecyclin D1 and c-Myc genes [c-Myc was observed in patient-derived CRC-SCs and tumor intestinal organoids upon treatment with ralimetinib.T112 is a key phosphorylation site in β-catenin N-terminus and is specifically targeted by 3-phosphoinositide-dependent protein kinase 1 (PRKD1) and casein kinase II (CKII) , 40. CKIyc genes . In addiyc genes . In CRC This study uncouples p38α cytoplasmic and nuclear functions in the Wnt signaling pathway in CRC cells. Our results identified two β-catenin residues that are phosphorylated by p38α and seem to play an important role in β-catenin transcriptional activation. Considering the role of β-catenin activation in CRC tumorigenesis, our data suggest that p38α pharmacological inhibition is a promising approach to develop new therapeutic strategies for colorectal carcinogenesis.The translational relevance of these findings is further strengthened by the fact that ralimetinib proved effective in CRC model systems such as patient-derived CRC-SCs and tumor intestinal organoids. Of note, ralimetinib displayed a tolerable safety profile, with evidence of antineoplastic activity, in a recent phase I clinical trial in patients with advanced or metastatic cancer (ID NCT01393990). Moreover, it has already been tested in combination with other agents in a phase I study in CRC patients (ID NCT02860780).Taken together, the data presented in this paper may help design prospective clinical trials of tailored interventions for CRC based on p38α inhibition.Additional file 1: Figure S1. p38α pharmacological inhibition or genetic ablation modulates p-GSK3-β levels. (A-B) Immunoblotting analysis of p-β-catenin (S33/37/T41)  and p-GSK3β (T390)  in HCT-116 CRC cells treated or not with the p38α inhibitor ralimetinib (10 μM) for 48 h (A) or silenced by genetic ablation for p38α (B). CTRL = Control. Results are representative of at least three independent experiments. Figure S2. Immunohistochemistry quantification of p38α and β-catenin protein expression in a CRC mouse model. Immunohistochemistry quantification of p38α and β-catenin protein expression in colon tissue from C57BL/6 and AOM-APCMin/+ mice treated as indicated in Fig. #P < 0.05 vs. DMSO. Figure S3. Quantification of p38α and β-catenin immunofluorescence data. Quantification of the immunofluorescence analysis of p38α and β-catenin co-staining experiments shown in Fig. #P < 0.05 vs. DMSO. Figure S4. Analysis of β-catenin genetic ablation efficiency. (A) Quantification results of the digital droplet PCR (ddPCR) assay (copies/µL) of c-Myc mRNA expression as processed by QuantaSoft. HCT-116 CRC cells were silenced by genetic ablation for β-catenin and then treated or not with the p38α inhibitor ralimetinib (10 μM) for 24 h. The error bars represent the maximum and minimum Poisson distribution for the 95% confidence interval generated by QuantaSoft. (B) Immunoblotting analysis of c-Myc protein amount in HCT-116 CRC cells treated as in A. β-actin was used as a loading control. Results are representative of at least three independent experiments. S33/37/T1 (phosph


Epoxy resins are highly valued for their remarkable mechanical and chemical properties and are extensively used in various applications such as coatings, adhesives, and fiber-reinforced composites in lightweight construction. Composites are especially important for the development and implementation of sustainable technologies such as wind power, energy-efficient aircrafts, and electric cars. Despite their advantages, their non-biodegradability raises challenges for the recycling of polymer and composites in particular. Conventional methods employed for epoxy recycling are characterized by their high energy consumption and the utilization of toxic chemicals, rendering them rather unsustainable. Recent progress has been made in the field of plastic biodegradation, which is considered more sustainable than energy-intensive mechanical or thermal recycling methods. However, the current successful approaches in plastic biodegradation are predominantly focused on polyester-based polymers, leaving more recalcitrant plastics underrepresented in this area of research. Epoxy polymers, characterized by their strong cross-linking and predominantly ether-based backbone, exhibit a highly rigid and durable structure, placing them within this category. Therefore, the objective of this review paper is to examine the various approaches that have been employed for the biodegradation of epoxy so far. Additionally, the paper sheds light on the analytical techniques utilized in the development of these recycling methods. Moreover, the review addresses the challenges and opportunities entailed in epoxy recycling through bio-based approaches. Epoxy polymers are widely used in various industries, e.g., as coatings, adhesives, and for lightweight construction due to their unique properties such as high strength, chemical resistance, and adhesion to various surfaces ,2. ThereIn this context, the overall production of plastic waste is projected to double within the next 20 years, with only 18% currently being recycled, leading to the deposition of around 12,000 Mt of plastic waste in landfills and the environment by 2050 see  5,6,7].,7.6,7]. 2 and other pollutants, but it also generally results in a reduction of the material properties. Particularly with regard to composites, the fibers often suffer from the extreme reaction conditions, resulting in a reduction in their quality  (TGDDM) and DGEBA were identified. The quantification of the compounds was realized using acetophenone as an internal standard to enable a comparison of the mass fractions of different batches of the resin.The analysis of single components of the epoxy matrix of a partially cured fiber-reinforced composite by RP-chromatography was described by Noël et al. . They exIn addition to liquid chromatography, gas chromatography with coupled mass spectrometry (GC-MS) was used for the analysis of solvolyzed CFRPs . The autThe contact angle is the angle formed by a liquid drop at the interface with a solid surface. It reflects the surface wettability of the solid, which is related to its surface energy and chemical composition . A high The application of contact angle measurement for epoxy degradation analysis was conducted by Pardi-Comensoli et al. . They usP. aeruginosa for 14 days. This decrease in contact angle suggests the formation of a biofilm on the surface, as reported by the authors.A similar approach was followed by Zhang et al. , who anaMechanical analysis involves measuring the mechanical properties of plastics, such as tensile strength, elongation, modulus, impact resistance, and hardness. These properties reflect the molecular structure and chain length of plastics, which can be altered by degradation processes. Mechanical testing can be performed by using standard instruments and methods, such as universal testing machines, impact testers, hardness testers, and dynamic mechanical analysis .The mechanical properties of epoxy- and melamine formaldehyde-modified PU films were measured before and after soil burial biodegradation for 180 days by Dutta et al. . The tenEpoxy resins have gained tremendous importance in the industrial sector as they possess superior mechanical and thermal properties, making them an ideal choice for various applications ,58. The While biological plastic recycling has mostly focused on thermoplastics, research on more recalcitrant plastics such as epoxy is limited . There hTo overcome these challenges, novel approaches need to be developed to extend the range of biodegradable polymers. One potential solution is to exploit the natural biodiversity, especially microorganisms that have not yet been cultivated or cannot be cultivated at all ,120. TheFurthermore, the targeted use of oxidative enzymes has emerged as a promising strategy for the degradation of non-hydrolyzable polymers. By studying reaction conditions and enzyme formulations, researchers have made progress in enhancing the degradation efficiency of recalcitrant plastics such as PE using oxidative enzymes ,123. TheApart from recycling approaches for conventional, fossil-based epoxy, research on bio-degradable epoxy and bio-reinforced resins is also of high importance. Novel bio-degradable epoxy resins have been developed, such as those based on soy protein and lignin ,125. TheIn conclusion, the recycling of epoxy resins with bio-based methods is an important area of research, as it offers a sustainable and environmentally friendly alternative to conventional methods. While challenges exist in terms of recalcitrant epoxy degradation and the need for robust biocatalysts, advancements in metagenome mining, directed evolution, and utilization of oxidative enzymes provide avenues for overcoming these hurdles. Additionally, research on bio-degradable epoxy and bio-reinforced materials contributes to a holistic approach towards a more sustainable epoxy industry. Continued interdisciplinary efforts in exploring and optimizing bio-based epoxy recycling methods will pave the way for a greener future and a circular economy.


Non-small-cell lung cancer (NSCLC) patients become resistant to targeted therapies such as tyrosine kinase inhibitors (TKIs) through mutations in the EGFR gene and by overexpression of proteins such as p120-catenin and PRMT-1. We studied EGFR wild-type and mutated EGFR in parental and Osimertinib-resistant cell lines. Our results showed overexpression of p120-catenin, PRMT-1, and Kaiso factor in Osimertinib-resistant cell lines compared to parental cell lines. We also found co-localization of PRMT-1 and Kaiso factor in resistant cell lines. Overexpression of p120-catenin and PRMT-1 was found in tumor samples recovered from smokers as compared to non-smokers. Further studies showed that inhibiting p120-catenin mediated increased Osimertinib efficiency and decreased wound healing compared to mock siRNA controls. These results indicate that p120-catenin and PRMT-1 could play a vital role in Osimertinib resistance and could be potential targets to increase TKI efficiency.Osimertinib, an irreversible tyrosine kinase inhibitor, is a first-line therapy in EGFR-mutant NSCLC patients. Prolonged treatment with Osimertinib leads to resistance due to an acquired C797S mutation in the EGFR domain and other mechanisms, such as epithelial-mesenchymal transition (EMT). In this study, we investigated the role of PRMT-1 and p120-catenin in mediating Osimertinib resistance (OR) through EMT. These studies found upregulation of gene and protein expression of PRMT-1, p120-catenin and Kaiso factor. Knockdown of p120-catenin using siRNA increased OR efficacy by 45% as compared to cells treated with mock siRNA and OR. After 24 h of transfection, the percentage wound closure in cells transfected with p120-catenin siRNA was 26.2%. However, in mock siRNA-treated cells the wound closure was 7.4%, showing its involvement in EMT. We also found high levels of p120-catenin expressed in 30% of smokers as compared to 5.5% and 0% of non-smokers and quit-smokers (respectively) suggesting that smoking may influence p120-catenin expression in NSCLC patients. These results suggest that biomarkers such as PRMT-1 may mediate EMT by methylating Twist-1 and increasing p120-catenin expression, which causes transcriptional activation of genes associated with Kaiso factor to promote EMT in Osimertinib-resistant cells. In the United States, lung cancer accounts for about 21% of all cancer-related deaths . PatientTyrosine kinase inhibitors such as Erlotinib and Gefitinib (first generation TKIs) are used as molecular targeted therapies and are used to treat patients harboring L858R activating mutations. However, patients often acquire a secondary mutation called the T790M mutation that makes them resistant to first-generation TKIs . Third-gProtein arginine methyltransferase I (PRMT-1) is a type I arginine methyltransferase which is responsible for approximately 75% of arginine methylation in cells . The uprp120-catenin is a protein that stabilizes the epithelial adherens junction (AJ), which is an E-cadherin-based complex that maintains cell-cell junction integrity . When E-Although several studies are dedicated to elucidating the role of EMT in cancer progression and tumorigenicity, the role of p120-catenin and PRMT-1 in TKI resistance is minimally studied. In this study, we investigated the role of PRMT-1 and p120-catenin in mediating resistance to Osimertinib in NSCLC. We found that expression of p120-catenin and PRMT-1 was upregulated in TKI-resistant cells and knockdown of p120-catenin increased efficacy of Osimertinib. In addition, we analyzed tumor sections from smokers and non-smokers and found that expression of p120-catenin was increased in smokers as compared to non-smokers. These studies indicate that p120-catenin and PRMT-1 may play a role in TKI resistance.Erlotinib hydrochloride was acquired from LC labs  and dissolved in dimethyl sulfoxide (DMSO) at 20 mM. Aliquots of 20 µL were stored at −20°. ZM 323881 hydrochloride (5-((7-Benzyloxyquinazolin-4-yl)amino)-4-fluoro-2-methylphenol hydrochloride) was purchased from Tocris Bioscience , dissolved in DMSO at 1 mM, after which aliquots were stored at −20 °C. Epidermal growth factor (EGF) was purchased from Peprotech , resuspended in PBS at a 15 ng/µL concentration, and stored at −20 °C.Rabbit monoclonal antibodies for E-cadherin, Claudin, and Vimentin were purchased from Cell Signaling Technologies  as a part of the EMT antibody sampler kit (Cat. No. 9782). Rabbit monoclonal PRMT-1 antibody was purchased from Cell Signaling Technologies (Cat. No. 2449S), mouse monoclonal p120-catenin/Delta catenin antibody was purchased from Invitrogen . Rabbit polyclonal antibody against Kaiso factor was used for immunofluorescence (H-154) (Cat. No. sc-98589) and mouse monoclonal antibody against Kaiso factor was used for immunoblotting (D-10) (Cat. No. sc-365428) and were both purchased from Santa Cruz Biotechnology . Mouse monoclonal antibody for β-Actin (Cat. No. A5441) was purchased from Sigma Aldrich . Anti-rabbit IgG (Cat. No. 31460) and anti-mouse IgG (Cat. No. A16078) secondary monoclonal antibodies were obtained from Invitrogen. All the primary antibodies were diluted to 1:1000 in 1% BSA made in 1X TBST. The secondary antibodies were made at 1:10,000 dilution with 1% blocking grade milk (Cat. No. 170-6404XTU) from Bio-Rad .2 at 37 °C following the protocol of the American Type Culture Collection (ATCC). The H2170 and H358 cell lines have EGFR wild-type status, while H1975 has two EGFR tyrosine kinase domain mutations—L858R and T790M . An. An25]. Next, we examined the ability of p120-catenin knockdown to overcome Osimertinib resistance in H358OR cells . KnockdoNext, we compared p120-catenin and PRMT-1 expression in smokers, quit-smokers, and non-smokers. This is clinically important as cigarette smoking is the major factor driving the pathogenesis and progression of lung cancer. Previous reports have shown that cigarette smoking plays a role in EMT induction ,16,17. AAlthough our results suggest reversal of EMT by upregulation of E-cadherin, a potential limitation of the study is a need to further validate the reversal by exploring the effect on other EMT-related proteins, such as Twist, which is modulated by PRMT-1 . AdditioOverall, this study demonstrated that the EMT biomarkers PRMT-1 and p120-catenin play an important role in activation of the EMT process, increasing the invasive characteristics of NSCLC and, thus, promoting Osimertinib-resistance. We further demonstrated that, by knockdown of p120-catenin, we were able to increase the efficacy of Osimertinib, suggesting that EMT may play a significant role in contributing to Osimertinib resistance. Finally, immunohistochemistry analysis of p120-catenin levels with the smoking status of NSCLC patients suggested that high expression of p120-catenin was mainly observed in smokers as compared to quit-smokers and non-smokers. This data on p120-catenin could potentially help in improving the prognosis of NSCLC patients. Thus, our study on EMT biomarkers opens new opportunities to explore Osimertinib resistance mechanisms and study novel potential therapeutic targets in NSCLC.


Protein arginine methyltransferase 5 (PRMT5) is a major type II enzyme responsible for symmetric dimethylation of arginine (SDMA), and plays predominantly roles in human cancers, including in ovarian cancer. However, the exactly roles and underlying mechanisms of PRMT5 contributing to the progression of ovarian cancer mediated by reprogramming cell metabolism remain largely elusive. Here, we report that PRMT5 is highly expressed and correlates with poor survival in ovarian cancer. Knockdown or pharmaceutical inhibition of PRMT5 is sufficient to decrease glycolysis flux, attenuate tumor growth, and enhance the antitumor effect of Taxol. Mechanistically, we find that PRMT5 symmetrically dimethylates alpha‐enolase (ENO1) at arginine 9 to promotes active ENO1 dimer formation, which increases glycolysis flux and accelerates tumor growth. Moreover, PRMT5 signals high glucose to increase the methylation modification of ENO1. Together, our data reveal a novel role of PRMT5 in promoting ovarian cancer growth by controlling glycolysis flux mediated by methylating ENO1, and highlights that PRMT5 may represent a promising therapeutic target for treating ovarian cancer. PRMT5 promotes ovarian cancer growth and Taxol response through enhancing glycolysis pathway, which mediated by regulating the symmetric dimethylation of arginine (SDMA) modification of ENO1. In this study, we found that PRMT5 is upregulated in ovarian cancer and promotes ovarian cancer cell glycolysis flux, tumor growth, and Taxol response. Further, we revealed that PRMT5 promotes glycolysis flux by symmetrically dimethylating ENO1, leading to the active ENO1 dimer formation. Given the important in ovarian cancer progression mediated by glycolysis flux, we applied previously designed and synthesized PRMT5 inhibitors, which block glycolysis flux, tumor growth, and enhance antitumor effects of Taxol in ovarian cancer. Taken together, our results showed that the PRMT5‐ENO1 axis drives ovarian cancer progression, and further suggested that this axis may be a novel therapeutic target for treating ovarian cancer.22.1To identify the key drive epigenetic regulators in ovarian cancer progression, we analyzed the expression levels of PRMT family (PRMT1‐PRMT9) based on GEO database (GSE14407). We found that the levels of PRMT5 were evidently increased in ovarian cancer Figure . FurtherTo explore the clinical relevance of our findings, we performed immunohistochemistry (IHC) staining to determine the PRMT5 protein levels in clinical sample tissues of ovarian cancer . We found that the expression of PRMT5 was positive in 22 (56.4%) of the 39 ovarian cancer patients Table , and it To explore the functional role of PRMT5 in ovarian cancer, we generated PRMT5 stable knockdown cell line by specific short hairpin RNAs (shRNAs), and found that knockdown PRMT5 dramatically decreased A2780, A2780‐Taxol, OV2008, and C13* cells proliferation by colony formation assay and cell number counting assay Figure . The dec2.2We next explored the possible underlying signaling mechanisms of PRMT5‐driven ovarian cancer cell growth. First, the data of ovarian cancer patients downloaded from the cBioPortal database were grouped according to the expression level of PRMT5.2.3To determine how PRMT5 impacts glycolysis pathway in ovarian cancer cells, we examined the effect of PRMT5 on metabolic enzymes involved in glycolysis pathway. First, we found that the expression levels of glycolytic enzymes were not changed in PRMT5 knockdown ovarian cancer cells compared with the control group Figure . So, we 2.4Next, we want to explore the underlying mechanism of PRMT5 in regulating ENO1 activity through methylating ENO1 directly. First, we incubated the recombinant GST‐tagged ENO1 purified from bacteria with Flag‐PRTM5, which pulled down from HEK239T cells, and we found that Flag‐PRMT5 WT, but not inactivating mutant (ΔM), methylated the recombinant GST‐tagged ENO1 Figure . In addihttps://www.phosphosite.org/proteinAction.action?id = 2610&showAllSites = true and https://www.phosphosite.org/siteTableNewAction?id = 2610&showAllSites = true).Proteomics analysis  revealed that there was a group of arginine methylation residues on ENO1 protein  is a natural anticancer drug, which exerts antitumor effect by inhibiting mitosis and has been widely used in clinical treatment of ovarian cancer.In this study, we identified PRMT5 as a driver for ovarian cancer in promoting ovarian cancer progression. Mechanistically, we revealed that PRMT5 enhances glycolytic flux through regulating the ENO1 activity mediated by SDMA, which is critically required for the formation of active ENO1 dimer. In summary, our study suggested the mechanisms and roles of PRMT5 in promoting ovarian cancer growth, and targeting PMRT5 could be provided new potential clues for the treatment of ovarian cancer.44.1The Biological Resources and Reagents are listed in KEY RESOURCES TABLE of Table 4.2HEK293T, HosEpic, A2780, and A2780‐Taxol cells were obtained from Dr. Zhi Shi . IOSE‐80, Hey‐T30, and SKOV3 cells were purchased from Procella . HEK293T, OVCAR3, OVCAR3‐Taxol, and HosEpic cells were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum . IOSE‐80, A2780, A2780‐Taxol, Hey‐T30, and SKOV3 cells were maintained in RPMI‐1640 medium  supplemented with 10% FBS. Human ovarian surface epithelial cancer cells OV2008 and C13* were gifts from Dr. Benjamin K. Tsang . OV2008 and C13* cells were cultured in RPMI 1640 medium with 10% FBS. Cells were cultured at 37°C in an atmosphere of 5% CO2/95% air (normoxic conditions) at 37°C. For glucose‐starved culture, cells were washed with phosphate buffer saline (PBS) and then incubated with glucose‐free RPMI‐1640 medium containing 1% FBS. For the cells that need to be treated with drugs, the cells were treated with the indicated concentration of drugs for corresponding time  after cell attachment, and then the subsequent experiments were carried out.4.3Thirty nine clinical ovarian cancer tissues and 22 normal tissues (Cohort 1) were collected from Tianjin Central Hospital of Gynecology Obstetrics  after surgical resection. The ovarian cancer tissues (Cohort 2 = 65 ovarian cancer tissues) were purchased from Shanghai Outdo Biotechnology Co., Ltd. . The related features were shown in Table 4.4Stable knockdown of endogenous PRMT5 or ENO1 was achieved using lentiviral vector harboring shRNA purchased from Transheep Biological Corporation . To generate cells with stable knockdown of PRMT5 or ENO1, lentiviral vector and packing plasmids (pMD2.G and psPAX2) were first transfected into HEK293T cells, and the lentiviral particles filtered through 0.45‐μm filter were infected into A2780 cells, followed by puromycin selection of the infected cells.siRNA targeted for PRMT5 were purchased from GenePharma  and were transfected into target cells using siRNA‐mate  according to the manufacturer's instructions.4.5Human PRMT5 cDNA (NM_006109.5) and ENO1 cDNA (NM_001428.5) were cloned into pLVX3 with 3X Flag‐tag at N‐terminus or pETM3C vector with his tag. The PRMT5 ΔM and PRMT5 slient mutation  were constructed in pLVX3 (or pETM3C) using the Fast Mutagenesis System . R9K, R15K, R50K, R183K, R372K, and R412K mutations were constructed in pLVX3 (or pETM3C) using the Fast Mutagenesis System  with GST tag at the N‐terminus of ENO1. The sequences of these primers used in these are given in Table 4.6We use an XF‐96 Extracellular Flux Analyzer  to analyze the real‐time bioenergy changes of the ECAR as described previously.4.7For lactate production assays, cells were seeded into six well plates and incubated for 24 h at 37°C. After aspirating the original medium from each well, cells were washed with PBS, and then incubated with phenol red‐free RPMI 1640 medium for 1 h. Lactate production in the medium was detected by the Lactic Acid assay kit  according to the manufacturer's instruction. Relative lactate levels were normalized to cell numbers by counting.4.8ENO1 activity was measured in the reaction buffer containing 50 mmol/L Tris‐HCl (PH 7.5), 100 mmol/L KCl, 25 mmol/L MgSO4, 1.3 mmol/L ADP , and 0.15 mmol/L nicotinamide adenine dinucleotide . In addition, 0.6–1 U of pyruvate kinase and 0.9–1.4 U of lactate dehydrogenase  were added to the reaction solution. After adding 1.9 mmol/L 2‐phosphoglycerate  and appropriate amount of ENO1 (1 μg of purified rENO1 or 10 μg of cell lysates), the absorbance in 340 nm as a measure of NADH was recorded at room temperature every 1 min for 10−30 min on a Microplate Photometer .4.9In vitro methylation reactions were performed in 50 μL reaction system with PRMT5, ENO1, and 16 μmol/L S‐adenosyl‐methionine , and supplemented with TBS . The reactions were incubated at 30°C for 1.5 h and stopped by the addition of sample loading buffer for western blot (WB).4.10After performing in vitro methylation reaction, purified recombinant ENO1 was incubated at room temperature for 5 min in the absence or presence of 0.0025% glutaraldehyde containing 5% glycerol, 20 mM Tris‐HCl (pH 7.5), 100 mmol/L NaCl, 0.2 mmol/L ethylene diamine tetraacetic acid, and 2 mmol/L dithiothreitol. Crosslink reaction should be terminated by adding SDS sample buffer, skip the boiling sample step, and followed by SDS‐PAGE. The same amounts of noncrosslinking proteins were loaded to ensure equivalent protein amounts.4.11Total RNA was isolated using TRIzol  reagent. cDNA was synthesized using Hifari III 1st Strand cDNA Synthesis SuperMix for qPCR . Real‐time quantitative PCR (RT‐qPCR) was performed using Hieff qPCR SYBR Green Master Mix (No Rox) .4.12NP40 lysis buffer with cells was incubated on ice for 20 min, and then centrifuged at 12,000 rpm for 10 min at 4°C. Flag (GST)‐conjugated beads or agarose preconjugated with the appropriate antibody for 2 h was added to the supernatant and incubated overnight at 4°C. Immunoprecipitates obtained by centrifugation were washed three times with prechilled PBS and subjected to western blotting using specific antibodies.4.13For immunohistochemical (IHC) staining, sections obtained from formalin‐fixed paraffin‐embedded samples were dewaxed and rehydrated following standard methods. Antigen retrieval was performed by boiling samples in citrate buffer for 15 min and endogenous peroxidase was blunted by incubating samples with 3% hydrogen peroxidase for 10 min. Sections were blocked with 10% normal goat serum (NGS)  for 20 min at room temperature and then incubated overnight at 4°C with primary antibodies (Ki67)  diluted in PBST (0.03% Triton X‐100 in PBS) containing 10% NGS. After rinsing with PBST and incubating with secondary antibodies, all paraffin sections were developed with DAB  and counterstained with hematoxylin. Finally, the sections were dehydrated through an ethanol series, sealed with neutral resin and photographed under a microscope.https://www.dentalearner.com/archives/2541.We used ImageJ for Immunohistochemical staining analysis, the methods refer to 4.146 A2780 cells harboring empty vector were injected into mice. The mice were performed according to the institutional ethical guidelines for animal experiments. Two perpendicular diameters were measured to calculate tumor growth using equation 4π/3 × (length/2) × (width/2).Nude mice  were purchased from Charles River . To establish A2780 cells CDX models, 5 × 10To investigate the effects of DW14761 on tumorigenesis In vivo, mice with A2780 xenografts were injected ip with DW14761  every 2 days, whereas tumor volume and mice weight were recorded. To explore the combination effect of DW14761 and Taxol on tumor growth of ovarian cancer, mice with A2780 xenografts were injected ip every 2 days with DW14761 (30 mg/kg), Taxol (10 mg/kg) , or in combination, and tumor volume and mice body weight were recorded during the treatment.4.15http://kmplot.com/analysis/index.php?p = background) was used to analyze overall survival. In addition, Survival and Survminer packages  in R were used to analyze patient survival and prognosis.We use the public datasets TCGA and GEO datasets to perform bioinformatics analysis. Kaplan–Meier plotter , Biomedical Ethics Committee of Nankai University (Ethics Document NO.NKUIRB2022022), and Tianjin Central Hospital of Gynecology Obstetrics with written informed consent from all subjects. All subjects were given a written informed consent in accordance with the Declaration of Helsinki.This work was supported by grants from National Natural Science Foundation of China (81973356 to C.S and 82273963 to S.Z), the Natural Science Foundation of Tianjin (21JCZDJC00060 to C.S), supported by the Open Fund of Tianjin Key Laboratory of human development and reproductive regulation (2020XHY02 to C.S) and Tianjin Key Medical discipline  Construction Project (TJYXZDXK‐043A), and the Fundamental Research Funds for the Central Universities of Nankai University.Supporting InformationClick here for additional data file.


Mouse models expressing site-specific recombinases (SSRs) or reporter genes are important tools providing genetic access to defined cell types and have been widely used to address CRH+ neurons and connected brain circuits. Here, we investigated a recently generated CRH-FlpO driver line expanding the CRH system-related tool box. We directly compared it to a previously established and widely used CRH-Cre line with respect to the FlpO expression pattern and recombination efficiency. In the brain, FlpO mRNA distribution fully recapitulates the expression pattern of endogenous Crh. Combining both Crh locus driven SSRs driver lines with appropriate reporters revealed an overall coherence of respective spatial patterns of reporter gene activation validating CRH-FlpO mice as a valuable tool complementing existing CRH-Cre and reporter lines. However, a substantially lower number of reporter-expressing neurons was discerned in CRH-FlpO mice. Using an additional CRH reporter mouse line (CRH-Venus) and a mouse line allowing for conversion of Cre into FlpO activity (CAG-LSL-FlpO) in combination with intersectional and subtractive mouse genetic approaches, we were able to demonstrate that the reduced number of tdTomato reporter expressing CRH+ neurons can be ascribed to the lower recombination efficiency of FlpO compared to Cre recombinase. This discrepancy particularly manifests under conditions of low CRH expression and can be overcome by utilizing homozygous CRH-FlpO mice. These findings have direct experimental implications which have to be carefully considered when targeting CRH+ neurons using CRH-FlpO mice. However, the lower FlpO-dependent recombination efficiency also entails advantages as it provides a broader dynamic range of expression allowing for the visualization of cells showing stress-induced CRH expression which is not detectable in highly sensitive CRH-Cre mice as Cre-mediated recombination has largely been completed in all cells generally possessing the capacity to express CRH. These findings underscore the importance of a comprehensive evaluation of novel SSR driver lines prior to their application.Corticotropin-releasing hormone expressing (CRH The combination of Cre-/Flp-expressing mouse strains with recombinase-dependent reporters or effectors has proven as a versatile and efficient approach offering a myriad of options ranging from manipulation of gene expression and cell activity to visualization of molecules, cells and circuits  are potent tools used in neuroscience research and crucial for understanding mechanisms underlying neurological and psychiatric disorders. circuits .Corticotropin-releasing hormone  is a neuropeptide playing a critical role in different aspects of the body’s stress response. CRH in the paraventricular nucleus of the hypothalamus (PVN) controls the neuroendocrine response by activating the hypothalamic–pituitary–adrenal (HPA) axis and regulating adrenocorticotropic hormone (ACTH) secretion from the pituitary which, in turn, triggers the synthesis and release of glucocorticoids from the adrenal glands as ultimate effectors of the stress response . ParvoceBeyond the PVN, CRH is widely expressed throughout the brain . Here, i+ soma somewhat difficult. Moreover, it is of note that even with the most efficient antiserum raised against CRH more than 4 decades ago in the Vale lab . For each mouse line 3–7 animals were analyzed of which representative images are included in the figures.All animal experiments were conducted with the approval of and in accordance with the Guide of the Care and Use of Laboratory Animals of the Government of Upper Bavaria, Germany. Mice were group- housed under standard lab conditions  and maintained under a 12 h light–dark cycle with food and water CRH-Cre (tm1(cre)ZjhCrh, Jackson Laboratory stock no. 012704) (CRH-FlpO (tm1.1(flpo)BsabCrh, Jackson Laboratory stock no. 031559; CRH-Venus  (Ai9 (tm9(CAG-tdTomato)HzeGt(ROSA)26Sor, Jackson Laboratory stock no. 007909) (Ai65 (tm65.1(CAG-tdTomato)HzeGt(ROSA)26Sor, Jackson Laboratory stock no. 021875) (CAG-LSL-FlpO (tm5(CAG-flpo)ZjhGt(ROSA)26Sor, Jackson Laboratory stock no. 028584) . This novel mouse line, reporting only Flp recombinase activity, was designated as Ai65F. In this study, the following double and triple transgenic lines were generated by cross-breeding of single transgenic lines: CRH-Cre::Ai9, CRH-FlpO::Ai65F, CRH-Venus::CRH-Cre::Ai9, CRH-Venus::CRH-FlpO::Ai65F, CRH-Cre::CRH-FlpO::Ai65, CRH-Cre::CRH-FlpO::Ai9, CRH-Cre::CAG-LSL-FlpO::Ai65 and CRH-Cre::CAG-LSL-FlpO::Ai9. All transgenic alleles involving the Rosa26 locus were heterozygous. Detailed breeding schemes, primer sequences and protocols for genotyping are available upon request.The following transgenic mouse lines were used in this study:  012704) , CRH-FlpRC09893) , Ai9 (Gt 007909) , Ai65 (G 021875)  and CAG- 028584) . A pure 2.2.Vibratome sections: mice were sacrificed by an overdose of isoflurane and transcardially perfused with 20 mL 0.1 M PBS (4°C) supplemented with heparin and subsequently with 20 mL of 4% paraformaldehyde (PFA) in 0.1 M PBS (4°C). After recovery, brains were post-fixed overnight in 4% PFA at 4°C and coronal 50 μm sections were prepared using a vibratome . Cryosections: perfused and dissected brains were post-fixed in 4% PFA for 6 h. After 48 h in 30% sucrose in 0.1 M PBS at 4°C, brains were snap frozen on dry ice and 40 μm sections were prepared using a cryostat . All sections were stored at −20°C in cryopreservation solution  until further use.2.3.Vibratome sections were blocked with 10% normal goat serum (NGS) and 0.15% Triton in PBS for 30 min at RT and then washed in 0.1 M PBS for 3 × 5 min. Subsequently, sections were incubated with anti-GFP primary antibody  diluted in 0.15% NGS and 0.15% Triton in PBS overnight at 4°C. After another washing step, sections were incubated with an Alexa Fluor 488 secondary antibody  while shaking for 2 h at RT. Following 3 more washing steps, sections were mounted on glass slides (epredia) with Fluoromount-G (SouthernBiotech).2.4.Freshly dissected brains were snap frozen on dry ice and stored at −80°C until further use. Brains were sectioned coronally at 20 μm using a cryostat. Sections were mounted onto SuperFrost slides and kept at −80°C until further use. ISH was performed as previously described . The fol2.5.CRH-FlpO mouse line to induce reporter gene expression in CRH+ cells following stress exposure, CRH-FlpO::Ai65F mice were exposed to acute restraint stress. On the day of the experiment, animals were placed for 15 min in a 50 ml conical tube with the bottom removed. Animals were sacrificed 1 week later and brains were used for the assessment of stress-induced expression of tdTomato.To test the capacity of the 2.6.Slidescanner: overviews of whole brain sections and region-specific scans were performed with an Olympus VS120 Slide Scanner. Image capturing and initial processing was conducted with the Olympus VS Software. Briefly, overview images of the whole slide were acquired in the batch scanning mode. Subsequently, region-specific scans were taken with the 20×, whole sections images with the 10× objective. The scan region was adjusted to the size of the region. Exposure time and focus were set to automated mode. Sections were acquired with the Cy3 (TdTomato) channel or the FITC (GFP) channel. Confocal microscopy: for higher resolution, confocal images of regions of interest were acquired with a Zeiss Axioplan2 fluorescence microscope using Axio Vision 4.5 software. Fluorescently labeled cells were excited at 488 nm (GFP Alexa-488) and 559 nm , respectively, using a 20× objective. After acquisition in virtual slide (.VSI file) or Carl Zeiss (.CZI) format, images were extracted and saved as Tagged Image File Format (.tiff) for further processing. Images were analyzed using FijiVibratome sections and cryosections were imaged using a slidescanner or confocal microscope. 3.3.1.CRH-FlpO mice were generated by targeted insertion of a FlpO expression cassette into the murine Crh locus immediately downstream of the stop codon. FlpO expression driven by the Crh locus is conveyed by an internal ribosomal entry site (IRES). As a selection marker, a self-excising neomycin cassette driven by the testis-specific angiotensin converting enzyme promotor (tACE) was used, which removed itself in the male germline leaving a single loxP2272 site behind (CRH-FlpO mice and performed an in situ hybridization (ISH) against FlpO. The spatial pattern of FlpO mRNA expression fully recapitulated the expression of endogenous Crh as indicated by the detection of distinct signals in all regions of salient CRH expression including the piriform cortex (Pir), IPACL, BNST, CeA, and PVN. The expression of FlpO appeared generally weaker, as CRH-FlpO mice were only heterozygous  and the inferior cerebellar peduncle (icp), the latter representing climbing fibers originating in the IO and innervating the cerebellum  and behavioral  stress response as well as on the CTX, because of its homogeneous and scattered distribution of CRH+ neurons. First, we tested the degree of overlapping Cre and FlpO expression, comparing the occurrence of tdTomato+ cells in the intersectional reporter mouse line CRH-Cre::CRH-FlpO::Ai65 with CRH-Cre::Ai9 mice  Cre caused ectopic CRH reporting by tdTomato, (ii) CRH expression remained below a certain threshold, preventing reliable visualization by antibody staining, or (iii) CRH expression occurred only transiently during development but had ceased in adulthood. To evaluate the influence of the Venus expression level on the number of reported neurons, we bred CRH-Venus mice to homozygosity, which resulted in a significantly enhanced Venus signal and an increased number of Venus+ cells in all regions of interest. This result indicated that tdTomato+/Venus− neurons were not a consequence of ectopic Cre expression but rather reflected their low level of CRH expression. Accordingly, the Venus levels of these cells were too low to be detected by antibody staining, whereas the similarly low amount of Cre recombinase was sufficient to activate tdTomato expression  is not limiting FlpO recombination activity in general  When using and comparing FlpO- and Cre-based reporter systems, the strength of the driving promoter has to be taken into account. (ii) In intersectional approaches, the number of reported co-expressing cells is limited by the efficacy of the FlpO. (iii) In approaches involving knockout or overexpression of a specific target gene, the efficacy is highly dependent on the used SSR. These results are essential to interpret and reconcile divergent scientific discoveries which will ultimately advance our comprehension of the physiological role of CRH in the central nervous system.In conclusion, we show a difference in the recombination efficiency of Cre and FlpO in CRHThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Government of Upper Bavaria, Germany.CZ, CR, and YD designed and performed all experiments, analyzed and interpreted data, drafted figures, and wrote the manuscript. JZ performed a subset of experiments and reviewed the manuscript. KS and KI provided materials, edited the manuscript and contributed scientific advice. JD designed the project, supervised all experiments and analyses, edited the manuscript, and provided scientific advice, guidance and support. All authors contributed to the article and approved the submitted version.The project was supported by the Max Planck Society (JD) and the Federal Ministry of Education and Research . JZ and YD received funding from the Chinese Scholarship Council. CR received funding from the International Max Planck Research School for Translational Psychiatry and the Gunter Sachs Donation.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.https://www.frontiersin.org/articles/10.3389/fnins.2023.1163462/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.