NTR), as a negative regulator of Wnt/β-catenin signaling in zebrafish embryos and in mammalian cells. Nradd significantly suppresses Wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Nradd is localized at the plasma membrane, physically interacts with the Wnt receptor complex and enhances apoptosis in cooperation with Wnt/β-catenin signaling. Our functional analyses indicate that the N-glycosylated N-terminus and the death domain-containing C-terminus regions are necessary for both the inhibition of Wnt signaling and apoptosis. Finally, Nradd can induce apoptosis in mammalian cells. Thus, Nradd regulates cell death as a modifier of Wnt/β-catenin signaling during development.Wnt/β-catenin signaling controls many biological processes for the generation and sustainability of proper tissue size, organization and function during development and homeostasis. Consequently, mutations in the Wnt pathway components and modulators cause diseases, including genetic disorders and cancers. Targeted treatment of pathway-associated diseases entails detailed understanding of the regulatory mechanisms that fine-tune Wnt signaling. Here, we identify the neurotrophin receptor-associated death domain (Nradd), a homolog of p75 neurotrophin receptor (p75 Wnt/β-catenin signaling is an evolutionarily conserved signaling pathway that controls a plethora of cellular processes including proliferation, migration, apoptosis, cell fate determination and tissue patterning during development and adult homeostasis ,2,3. DueWnt/β-catenin signaling is thought to operate with its core components as follows: If there exist no active Wnt ligands in the environment, the so-called Wnt-off state, the β-catenin destruction complex containing Axin, adenomatous polyposis coli (Apc), glycogen synthase kinase 3 (Gsk3) and casein kinase 1 (Ck1) bind to β-catenin and leads to its proteasomal degradation after phosphorylation ,13. The Due to the deleterious consequences of aberrant signal transduction, it is not surprising that Wnt/β-catenin signaling is controlled by a large number of positive and negative regulators. Tight regulation of the pathway at the plasma membrane is achieved by a complex network of extracellular or membrane-bound modulators ,20,21. Mneurotrophin receptor-associated death domain (nradd) gene, encoding for a single-pass transmembrane protein with a C-terminal death domain, as a transcriptional target of Wnt/β-catenin signaling during the embryonic development of zebrafish. Nradd exhibits a high degree of homology with the p75 neurotrophin receptor (p75NTR), which has been characterized as a receptor for members of the neurotrophin family [NTR, a member of the tumor necrosis factor receptor superfamily (TNFRSF) [NTR and sortilin [Here, we identify the n family ,28. The n family ,30. Thern family . Neurotr(TNFRSF) ,33,34. B(TNFRSF) ,35. Pronsortilin ,37.NTR, respectively [NTR can enhance neurotrophin binding to Trk receptors and Trk signal transduction through protein kinase B (PKB or AKT) and MAPKs, depending on the circumstances. They can either enhance survival by the nuclear factor-κB (NF-κB) pathway or antagonize the action of Trk through the activation of the JUN N-terminal kinase (JNK) and Rhoa pathways [NTR signaling [Neurotrophins induce neuronal survival or cell death by activating Trk receptors and p75ectively . The binectively . On the pathways ,42,43,44ignaling . Mouse Nignaling . HoweverWe describe Nradd as a feedback inhibitor of Wnt/β-catenin signaling during zebrafish development and in mammalian cells. Nradd localizes to the plasma membrane and interacts with the Fz8 and Lrp6 receptors. Our functional analyses show that Nradd acts together with Wnt/β-catenin signaling to promote apoptosis during development and that both the death domain and N-glycosylated N-terminus are essential for its apoptotic function. Finally, Nradd can also enhance apoptosis in human embryonic kidney and neuroblastoma cell lines. Overall, Nradd acts as a negative modulator of Wnt/β-catenin signaling and a key player in the regulation of apoptosis during development.Danio rerio) lines Tg(hsp70l:wnt8a-GFP)w34 and Tg(hsp70l:Mmu.Axin1-YFP)w35 were outcrossed to wild type (wt) AB zebrafish [ia transgenic line was used as a reporter of Wnt/β-catenin signaling, outcrossed to wt zebrafish and embryos were sorted as described previously [Transgenic zebrafish  using a Direct-Zol RNA kit  and cDNA was synthesized with an iScript reverse transcription kit  using a 1:1 mixture of oligo (dT) and random primers. iScript reverse transcriptase (RT) was replaced with water for (-RT) controls. The following products were amplified with the corresponding primer pairs using 1 µL of cDNA. Zebrafish wt Nradd-EGFP: forward primer 5′-AAAAAGGATCCCCACCATGAAAGGAGCAACTGAAGC-3′ and reverse primer 5′-AAAAAGAATTCGACCACTGATACTCCTTGCG-3′; Nradd death domain deleted (DDD)-EGFP: forward primer 5′-AAAAAGGATCCCCACCATGAAAGGAGCAACTGAAGC-3′ and reverse primer 5′-AAAGAATTCATCCTGTTTGCTGTCCCGTTTA-3′. Both PCR products were digested with BamHI and EcoRI and ligated into a pCS2P+ vector that has EGFP. The Nradd DDD construct was generated through the deletion of a 95 amino acid region between the 318th and 413th amino acids at the C-terminal region. Nradd glycosylation site deleted (GSD)-EGFP was generated by site-directed mutagenesis at wt Nradd-GFP using overlap extension PCR. The Nradd GSD construct was generated through the deletion of a 101 amino acid region between the 22nd and 123rd amino acids at the N-terminal region. The first round of PCR was conducted with the two primer pairs forward1 5′-AAAAAGGATCCCCACCATGAAAGGAGCAACTGAAGC-3′, reverse1 5′-ACACTCCCCATCCTGGCCCACAGCCAAAGGCCATCTT-3′ and forward2 5′- AAGATGGCCTTTGGCTGTGGGCCAGGATGGGGAGTGT-3′, reverse2 5′-AAAAAGAATTCGACCACTGATACTCCTTGCG-3′. The second round of PCR was performed by using the purified PCR product of the first round as the template and the primers forward1 and reverse2. The purified PCR product of the second round of PCR was again digested with BamHI and EcoRI and ligated into a pCS2P+ vector that has EGFP. For Fz8a-EGFP, zebrafish Fz8a was amplified with the forward primer 5′-AGAATTCAACCACCATGGAGTGCTACCT-3′ and the reverse primer 5′- GGATCCTCAGACTTGGGACAAAGGC-3′. The PCR product was digested with EcoRI and BamHI and ligated into a pCS2P+ vector that has EGFP. For Fz8a-mRuby3, mRuby was amplified with the forward primer 5′-AAGGCGCGCCTATGGTGTCTAAGG-3′ and the reverse primer 5′- AATCTAGATTACTTGTACAGCTCGTCCATGCCAC-3′ from the mRuby3 plasmid. The PCR product was digested with AscI and XbaI and ligated into an Fzd8a-EGFP plasmid after excising EGFP with the same restriction enzymes. For Nradd-mRuby3, mRuby3 was amplified with the forward primer 5′-AAATCTAGAATGGTGTCTAAGGGCGAAGA-3′ and the reverse primer 5′- AAAGATATCTTACTTGTACAGCTCGTCCAT-3′ from the mRuby3 plasmid. The PCR product was digested with XbaI and BsaBI ligated into a wt Nradd-EFGP plasmid after excising EGFP with the same restriction enzymes. Successful cloning was verified by sequencing, restriction digestion and agarose gel electrophoresis.nradd and 20 pg for Wnt8. Embryos were fixed at indicated stages  in 4% paraformaldehyde (PFA) overnight. WMISH was performed with nradd, mCherry, goosecoid, otx2, hoxb1b, foxg1a, her5 and krox20 antisense RNA probes as described previously [Capped sense RNAs of GFP, wt Nradd, Nradd-GFP, Nradd DDD-EGFP and Nradd GSD-EGFP were synthesized using an mMessage mMachine Kit . mRNAs were injected into 1-cell zebrafish embryos as 250 pg for eviously .rpl13a or β-actin were used as the reference genes for normalization to determine the relative gene expression levels. qPCR was performed in triplicate using GoTaq qPCR Master Mix  in an Applied Biosystems 7500 Fast Real Time PCR machine . The data were analyzed using GraphPad Prism 8 software . The values are mean ± standard deviation (SD) of three samples. The following primers were used: mCherry forward 5′-GAACGGCCACGAGTTCGAGA-3′ and reverse 5′-CTTGGAGCCGTACATGAACTGAGG-3′, zebrafish sp5l forward 5′-GCTTCACGCAGGTGTGGAT-3′ and reverse 5′-TTCTGGAGATGAGCTGGGAGT-3′, zebrafish axin2 forward 5’-TAGTTTTGCCCCTGCCACG-3’ and reverse 5’-TCCCAGCTTGTAAGGAGGAATG-3’, zebrafish cdx4 forward 5’-CCAGAGAAAATCAGAGCTGGCA-3’ and reverse 5’-TTGCACCGAGCCTCCACTATT-3’, zebrafish nradd forward 5’-AAGTGTCAGCCATGCCAAGA-3’ and reverse 5’-AGGAATACCGATGGGGCAATG-3’, zebrafish rpl13a forward 5′-TCTGGAGGACTGTAAGAGGTATGC-3′ and reverse 5′-AGACGCACAATCTTGAGAGCAG-3′, zebrafish β-actin forward 5′-GAAGGAGATCACCTCTCTTGCTC-3′ and reverse 5′-GTTCTGTTTAGAAGCACTTCCTGTG-3′.cDNA was synthesized from RNA using a ProtoScript II First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. Zebrafish 2 humidified environment. All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin.Human embryonic kidney 293T (HEK293T), neuroblastoma SH-SY5Y and bone osteosarcoma U2OS cell lines were purchased from ATCC . HEK293T and U2OS cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) and SH-SY5Y cells in DMEM F-12 at 37 °C in a 5% (v/v) COt-test. Error bars represent SD, where * and ** indicate p < 0.05 and p < 0.01, respectively.HEK293T were seeded on 24-well plates and transfected in triplicate with 20 ng of Wnt8, with and without 300 ng of Nradd. SH-SY5Y cells were seeded on 24-well plates and transfected in triplicate with 250 ng of Nradd and stimulated with Wnt3a conditioned media (CM) for 6h. All cells were co-transfected with 20 ng of firefly luciferase reporter pGL3 BAR  and 5 ngZebrafish embryos were injected with 250 pg Nradd-EGFP and 250 pg RFP-GPI at the 1-cell stage, dechorionated at 3 hpf, mounted in 3% methylcellulose, and imaged using a Zeiss LSM 880 confocal microscope . U2OS cells were seeded in a 6-well plate on a 35 mm glass-bottom dish with a 10 mm micro-well #1.5 cover glass (0.16–0.19 mm) and transfected with Fz8a-EGFP and Nradd-mRuby3 plasmids (500 ng each) using Lipofectamine 3000 reagent at 24 h. The next day, cell membranes were stained with CellMask Deep Red at a concentration of 5 µg/mL for 5 min. The cells were imaged in Leibovitz’s L-15 medium without phenol red using a Zeiss LSM 780 confocal microscope.2, pH 7.4) and incubated for 1 h at 37⁰C with GPMV buffer including 2 mM N-ethyl maleimide (NEM). After 1 h, the supernatant containing vesicles was collected, incubated with Atto647N-labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (Atto647N-PE) for membrane staining and imaged using a Zeiss LSM 780 confocal microscope.U2OS cells were seeded in a 6-well plate on a 35 mm glass-bottom dish with a 10 mm micro-well #1.5 cover glass and transfected with 500 ng Nradd-EGFP using Lipofectamine 3000 reagent at 48 h. At ~70% confluence, the cells were washed twice with GPMV buffer . Lysate was centrifuged at 300 g for 5 min at 4 °C. Supernatant was precipitated with a SureBeads™ Protein G Magnetic Beads Co-Immunoprecipitation Kit  according to the kit protocol. For western blotting, samples were dissolved in 5X loading dye and separated by SDS gel electrophoresis on a 10% acrylamide-bis acrylamide gel. Proteins were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane . Membranes were blocked in 5% milk powder for 45 min at room temperature (RT) and incubated with the following antibodies at the corresponding dilutions. Primary antibodies: rabbit anti-GFP ((D5.1) XP, 1:1000; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-HA . Secondary antibodies: anti-rabbit IgG, horseradish peroxidase (HRP)-linked and anti-mouse IgG, HRP-linked .The molecular interaction between Nradd protein and the components of the Wnt–receptor complex was examined by comparing the diffusion characters on the membrane using a fluorescence cross-correlation spectroscopy (FCCS) technique. FCCS measurements were performed on a Zeiss LSM 780 confocal microscope equipped with 63X oil immersion objective (1.4 NA). GFP- and mCherry-labeled proteins were excited with 488 and 561 nm lasers, respectively. Detection intervals of 500–550 nm and 600–690 nm were used to detect green and red signals, respectively. Curves were analyzed using the FoCuS-point software package [hsp70l:wnt8a-GFP)w34 were injected with the capped sense RNAs of control GFP (100 pg), wt Nradd-GFP (250 pg), Nradd-DDD (250 pg) or Nradd-GSD (250 pg) at the 1-cell stage. Injected embryos were heat shocked at 60% epiboly, dechorionated at 24 hpf by adding 100 μL of 10 mg/mL pronase in 10 mL E3 medium  and fixed in 4% PFA in PBST  overnight at 4 °C. Embryos were incubated in ice-cold methanol at -20 °C for 24 h. Methanol was removed and embryos were washed twice with 1 mL 1× PDT  for 30 min by rocking gently at RT. Next, embryos were incubated with 500 μL blocking buffer  at RT for 1 h and incubated with rabbit anti-cleaved caspase 3 antibody  at 4 °C overnight. The next day, embryos were washed twice with 1× PDT, incubated with rhodamine (TRITC)-AffiniPure donkey anti-rabbit IgG  for 2 h at RT, re-washed with 1× PDT, mounted in 50% glycerol in PBS and imaged using a Zeiss LSM 880 confocal microscope.Zebrafish embryos produced by crossing wt AB and Tg. Flow cytometry was performed using a BD Fortessa and data were analyzed with FlowJo 8.8.6 software . Experiments were performed in three biological replicates and data are representative of at least three independent experiments.HEK293T or SH-SY5Y cells were seeded in 6-well plates. For the control and Nradd, cells were transfected with firefly luciferase reporter pGL3 BAR (100 ng) and the plasmid containing Nradd without GFP tags (250 ng), respectively, according to the manufacturer’s protocol. For the positive controls, cells were treated with 0.3 mM Hnradd) that has no previously defined function in Wnt/β-catenin signaling and in vertebrate embryonic development. At late gastrula stages, nradd was expressed at the marginal zone and later became concentrated at the posterior sites of the embryo w35 (hs:Axin1) and Tg(hsp70l:wnt8a-GFP)w34 (hs:Wnt8) by overexpressing pathway inhibitor Axin1 and pathway activator Wnt8, respectively. In all three developmental stages, the inhibition of Wnt/β-catenin signaling significantly suppressed nradd expression, while its activation upregulated nradd ia (7xTcf:mCherry). Reporter expression dramatically decreased in nradd-overexpressing embryos at late gastrula, somitogenesis and organogenesis stages , which generated class 3 and 4 phenotypes at 24 hpf  expression [nradd can relieve wnt8-mediated repression of the dorsal organizer. Wnt/β-catenin signaling is also required for the induction of posterior neural fates during gastrulation. nradd overexpression caused an extension of the early forebrain and midbrain region marked by otx2 [nradd in later patterning of the neuroectodermal fates, we tested whether it can rescue wnt8-induced posteriorization of the zebrafish embryo brain. nradd overexpression rescued the anterior neuroectodermal fates, shown by the forebrain marker foxg1a, which completely disappeared in wnt8-overexpressing embryos during mid-somitogenesis  in zebrafish embryos during the early blastula stage . To test whether zebrafish Nradd is functional in humans, first we transfected human embryonic kidney 293T (HEK293T) cells with the zebrafish Nradd. Nradd significantly induced apoptosis but did not act together with Wnt3a  is a Wnt/β-catenin target during zebrafish development, ii) can suppress Wnt/β-catenin signaling during development and in mammalian cells, iii) efficiently inhibits Wnt8-mediated ventralization of the mesoderm and posteriorization of the neuroectoderm, iv) localizes at the plasma membrane and physically interacts with the Wnt–receptor complex, v) promotes apoptosis by acting together with Wnt/β-catenin signaling during development and vi) can also enhance apoptosis in mammalian cells.Owing to its essential roles in embryonic development and the maintenance of tissue homoeostasis, Wnt/β-catenin signaling is tightly regulated by various pathway modulators. Here, we characterize the functional role of Nradd, a poorly characterized homologue of p75and downstream executioner caspases including caspase 3 . Aberran neurodevelopmental disorders, neurodegenerative conditions such as traumatic brain injury, amyotrophic lateral sclerosis and stroke and psychiatric disorders ,63. p75NTR, a pleiotropic signaling molecule, can act as a co-receptor for various receptors, including Trk, sortilin and Nogo, and mediate a variety of cellular functions from survival and axonal growth to apoptosis through several signaling pathways [NTR homolog, in the regulation of apoptosis during embryonic development. Wnt signaling regulates the early and late stages of apoptosis during the development of various organs, including the brain, the limbs and the heart [has been associated withneurotrophin signaling in the coordination of neuronal development and differentiation [Neuronal survival and innervation rely on the presence of developing neuronal populations that receive accurate trophic factors from neighboring cells and activate intracellular signaling pathways . Neverthpathways ,65,66,67he heart ,69,70,71he heart . Wnt signtiation ,74. Wnt/ntiation . Our resNTR has been shown to promote the apoptosis of neurons and oligodendrocytes in development, regeneration and pathological conditions [NTR structural domains in the activation of death receptor signaling. The extracellular domain of p75NTR appears to be responsible for the conformational changes that propagate the signal to its death domain, which further recruits the interactors of various signaling pathways [NTR-induced neuronal apoptosis [p75nditions ,78,79,80pathways ,82,83. Tpoptosis . The indpoptosis . Our funNRADD gene carries inactivating mutations that have converted it into a pseudogene in humans. Strikingly, our data show that zebrafish Nradd can not only inhibit Wnt/β-catenin signaling but also promote apoptosis in human embryonic and cancer cell lines, indicating that Nradd has an evolutionarily conserved function in the regulation of Wnt signaling and apoptosis. It is possible that human p75NTR homologs might compensate for the absence of Nradd in humans. While Nradd appears to act together with Wnt signaling to enhance apoptosis during development, Wnt activation does not further enhance Nradd-mediated apoptosis in human cells. The activation of Wnt/β-catenin signaling could, however, sensitize human melanoma cells to apoptosis induced by the TNF-related apoptosis-inducing ligand (TRAIL/APO2L) [Mouse NRADD has been reported to induce cell death in primary neuronal cells and neuroblastoma cell lines ,85,86. OL/APO2L) . In contL/APO2L) . FurtherL/APO2L) . Thus, wThe role of Wnt/β-catenin signaling in neural development starts at the early induction of the neural plate and continues throughout subsequent patterning of the neuroectoderm during CNS development . Our resOur study reveals the death receptor-encoding nradd, a homolog of p75NTR, as a Wnt target during development and an inhibitor of the Wnt/β-catenin signaling pathway in zebrafish embryos and mammalian cells. Nradd can potently suppress canonical Wnt-mediated phenotypes in mesodermal and neuroectodermal patterning during zebrafish gastrulation. By physically interacting with the Wnt–receptor complex at the plasma membrane, Nradd acts together with Wnt/β-catenin signaling to promote apoptosis during development. Moreover, human embryonic and neuroblastoma cells undergo apoptosis upon Nradd overexpression. Thus, by both fine-tuning pathway activation at the plasma membrane and regulating cell death, the Wnt modulator Nradd might serve as an attractive target for the discovery of therapeutic interventions against Wnt-related human diseases.


Morusin on inhibiting osteoclast differentiation in vitro. However, whether Morusin can regulate the osteogenic differentiation in addition to the proliferation of BMSCs remains unclear.Osteoporosis (OP) is a metabolic bone disease due to the imbalance of osteogenesis and bone resorption, in which, bone marrow mesenchymal stem cells (BMSCs) have a significant effect as the seed cells. Recent research has shown the function of Morusin for 3, 5, 7, and 14 days. The proliferation of BMSCs was detected by MTT assay. The effect of Morusin on osteogenic differentiation of BMSCs was detected by RT-qPCR, Western blotting, ALP, and Alizarin Red staining. The effect of Morusin on Wnt/β-catenin signaling pathway was analyzed by RT-qPCR, Western blotting, and immunofluorescence. Finally, in the ovariectomy-induced osteoporosis model, the anti-osteoporosis activity of Morusin was determined by micro-CT, HE, and immunohistochemistry.BMSCs were isolated from 4-week-old Wistar rats and then treated with different concentrations of Morusin in the promotion of the proliferation in addition to osteogenic differentiation of BMSCs. Moreover, it also has an impact in activating the Wnt/β-catenin signaling pathway via inhibition of β-catenin phosphorylation as well as promotion of its nuclear translocation. Upon Dickkopf-related protein-1  was added to the Morusin, Morusin had a decreased stimulatory osteogenic effect on BMSCs. Finally, in the rat OP model, we found that Morusin could also exert anti-osteoporosis activity in vivo.The results showed the function of 2.5–10 μM Morusin in the promotion of osteogenic differentiation of BMSCs via the activation of Wnt/β-catenin signaling pathway and also shows the potential of Morusin to be an agent for osteoporosis treatment.This study indicates the ability of  Osteoporosis (OP), a highly prevalent metabolic bone disease, is featured with reduced bone mineral content in addition to structural degeneration of the skeletal system , 2. GeneThe Wnt/β-catenin signaling pathway is a significant classical pathway in osteogenic differentiation of BMSCs . As a meMorusin -Akt signaling pathway  from the Experimental Center of the Hubei Medical Scientific Academy . All animal experiments were conducted in the Center for Animal Experiment of Wuhan University , and all protocols were authorized by the Animal Welfare Committee in Wuhan University (License number: 14016).& Morusin (40 mg/kg) group . Then, immunohistochemistry was performed to determine the level of RUNX2 protein in the femur. The dilution of RUNX2 primary antibody was 1:100. An optical microscope  was implemented for the staining result observation and photographing. Then, we analyzed the stained images with image J software (version 6.0) before measurement of the mean optical density (MOD) of 5 random fields in every section for determining the staining intensity.Femurs fixed in 75% alcohol were scanned and analyzed with skyscan1276 μCT system . The sample was placed in a transparent cylindrical groove and secured with paper tape. The scanning mode was set to 0.5-mm filter, 450-V scanning voltage, and 5-μm scanning resolution. After scanning data reconstruction, the region of interest was selected for non-volume-related parameter analysis  and reconstructed to obtain 3D images .t test to determine the statistical significance. For comparison between more than two groups, we implemented one-way ANOVA. A p < 0.05 showed statistically significant difference.We indicated all experimental values as mean ± standard error of mean (SEM) of the experimental values (we repeated each experiment for three times). For comparison between the two groups, we performed double-tailed Student Morusin at diverse concentrations (0–50 μM) was treated for 3–14 days. Then, we executed MTT assay. As per the results, there was promoted proliferation detected in the osteogenic BMSCs treated with Morusin at 2.5, 5, and 10 μM  on BMSC differentiation and detected the levels of osteogenic marker genes including bone sialoprotein (Bsp), Col1a1, Runx2, and osteocalcin (Ocn). Morusin was found to promote osteogenic marker genes in terms of the expression at 2.5, 5, and 10 μM, while 10 μM of Morusin revealed the strongest effect , and dishevelled-1 (Dvl-1) mRNAs were detected in BMSCs treated with Morusin (0–10 μM) on the 3rd, 5th, and 7th days of differentiation, respectively  about the gene expression of β-catenin, Lef-1, and Dvl-1  heightened the expression of β-catenin (red) in the nuclei, while DKK-1 (500 ng/ml) partially reversed Morusin’s effect on β-catenin translocation  had the effect in inhibiting the phosphorylation of β-catenin. Namely, with the increase of Morusin concentrations, the phosphorylated β-catenin decreased in terms of the protein expression, while total β-catenin increased  was partially reversed by DKK-1 (500 ng/ml)  Fig. c. These Morusin (40 mg/kg) was admitted to the OVX rats to evaluate the effect of Morusin on OP. Micro-CT scan results showed that Morusin (40 mg/kg) could alleviate the bone loss caused by estrogen deficiency to some extent, which was manifested in increased Tb.N, Tb.Th, BV/TV, and Conn.Dn, while a decrease in Tb.Sp and SMI , while DKK-1 can partially block the promoting effect of Morusin on osteogenic differentiation of BMSCs. When Wnt/β-catenin signaling pathway is activated, it can lead to the disintegration of APC, Axin, and GSK3β complex. Then, inhibition of β-catenin phosphorylation raises the levels of β-catenin in the cell and allows β-catenin to enter the nuclei to bind to the transcription factor TCF/LEF, thereby initiating transcription of downstream target genes [Morusin had an impact of inhibition about the phosphorylation of β-catenin but promoted its nucleus translocation. These findings revealed that Morusin had the ability in advancing osteogenic differentiation of BMSCs via activating Wnt/β-catenin signaling pathway.Although eoblasts –39. Theret genes . Thus, wMorusin on bone loss in rats was further investigated. In this research, OVX rats were used as the osteoporosis model, which has been widely regarded as a model with low osteogenic capacity of the BMSCs [Morusin treatment, the bone loss of OVX rats was reduced, the number of osteoblasts was increased, and the protein expression of osteogenic marker genes was also increased. These evidences show that Morusin can promote osteoblastic proliferation and bone formation in OVX rats, which is consistent with vitro experiment.The impact of he BMSCs , 41. MicMorusin in promoting the osteogenic differentiation in addition to proliferation of BMSCs via activation of Wnt/β-catenin signaling pathway in vitro, and also presented anti-osteoporosis activity in OVX rats. This study also suggested the potential of Morusin to be a therapeutic agent for osteoporosis. Further researches are needed before its clinical application.It could be concluded that our current study demonstrated the ability of


The ability to infer how confident other people are in their decisions is crucial for regulating social interactions. In many cooperative situations, verbal communication enables one to communicate one's confidence and to appraise that of others. However, in many circumstances, people either cannot explicitly communicate their confidence level  or may be intentionally deceitful . It is currently unclear whether one can read others’ confidence in the absence of verbal communication, and whether one can infer it as accurately as for one’s own confidence. To explore these questions, we used an auditory task in which participants either had to guess the confidence of someone else performing the task or to judge their own confidence, in different conditions . Results demonstrate that people can read the confidence someone else has in their decision as accurately as they evaluate their own uncertainty in their decision. Crucially, we show that hetero-metacognition is a flexible mechanism that relies on different cues according to the context. Our results support the idea that metacognition leverages the same inference mechanisms as those involved in theory of mind. Metacognition—‘cognition about cognition’—is typically characterized as involving two distinct but interconnected processes: evaluation and control. Metacognitive evaluation involves monitoring the quality of first-order processing, such as memory, perception, language, reasoning and so on –10. MetaOver the last decade, the study of metacognition has essentially focused on the processes and on the mechanisms that underpin intra-personal metacognition . However, as is the case for any cognitive information, metacognitive information may be shared with others in order, for instance, to improve collective decision making. For example, when one feels sick, one may lack confidence in one’s ability to self-diagnose accurately and hence decide to go see a doctor to share this uncertainty and have a professional opinion. Recently, different key studies have explored the potential benefit of sharing one’s uncertainty in the context of perceptual decision-making.Many Cooks Spoil the Broth effect. This effect shows that adding more and more expert individuals to a group may eventually undermine both group and individual performance [Indeed, recent evidence shows that people communicate their metacognitive representations, namely their confidence in their perceptual decisions, and that, under certain conditions, the communication of such metacognitive information leads to improved joint perceptual decisions —this is formance , 15.These results have led some authors to propose that the function of sharing metacognitive representations is precisely to regulate group behaviour , 17, a pcommunicating confidence may influence group decision. However, people’s ability to read other people’s confidence has so far received little attention. Of course, in many situations, sharing confidence is just a matter of verbal communication: subject A says to subject B how uncertain she is about such or such decision. In many other situations, however, verbal communication cannot be carried out as easily or even trusted. Imagine for instance that you are competing with someone or playing poker. While neither of you wants to share information and will in fact deploy efforts to hide information, the ability to read your opponent’s confidence remains nevertheless crucial for your own performance. Similarly, in other daily life settings, such as a romantic date or a job interview, one may not be able to rely as much on verbal communication as on other cues. Likewise, teachers need to be able to carry out online evaluations about whether their students are keeping up with the pace. This potential ability to read others’ uncertainty mental states is in line with substantial research dedicated to the theory of mind ability to read others’ emotions or doxastic states [So far, research on metacognition and group decision has thus essentially focused on how c states . In thisc states .In a significant paper, Patel et al.  have shoIn the present study, we focused on three main questions: First, are people able to read others’ confidence in the absence of verbal communication and, if this is indeed the case, what are the cues through which this is accomplished? In particular, we surmised that movement cues are not the only cues that people may use when assessing someone else’s confidence, especially when the participant observing the other person also has access to the stimuli. In addition, we hypothesized that observers may also use task difficulty and others’ response time to infer their confidence. Both of these cues have indeed been shown to be important when evaluating one’s own confidence , 25 and The second question we addressed is whether assessing one’s own confidence is more accurate than assessing someone else’s. In other words, is there a first-person perspective benefit when assessing confidence, or does assessing one’s own confidence leverage exactly the same machinery as that involved when assessing someone else’s confidence  per se. This is in line with recent work distinguishing confidence in a response from general (un)certainty that does not refer to a particular response [To address these issues, we designed an auditory pitch discrimination task in which participants had to decide which of two pure tones presented successively had the higher pitch—a first-order decision—and to rate their confidence in their response—a second-order decision. Pairs of participants were tested together. In one condition, participants performed the task separately (Baseline condition); in another condition, while one participant was performing the task, the other was observing her doing it and had access to the stimuli (Full-Observation condition). In what follows the term ‘observer’ denotes the participant observing the other participant performing the task, whom we will call the ‘agent’. In the Full-Observation condition, the observer was to guess the confidence of the agent on each trial . In the Partial-Observation condition, the observer was also to guess the confidence of the agent, but she did not have access to the stimuli. Finally, in the Self-Observation condition, each participant observed herself doing the task from a video recording of their Baseline condition, but did not have access to the stimuli themselves. In the three observation conditions, we asked observers to guess the confidence of the agent. Note, however, that due to the lack of stimulus access in some of the conditions, it is not possible to define confidence in a judgement/response. It remains possible that in these cases, observers instead report how uncertain or how fast the agent is instead of how confident they are response .versus someone else , in the absence of the stimuli.We hypothesised that participants would be able to judge the confidence of the agent and that it would be easier for observers to judge agents’ confidence in the Full-Observation condition than in the Partial-Observation condition because of the strong cue that task difficulty constitutes when judging confidence. However, if reading others’ confidence in the absence of verbal communication is indeed possible, the performance of observers should also be predictive of the actual confidence reported by the agent in the Partial-Observation condition. We additionally hypothesised that, in this condition, agents’ response times might constitute a strong cue for observers. As indicated above, response times are an important cue used to infer one’s own confidence , 25. FurFifty participants were recruited . As the experimental task involved pairs of participants, we recruited only female participants so as to avoid gender effects. All participants reported no history of hearing disorder and no history of psychiatric or neurological disorders. Participants received a monetary compensation (10€ per hour). They were naive to the purpose of the study and gave informed consent, in accordance with institutional guidelines. The study was approved by the local ethical committee of the Université libre de Bruxelles.All experimental sounds were sinusoidal pure tones, with 5 ms rise/fall time and 44100 Hz sampling rate, generated using MATLAB  with the Psychophysics toolbox –30. AudiParticipants were paired two by two and did not know each other. Upon arrival, one participant was randomly assigned to first take the role of the agent and the other the role of the observer. They were instructed not to talk to each other.The experiment was divided in two sessions of approximately 2 hours each. The second session took place between 24h and 48h after the first session. Each session corresponded to two experimental conditions consisting of 250 trials each . In each condition, the agent had to perform the pitch discrimination task and press the left or right arrow on the keyboard with their right hand to indicate which sound had the highest pitch. Participants then had to express how confident they were in their response by pressing a key with their left hand on a separate keypad using a scale ranging from 1 (guess) to 4 (sure). At the end of the session, participants switched roles so that the agent became the observer and the observer became the agent. For each new condition, they returned to their original role assignment.In the Baseline condition , particiIn the Partial-Observation condition  the dispFinally, in the Self-Observation condition , particiThe Baseline condition always took place first and the Self-Observation condition always took place last so as to minimize any memory effect in the Self-Observation condition and to ensure that all participants knew the task before judging the confidence of the agent in the ensuing conditions. The order of the Full-Observation condition and the Partial-Observation condition was randomised over pairs of participants. At the end of the third session participants completed the Berkeley expressivity questionnaire  in orderData preprocessing and analyses were performed with R (2016), using the afex , lme4 33, lmerTeIn order to compare observers’ ability to assess the confidence of the agent as well as her own confidence in the different conditions, we performed mixed model analyses. We fitted a linear mixed-effects model of the guess of the observer, with confidence of the agent, condition  and their interaction as fixed and random effect.In order to test whether the influence of the confidence of the agent on the guess of the observer was mediated by the response times of the agent, we performed mediation analyses. In each condition, a mediator mixed model was first fitted to predict the response times of the agent by the agent’s confidence. Then, an outcome mixed model was fitted to predict the guess of the observer by the response times and the confidence of the agent. The mediation analysis was performed with these two models .ROC, [ROC. This is what we further refer to as the metacognitive sensitivity of the observer regarding the agent.Metacognitive sensitivity was estimated through the area under the type-II Receiver Operating Characteristic curve . HoweveIn addition, we used within-subject repeated measures analysis of variance to test for differences in first-order performances  and second-order responses (confidence ratings) and second-order performances (metacognitive sensitivity) followed by paired and one sample t-tests to determine the direction of differences. In all ANOVAs, degrees of freedom were corrected using the Greenhouse-Geisser method.Finally, we used Bayesian statistics to assess the likelihood that data were in favor of the alternative or null hypothesis using the default medium prior of the BayesFactor R package . This isIn order to compare agents’ performance at the first- and second-order level we carried out 6 ANOVAs, as follows see also .d’)  = 0.61, p > 0.4, ηp2 = 0.01, BF = 0.12) or on criterion  = 2.45, p = 0.10, ηp2 = 0.05, BF = 0.56). However, a repeated-measures ANOVA showed differences in mean response times across the different conditions  = 28.22, p < 10−3, ηp2 = 0.38). Specifically, paired t-tests indicated that response times were shorter in the Full-Observation  and Partial-Observation  conditions than in Baseline condition  (Full-Observation vs. Baseline: t(47) = 5.37, p < 10−5, Partial-Observation vs. Baseline: t(47) = 5.76, p < 10−6), but there was no difference between the Partial- and Full-Observation condition (t(47) = -1.02, p > 0.3, BF = 0.26)  = 0.12, p > 0.3, ηp2 = 0.003)).First, regarding the first-order task , repeated-measure ANOVAs showed no effect of condition on type 1 sensitivity  or confidence ratings variability using standard deviation as a measure of variance  = 0.44, p > 0.4, ηp2 = 0.03, BF = 0.10). This indicates that the first-order performance and confidence estimates of the agent were not impacted by the different conditions.Second, with respect to the second-order task, we found no effect of condition on mean confidence ratings  = 0.87, p > 0.3, ηROC as a measure of type 2 sensitivity. A repeated-measure ANOVA showed no difference between conditions  = 1.24, p > 0.3, ηp2 = 0.03, BF = 0.19).Third, we compared the metacognitive sensitivity of the agent across the different conditions using Ap2 = 0.02, BF = 0.17, p2 = 0.02, BF = 0.17).In order to compare observers’ confidence level and confidence variability between conditions we performed 2 within-subjects ANOVAs. The mean confidence level of the observer did not differ between conditions  = 1.09, p > 0.3, ηIn order to compare the relationships between the agent’s and observer’s confidence judgements between the different conditions, we fitted a linear mixed-effects model of the guess of the observer, with confidence of the agent, condition  and their interaction as fixed and random effects.−3).The first row in −3), indicating that the observer can track the confidence of the agent.The second row shows the estimation of the regression slope between the guess of the observer and the confidence of the agent in the Full-Observation condition, and shows that this relation is statistically significant , and between the Partial-Observation condition and the Full-Observation condition .Crucially, the fifth and the sixth row indicate that the relationship between the guess of the observer and the confidence of the agent was smaller in the Self-Observation compared to the Full-Observation condition  and that there was no difference between the Partial-Observation and the Full-Observation condition .t = - 0.90, p > 0.3) between the guess of the observer and the confidence of the agent in the Self-Observation condition compared to the Partial-Observation condition.Another linear mixed-effects model comparing only the Self-Observation condition (in which participants were judging their own performances in the baseline condition by means of video recording) to the Partial-Observation condition revealed no difference in regression slopes  and the outcome mixed model showed a significant relationship between the guess of the observer and the response times of the agent  and between the guess of the observer and the confidence of the agent .We used causal mediation analyses to test whether the effect of the confidence of the agent on the guess of the observer was mediated by the response times of the agent , there was 42%  thaThese findings suggest that the response time of the agent is a crucial mediator between the confidence of the agent and the guess of the observer in the Partial-Observation condition, in which participants did not have access to the stimuli. However, this mediation was partly reduced in the Full-Observation condition, and considerably reduced in the Self-Observation condition. We can draw this inference based on the fact that the 95% confidence interval in the Self-Observation condition does not overlap with the confidence intervals of the other two conditions. In other words, the observer relied less on the response times of the agent to estimate their confidence in the self-observation condition.ROC based on the confidence responses given by the observer and the first-order responses of the agent (which corresponds to the same subject in the Baseline condition).Thus far, we have shown that participants are able to evaluate other people’s confidence even when they do not have access to the stimuli the agent is judging. In addition, mediation analyses indicated that, when judging other people’s confidence, participants use the agent’s response times, especially when they do not have access to the stimuli. Finally, regression analyses showed a difference between the Partial- and Self-Observation conditions, with a stronger relationship between the confidence of the agent and the guess of the observer in the latter than in the former. This suggests that we do have some kind of privileged access to our own confidence. However, the cues the cognitive system is using differ between conditions, as mediation analyses show that in the Self-Observation condition, response times mediate to a lesser extent the relationship between the confidence of the agent and the guess of the observer. To further corroborate the results of regression analyses between the confidence of the agent and the guess of the observer, we performed type-II signal detection theory analyses (SDT) . Type-IIROC of participants judging themselves in the Baseline condition were significantly higher than 0.5  = 7.93, p < 10−9). In the Full-, Partial- and Self-Observation conditions the AROC were also significantly higher than 0.5  = 7.19, p < 10−8; Partial-Observation condition: Mean = 0.53, SD = 0.07, t(47) = 3.58, p < 10−3, Self-Observation condition: Mean = 0.53, SD = 0.06, t(47) = 3.01, p < 0.01), suggesting that the metacognitive sensitivity of the observer regarding the agent (or herself through a video recording in the Self-Observation condition) was also higher than chance.As expected, a one-way one sample t-test showed that the A−4, ηp2 = 0.24), with higher AROC in the Baseline condition compared to the Partial-Observation condition (paired t-test: t(47) = -4.09, p < 10−3) and to the Self-Observation condition (paired t-test: t(47) = -6.60, p < 10−7). In the Full-Observation condition we also found higher AROC compared to the Partial-Observation condition (paired t-test: t(47) = 4.88, p < 10−4) and to the Self-Observation condition (paired t-test: t(47) = 4.55, p < 10−4)  = 15.25, p < 10 < 10−4) . HoweverROC analysis largely corroborates the mixed model analysis demonstrating that participants are able to guess others’ confidence in absence of verbal communication. Participants have higher AROC in the Full-Observation or Baseline condition than in the Partial- or Self-Observation condition .In sum, the AWe found no significant correlations between the emotional expressivity of participants as assessed by the Berkeley expressivity questionnaire and the relationship between the confidence of the agent and the guess of the observer .In this study, we investigated the extent to which one can evaluate the confidence of others in the absence of verbal communication. We also asked whether one has privileged access to confidence when performing the task directly compared to observing someone else, or when observing oneself compared to observing someone else. We found that people are able to read the confidence of others, even in the absence of verbal communication. We also found that people can guess the confidence of someone else even when they do not have access to the stimuli. Finally, we found that one is not better at self-evaluating one’s own confidence than at evaluating other people’s confidence. Below, we expand on these different results in turn.ROC) computed from the guess of the observer and the performance of the agent was significantly above chance in the Full-Observation and Partial-Observation conditions.First, in line with the study from Patel, Fleming & Kilner  see Int, mixed rSecond, participants (observers) were as well fairly good at tracking the confidence level of agents in the Partial-Observation condition in which stimuli were not accessible. The latter finding suggest two, not necessarily exclusive, mind reading processes for confidence: 1) In the Full-Observation condition one could argue that participants are not simply performing the task mentally and inferring the confidence of others based on their own implicit judgements but also base their inferences on the observation of the agent behaviour (see below); 2) One could also suggest that when stimuli are not available  to participants, they switch to other cues. Mediation analyses suggest that response times had a stronger mediating role in the Partial-Observation than in the Full-Observation condition. However, one has to be careful because confidence intervals of the mediation values overlap between conditions; except with the more conservative dataset in Supplementary Material for which there is no overlap between the Full-Observation and the Partial-Observation conditions. One thus can conjecture that there is a shift in strategy from the Full-Observation condition to the Partial-Observation condition. This phenomenon might be especially important with respect to collective decision, as one may conjecture that response times can thus serve as a competence signal, so that the first person to respond can be the one that will dominate the collective decision.Note, however, that in the Full-Observation condition, response times are still mediating part of the variance between the actual confidence of the agent and the inferred confidence by the observer. This may reflect two possibilities: First, even in the Full-Observation condition participants do not base their inference of others’ confidence level entirely on their own implicit judgments; second, participants base their inference in relying on their own internal response times explaining the mediating role of agents’ response times and confidence responses of the observers. Future research could further disentangle these possibilities by experimentally manipulating agents’ response times. For instance, one could use confederates playing the role of the agent and purposely responding with incongruent or random response times. This would allow us to see whether the observers still take into account these response times or if they are not disturbed by it under the hypothesis that they are only relying on their own internal response times.plus having an access to stimuli, as shown by similar metacognitive sensitivity (AROC). Therefore, it seems that, at least in the current experimental design, performing the task oneself does not entail a privileged access in assessing confidence in comparison to observing someone else performing the task. In other words, a first-person perspective does not benefit participants. This could even suggest that one evaluates one’s own confidence like an external observer, that is as when one observes someone else. This finding is in line with current theoretical work [The third important result is that there is no difference in accuracy in assessing confidence level between the Baseline condition, in which participants were performing the task, and the Full-Observation condition in which they were only observing the agent performing the task cal work .versus someone else, we should find that participants are better in the latter than in the former condition. However, both mixed modelling and Type 2 signal detection theory analyses showed no difference between the metacognitive sensitivity of participants in the Self-Observation condition compared to the Partial-observation condition. In addition, using mediation analyses, we found that the mediation effect was the smallest in the Self-Observation condition , with only 29% of the relationship between the confidence of the agent and guesses of the observer mediated by response times. This indicates that when judging themselves, participants used other cues than response times to judge their past confidence. Taken together, these results highlight the fact that metacognitive monitoring (of oneself or someone else) is a flexible process integrating multiple cues and that is responsive to situational demands [However, it might be that the potential first-person perspective advantage is obscured by the fact that task difficulty constitutes a strong cue in assessing confidence. The access participants (observers) have to stimuli in the Full-Observation condition would equalize confidence accuracy between the latter condition and the Baseline condition. In order to disentangle this point, we compared the Partial-Observation condition to the Self-Observation condition. If there is any advantage at assessing oneself  demands , 43.Altogether, the present study demonstrates that we can successfully read the confidence of others in the absence of verbal communication and without having access to the information the agent is evaluating. It seems that we can switch between different cues depending on the situation we are in. From an evolutionary perspective, this may be a crucial ability, allowing us to evaluate the confidence of our peers in various situations , 17. A dS1 Fig(TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


We previously demonstrated that the pleomorphic adenoma gene like-2 (PLAGL2) is involved in the pathogenesis of Hirschsprung disease. Enhanced PLAGL2 expression was observed in several malignant tumours. However, the exact function of PLAGL2 and its underlying mechanism in colorectal cancer (CRC) remain largely unknown.Immunohistochemical analysis of PLAGL2 was performed. A series of in vitro and in vivo experiments were conducted to reveal the role of PLAGL2 in the progression of CRC.Enhanced PLAGL2 expression was significantly associated with EMT-related proteins in CRC. The data revealed that PLAGL2 promotes CRC cell proliferation, migration, invasion and EMT both in vitro and in vivo. Mechanistically, PLAGL2 promoted the expression of ZEB1. PLAGL2 enhanced the expression and nuclear translocation of β-catenin by decreasing its phosphorylation. The depletion of β-catenin neutralised the regulation of ZEB1 that was caused by enhanced PLAGL2 expression. The small-molecule inhibitor PNU-74654, also impaired the enhancement of ZEB1 that resulted from the modified PLAGL2 expression. The depletion of ZEB1 could block the biological function of PLAGL2 in CRC cells.Collectively, our findings suggest that PLAGL2 mediates EMT to promote colorectal cancer metastasis via β-catenin-dependent regulation of ZEB1. During EMT, differentiated epithelial cancer cells from the primary tumour, lose their epithelial characteristics and assume a mesenchymal phenotype, which promotes the formation of an invasive phenotype and enhances cancer cell metastasis. The molecular characteristics of EMT include the suppression of epithelial markers, including E-cadherin, and the concomitant promotion of mesenchymal markers such as N-cadherin and vimentin.7 In the initiation of EMT, E-cadherin depletion is a crucial initial step.4 Various EMT-inducing transcription factors, including Snail, Twist and ZEB protein families, and corresponding intracellular signalling pathways can initiate the EMT process.8 There are the most consistent negative correlations between the expression levels of ZEB1 and E-cadherin in various cancers.9 In EMT activation, ZEB1 not only suppresses epithelial gene expression but also upregulates mesenchymal markers such as N-cadherin. ZEB1 expression is also associated with worse clinical outcomes across different types of tumours.Colorectal cancer (CRC) is the third most frequent cancer worldwide, ranking second in cancer-related mortality.8 One of the most important signalling pathways in the induction of EMT is the Wnt/β-catenin signalling pathway, which promotes the nuclear translocation of the oncoprotein β-catenin. The β-catenin nuclear accumulation can be observed in approximately 80% of CRC specimens.10 β-Catenin that is located in the nucleus functions as a coactivator of T-cell and lymphoid enhancer factors (TCF–LEFs) to transcriptionally activate downstream genes.9 The abnormal activation of β-catenin/TCF signalling has been implicated in various tumours, most notably CRC. Due to the inactivated Wnt ligand, cytoplasmic β-catenin is phosphorylated by a complex with GSK-3β, APC and Axin, and is degraded by the proteasome and then prevented from reaching the nucleus.11 Due to a lack of nuclear β-catenin, TCF–LEFs instead act as transcriptional repressors.9Signals, such as the TGFβ and Wnt/β-catenin pathways, induce EMT by triggering the expression of Snail1 and ZEB1.16 In addition, the PLAG family proteins , have highly homologous N-terminal zinc finger structures.13 PLAGL2 and PLAG1 are oncogenes involved in various malignancies, whereas PLAGL1 functions as a tumour suppressor.13 Aberrant PLAG1 expression is involved in the development of uterine leiomyomas17 and salivary gland tumours.18 The overexpression of PLAGL2 contributes to the development of malignant gliomas by strongly impeding their differentiation and by promoting their self-renewal capacity.12 Growing evidence has demonstrated that enhanced PLAGL2 expression functions as a dominant oncogene in gastrointestinal cancers.19 In CRC, PLAGL2 is one of the top 20 overexpressed genes at 20q11. Several studies have focused on various fundamental cellular processes of PLAGL2 and its crucial mechanism in tumorigenesis, but the exact role of PLAGL2 and underlying mechanism in CRC yet remain largely unknown.PLAGL2, containing a C2H2 zinc finger, serves a carcinogenic function and is involved in the pathogenesis of numerous tumours.Our study revealed that enhanced PLAGL2 expression in CRC tissues is positively correlated with the expression of mesenchymal markers but is inversely correlated with the epithelial marker expression. The data in this study also demonstrated the crucial effects of PLAGL2 on the proliferation, migration and invasion of CRC cells both in vitro and in vivo. Moreover, these results demonstrated that PLAGL2 triggers EMT, contributing to CRC metastasis via β-catenin -dependent regulation of ZEB1. Our findings illustrate that PLAGL2 serves as a crucial regulatory factor of the β-catenin-ZEB1 molecular mechanisms, and may be a promising therapeutic target for CRC anti-metastatic strategiesForty-two pairs of CRC specimens and matched para-carcinoma samples, were randomly selected from patients who had not received chemotherapy or radiotherapy before excision. All samples were gathered from patients who underwent surgery at the Union Hospital . The diagnosis of CRC in each case was confirmed by the original histopathological report. Our study protocol (S-082/2019) was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology .2 at 37 °C and cultivated in the recommended growth medium, supplemented with 10% foetal bovine serum (FBS), 100 mg/ml streptomycin and 100 U/mL penicillin . The GSK-3β inhibitor CHIR-98014, the Akt inhibitor MK2206 and the small-molecule inhibitor PNU75654 were purchased from Selleck . The Akt activator SC-79 was purchased from MedChem Express .The CRC cell lines  and the normal colon epithelial cell line FHC were purchased from American Type Culture Collection  and were checked and authenticated for genotypes by DNA fingerprinting within 6 months. The cell lines were incubated in a humidified atmosphere with 5% CO20 Antibodies for the WB analysis are shown in Supplementary Table The WB analysis was performed as previously described.Total RNA from CRC cells and specimens was extracted with RNAiso Plus . The SYBR® Premix Ex Taq (TaKaRa) was utilised for the qRT-PCR assay. The primers were listed in Supplementary Table 3 cells were seeded into 96-well plates and observed for 120 h. In each sample, the medium with 10% CCK-8 reagents  was used to replace the original medium at the scheduled time points . After incubation at 37 °C for 2 h, the absorbance of each sample was then detected at 450 nm. An EdU cell proliferation assay kit  was also used to further assess the cell growth. 1 × 105 cells were planted in 96-well plates. Briefly, the cells were incubated with 50 µM EdU at 37 °C for 2 h before fixation, permeabilisation and EdU staining. Hoechst 33342 was utilised to counterstain the nuclei at room temperature for 30 min. Cell proliferation was investigated by counting the cells with incorporated EdU and there were 5 samples per group.2 × 10Transfected SW480 and LOVO cells were planted in 6-well plates (500 cells/well) and cultured in the recommended growth medium for 2 weeks. Changing culture medium was performed every 3–4 days. We fixed the cell colonies with 4% paraformaldehyde for 15 min, stained the colonies with 1% crystal violet, and then counted the colonies.The transfected cells were harvested for cell cycle analysis, washed with cold PBS, and fixed with 75% cold ethanol. Before analysis with a BD FACS Flow Cytometer, the cells were incubated with propidium iodide (PI)  for 30 min.The transfected cells were cultured in 6-well plates. After the cells reached 90% confluence, a standard 200 μl pipette tip was subsequently utilised to scratch linear wounds. In addition, the cell monolayers were cultivated in FBS-free medium. After scratching, the images of the wound closure were captured at 0, 24 and 48 h.4 cells, suspended in medium without FBS, were seeded into transwell chambers , with or without Matrigel (Sigma-Aldrich) coating. The lower chamber contained medium with 10% FBS as chemokine. Twenty-four hours later, the migratory or invasive cells on the lower surface of the chamber were photographed and counted in 10 random microscopic fields after crystal violet staining.8 × 1021 Primary antibodies specific for E-cadherin (1:100), Vimentin (1:100), N-cadherin (1:100) and ZEB1 (1:100) were obtained from Proteintech . The primary antibody specific for β-catenin (1:150) was obtained from CST . The Fluorescence images were captured .The IF assay was carried out as described previously.22 The IHC staining results were evaluated by two independent pathologists (double-blinded). Briefly, the percentage of stained tumour cells  and staining intensity scores  were summed. The CRC tissues were categorised into four groups: negative, ≤5% cells stained, regardless of intensity; weak expression, 1–2 points; moderate expression, 3–4 points; and strong expression, 5–6 points. The total score ≥3 was classified as significant overexpression and was considered as positive expression. Antibodies for the IHC analysis are shown in Supplementary Table IHC analysis was conducted as described elsewhere.Lentiviral vectors with PLAGL2 shRNA and negative control shRNA were acquired from Genechem  and utilised in our study: PLAGL2 shRNA 5′-GACCCATGATCCTAACAAA-3′. SW480 was transduced with a lentiviral vector with PLAGL2 shRNA. LOVO was then transfected with a lentivirus carrying full-length PLAGL2 or control sequences . The knockdown or overexpression of PLAGL2 was detected by qRT-PCR and WB analysis. Short interfering RNAs for ZEB1 and β-catenin were purchased from RiboBio. The sequences for the siRNAs were as follows: siZEB1, 5′-CCTAGTCAGCCACCTTTAA-3′, siβ-catenin, 5′-AUUACAAU CCGGUUGUGA ACGUCCC-3′.The ChIP assay was performed with a Simple ChIP Plus Enzymatic Chromatin IP Kit (CST), with anti-β-catenin  and anti-TCF4  antibodies. The bound DNA fragments were amplified by qRT-PCR, and then the products of qRT-PCR were examined by gel electrophoresis on 2% agarose gels. Input and IgG were used simultaneously to ascertain that the captured signals were derived from specific bonding. The PCR primers for ZEB1 were follows: forward primer (5′-ATGGACCAATAAATAACG-3′), reverse primer (5′-TCTTCAAACCTTTCAA CT-3′).6 cells, suspended in 150 μl PBS, were implanted subcutaneously into the groin of the mice. There were 7 mice in each group. Tumour size was measured every 4 days using the following formula: V = 0.5 × (length) × (width)2. Mice were sacrificed at 28 days after implantation. To assess the tumour metastasis in vivo, 3 × 106 cells were injected into the tail vein of mice, and all mice were sacrificed after 6 weeks. The care and handling of the mice were approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology.Lentivirus carrying specific DNA sequences were transduced into SW480 and LOVO cells. Five-week-old BALB/c male nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . To assess the proliferation in vivo, 5 × 10t-test for the comparison between groups. The χ2 test was utilised to evaluate the association between the protein levels and clinical characteristics. The correlations in the gene expression levels were analysed by Spearman’s rank correlation coefficients. Differences were thought to be significant at *p < 0.05, **p < 0.01 and ***p < 0.001. n.s: no significance. The results were analysed with SPSS 19.0 software . All in vitro experiments were repeated at least three times.The data analysis were conducted using a Student’s https://www.oncomine.org/resource/main.html)23 and the GEPIA database (http://gepia.cancer-pku.cn/detail.php)24 24 Fig. . Interes)24 Fig. . In addi)24 Fig. . AssociaAll five CRC cell lines exhibited higher PLAGL2 expression than that in the normal colon epithelial cell line FHC Fig. . To examTo evaluate the in vivo effects of PLAGL2 on CRC cell growth, the transfected cells were implanted subcutaneously into the groin of nude mice. The elevated PLAGL2 expression markedly promoted tumour growth in vivo compared to that of the controls Fig. . The expIn consideration of the correlation between enhanced PLAGL2 expression and EMT-related proteins in CRC samples, we wondered whether EMT could account for the PLAGL2-mediated phenotypic changes described above. We demonstrated that PLAGL2 significantly diminished the expression of E-cadherin and elevated the levels of N-cadherin and vimentin compared to those of the controls Fig. , which wGiven the significant inverse association between ZEB1 and E-cadherin across various carcinomas, we also wondered whether ZEB1 was involved in PLAGL2-induced EMT. A significantly positive correlation between PLAGL2 and ZEB1 was observed in CRC tissues Fig. . Besides25 ZEB1 repressed GRHL2 expression by directly binding to the GRHL2 promoter.27 The GRHL2-ZEB1 bidirectional negative feedback loop drives EMT or MET in response to extracellular signals.28 It was worth exploring Whether GRHL2-ZEB1 reciprocal feedback loop was involved in PLAGL2-mediated EMT process and tumorigenesis of CRC. We demonstrated that GRHL2 was significantly overexpressed in CRC specimens relative to that in matched tissues, which is consistent with the results derived from the GEPIA database  is a broad suppressor of oncogenic EMT by inhibiting the TGF-beta signalling pathway and directly inhibiting ZEB1 expression.β-Catenin is a hub molecule of the Wnt/β-catenin signalling pathway, which is involved in EMT and cancer cell metastasis. PLAGL2 was positively correlated with β-catenin expression Fig. . BesidesGSK-3-mediated β-catenin phosphorylation and degradation is the main method of regulating β-catenin expression levels. AKT phosphorylation can inactivate GSK-3β. Therefore, the phosphorylation status of β-catenin, AKT and GSK-3β was detected. Compared to the controls, enhanced expression of PLAGL2 promoted AKT and GSK-3β phosphorylation, impeding β-catenin phosphorylation. No significant difference was observed in the total AKT and GSK-3β levels in response to enhanced PLAGL2 expression compared to those in the controls Fig. . To furtBesides, the depletion of β-catenin could diminish the effect of PLAGL2 on CRC cells proliferation, migration and invasion Fig. . The dowPLAGL2 induces the expression of ZEB1 and β-catenin. The β-catenin-TCF complex modulates transcriptionally ZEB1 expression. These results prompted us to ascertain whether PLAGL2 modulates ZEB1 expression through a β-catenin- dependent pathway.To prove this hypothesis, we examined the correlation between β-catenin and ZEB1 expression. β-Catenin was also positively correlated with ZEB1 expression Fig. . In addi30 EMT is thought to initiate the early steps of these processes and has traditionally been deemed to a binary process involving a complete conversion from epithelial to mesenchymal state. Nevertheless, it has been increasingly accepted that EMT also includes a range of hybrid states, a phenotype known as “partial EMT’’ (P-EMT).33 Because p-EMT is not well defined at the molecular level,34 it is not clear whether this hybrid status represents an intermediate phase in the process of a mesenchymal transition or its own terminal state.31 Therefore, our research mainly focused on the molecular mechanisms of PLAGL2 inducing EMT and an aggressive phenotype in CRC. The EMT subtype has not been thoroughly explored and further research is needed. In this study, we discovered that PLAGL2 was remarkably overexpressed in CRC samples compared to the expression in control samples, and was correlated with worse clinical outcomes. Enhanced PLAGL2 expression was significantly associated with the expression of EMT-related proteins in CRC. Specifically, PLAGL2 promoted the expression of mesenchymal markers and impeded the expression of an epithelial marker (E-cadherin). These results were confirmed by a xenograft tumour assay, indicating that PLAGL2 modulates EMT in vivo. Previous studies have demonstrated that PLAGL2 impeded differentiation in neural stem cells and gliomas.12 PLAGL2 modified the proliferation of haematopoietic progenitor and leukaemia cells,15 and promoted cell migration and invasion in various cancers.35 The data in our present study showed that PLAGL2 promoted the proliferative and metastatic properties of CRC cells in vitro. Similarly, the xenograft assay also indicated that PLAGL2 enforced the metastasis and tumorigenesis of CRC.Here we identify the signalling mechanism through which elevated PLAGL2 expression involved in CRC progression. The data in this study illustrate that PLAGL2 induces EMT and an aggressive phenotype in CRC through β-catenin-dependent regulation of ZEB1. Tumour invasion and metastasis, regarded as the most important feature of malignant tumours, are complex and multistep processes.37 ZEB1 also induced tumorigenesis by impeding the expression of stemness-inhibiting microRNAs.38 In addition, ZEB1 was involved in 5-fluorouracil resistance in CRC cells.39 Given that ZEB1 plays a crucial role in CRC progression, it is urgent to reveal the potential signalling pathway that regulate the expression of ZEB1. Many oncogenes have been implicated in the regulation of ZEB1 expression. Myocyte enhancer factor 2D(MEF2D) transduced multiple signals that activated ZEB1 expression and EMT, promoting metastasis in CRC.40 Polypyrimidine tract binding protein 3(PTBP3) promoted EMT in breast cancer by regulating the ZEB1 mRNA stability.41 GRHL2 protein was the first direct transcriptional repressor of the ZEB1 gene to be reported.25 In different biological backgrounds, the function of GRHL2 is not consistent, sometimes even completely opposite. The GRHL2/ZEB1 feedback loop has been reported to control EMT/MET primarily in breast cancer,28 but no statistically significant correlation between GRHL2 and ZEB1 could be observed in the present study. In this study, a positive correlation between PLAGL2 and ZEB1 was observed in CRC samples, but we have not seen a statistically significant correlation between PLAGL2 and any other EMT transcription factor. We demonstrated that the expression of ZEB1 was diminished by the depletion of PLAGL2. The data also illustrated that ZEB1 is required for PLAGL2 to function as an oncogene in CRC. These results indicate that ZEB1 is a vital target gene of PLAGL2.Recent studies have described ZEB1 as a vital activator of the EMT process that mediates the EMT-related protein expression.42 cell migration43 and cell autophagy.44 Mutations in crucial regulatory factors of the β-catenin signal mechanism have been widely noted in CRC. The nuclear translocation of β-catenin impeded the expression of E-cadherin and subsequently activated the EMT process. The effects of EMT-TFs on the induction of EMT could be blocked by the depletion of β-catenin. Microtubule-associated serine/threonine kinase like (MASTL) induced CRC progression and chemoresistance by activating the β-catenin signalling pathway.45 The ring finger protein 6(RNF6)-mediated degradation of transducin-like enhancer of split 3 (TLE3) remarkably impeded the binding of TLE3 with TCF4/LEF, which promoted the recruitment of β-catenin to TCF4/LEF and activated β-catenin signalling in CRC.46 Our study demonstrated that PLAGL2 was significantly associated with the expression of β-catenin. In addition, the elevated expression of PLAGL2 resulted in the overexpression of β-catenin and in the nuclear translocation of β-catenin. Moreover, PLAGL2 induced AKT phosphorylation, thereby enforcing GSK-3β phosphorylation and inactivating its activity. Due to the inactivation of GSK-3β, β-catenin degradation was diminished, and the cellular β-catenin levels increased. Taken together, PLAGL2 induced β-catenin expression by regulating AKT/GSK-3β signalling. However, further investigation is warranted to identify the molecular mechanisms driving the PLAGL2 regulation of AKT phosphorylation.The β-catenin signalling pathway plays a significant role in the EMT process and is activated by the overexpression or by β-catenin nuclear translocation. The dysregulation of this signalling pathway involves in numerous biological processes, including cell apoptosis,9 TP53BP2 prevented β-catenin from activating the expression of ZEB1 by forming a TP53BP2-β-catenin- E-cadherin complex, maintaining the plasticity of epithelial cells and suppressing metastasis.8 Our study showed that β-catenin was also positively correlated with ZEB1 expression. The depletion of β-catenin neutralised the regulation of ZEB1 expression that was caused by modified PLAGL2 expression. The inhibitor PNU-74654 had almost identical effects as β-catenin depletion on the regulation of the expression of ZEB1.Interestingly, ZEB1 was demonstrated to be a vital activator in the EMT process, and the β-catenin pathway was involved in EMT. Previous studies have shown that β-catenin forms a transcriptional activation complex with TCF4, then binding to the ZEB1 promoter region and inducing its expression.Overall, this study illustrates that enhanced PLAGL2 expression in CRC is positively associated with overexpressed N-cadherin and Vimentin, and is inversely correlated with the expression of E-cadherin. PLAGL2 increases AKT and GSK-3β phosphorylation. The inactivation of GSK-3β reduces β-catenin degradation and elevates β-catenin levels, promoting β-catenin nuclear translocation. Elevated β-catenin forms a transcriptional activation complex with TCF4, then binding to the promoter region of ZEB1 and inducing its expression. Thus, PLAGL2 induces EMT and an aggressive phenotype in CRC through β-catenin-dependent regulation of ZEB1.the supplementary files


The overexpression of PRMT5 is highly correlated to poor clinical outcomes for colorectal cancer (CRC) patients. Importantly, our previous work demonstrated that PRMT5 overexpression could substantially augment activation of the nuclear factor kappa B (NF-κB) via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. Here, we report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1β (IL-1β) stimulation. Interestingly, we identified for the first time that the oncogenic kinase, PKCι could catalyze this phosphorylation event. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A), in either HEK293 cells or CRC cells HT29, DLD1, and HCT116 attenuated NF-κB transactivation compared to WT-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, the S15A mutant when compared to WT-PRMT5, could downregulate a subset of IL-1β-inducible NF-κB-target genes which correlated with attenuated promoter occupancy of p65 at its target genes. Additionally, the S15A mutant reduced IL-1β-induced methyltransferase activity of PRMT5 and disrupted the interaction of PRMT5 with p65. Furthermore, our data indicate that blockade of PKCι-regulated PRMT5-mediated activation of NF-κB was likely through phosphorylation of PRMT5 at S15. Finally, inhibition of PKCι or overexpression of the S15A mutant attenuated the growth, migratory, and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, we have identified a novel PKCι/PRMT5/NF-κB signaling axis, suggesting that pharmacological disruption of this pivotal axis could serve as the basis for new anti-cancer therapeutics. Colorectal cancer (CRC) remains one of the leading causes of cancer-related deaths worldwide. Unfortunately, the incidence and mortality rate of this cancer is expected to rise even further, creating an urgent need to identify specific molecular pathways underlying CRC malignancy that can be targeted for the development of new therapeutic approaches. Recently, protein arginine methyltransferase 5 (PRMT5) has emerged as a key positive modulator of CRC malignancy and its overexpression is highly correlated to poor patient prognosis . As a meAberrant activation of the NF-ĸB signaling pathway remains one of the most prominent etiological factors in CRC malignancy ,8. As a Although we have previously shown that PRMT5 can dynamically regulate p65 via IL-1β-inducible methylation at R30, the precise mechanisms governing this novel PRMT5/p65 signaling axis are still not fully understood. In the present study, we have now uncovered a novel mechanism that PRMT5-mediated activation of NF-κB is dependent on signal-induced phosphorylation of PRMT5 at serine 15 (S15) and propose that blocking this posttranslational modification (PTM) could serve the dual purpose of impairing the tumor-associated functions of PRMT5 via attenuating its activity towards NF-κB as a potential treatment strategy for CRC ,12. CompPTMs such as phosphorylation remain some of the key mechanisms employed by cells to not only increase the functional diversity of the proteome but to ultimately influence various aspects of pathogenesis . Using mPreviously, we discovered that overexpression of PRMT5 dramatically enhanced NF-ĸB activation while knockdown showed the opposite effect . MoreoveNext, we sought to identify the signature gene networks associated with the subset of genes differentially regulated by S15A using Ingenuity Pathway Analysis (IPA). Interestingly, we observed an enrichment of terms associated with key “Biological Functions” such as “migration of tumor cells”, “proliferation of tumor cells”, and “colony formation” . CollectWe also wondered whether the reduced transcriptional activation of NF-ĸB by S15A may be due to other factors such as altered subcellular localization of S15A compared to WT-PRMT5, modulation of the degradation pattern of IĸBα, or blockade of the nuclear translocation of p65. Our data showed no observable difference in the subcellular distribution pattern of the WT-PRMT5 and S15A proteins . MoreoveTo further explore the possible mechanisms underlying the downregulation of NF-ĸB activity by S15A, we wondered whether this mutant altered interaction between PRMT5 and p65 and thus would impair the downstream transcriptional competence of p65. As shown in To further elucidate the mechanistic underpinnings for the observed S15A-mediated downregulation of NF-ĸB target genes, we conducted chromatin immunoprecipitation (ChIP)-PCR analysis to determine the proximal promoter occupancy of p65 at the IL8 gene, a prototypical target gene of NF-ĸB that harbors a ĸB element in its promoter B. ExcitiThe aforementioned microarray and IPA data suggested that S15A-downregulated genes were involved in cancer-associated biological functions D. MoreovBased on the domain architecture of human PRMT5, we determined that the S15 residue is located within the unique N-terminal triose phosphate isomerase (TIM) barrel A. Interehttp://www.hprd.org/ PhosphoMotif finder) to predict PKC and PKA as potential kinases related to S15 phosphorylation based on site-specific consensus phospho-motifs (To determine which kinase(s) phosphorylates PRMT5 at S15, we utilized the Human Protein Reference Database  and thus constitute pivotal mechanisms by which PRMT5 can fine-tune NF-κB activation and target gene expression. Furthermore, S15 phosphorylation mediates IL-1β-induced PRMT5 activity. Together, these potentially serve to facilitate the tumor-associated functions exerted by this PKCι-mediated PRMT5-dependent NF-κB activation, including the enhanced proliferation, anchorage-independent growth, and migration associated with PRMT5 overexpression .A wealth of reported findings supports the essential biological functions of PRMT5 in cell proliferation, transcriptional activation, signal transduction, and cell differentiation through methylation of its substrate proteins . HoweverPhosphorylation of PRMT5 on other types of amino acids, such as tyrosine and threonine was previously reported. For instance, the Liu group identified tyrosine phosphorylation  of PRMT5 by mutant Jak2 playing an inhibitory role on PRMT5′s dimethylation of histone substrates. This inhibition also correlated with an enhanced myeloproliferative phenotype . In a quFurthermore, the discovery of PKCι’s link to phosphorylation of PRMT5 on S15 and NF-κB activation is significant. PKCι is an oncogene that has been implicated in the initiation and progression of CRC via regulation of epithelial cell integrity and polarity in a model of colitis-associated colon cancer . MoreoveFrom a structural perspective, PRMT5 is commonly found complexed with other proteins and these interactions have been shown to dictate both its catalytic activity and substrate specificity ,22. InteIn conclusion, both NF-κB and PRMT5 are individually known to play critical roles in cancer progression, and the mechanisms underlying the cooperativity between the two are inherently critical and complicated. Based on our results, we presented a hypothetical model in which IL-1β stimulation activates the NF-κB pathway and induces PKCι-mediated phosphorylation of PRMT5. Insights from this study are, therefore, of particular importance in terms of enhancing our understanding of the role of phosphorylation sites such as S15 on PRMT5 in modulating the interaction between PRMT5 and NF-κB. In the future, these findings may also open up novel therapeutic avenues for CRC patients based on disrupting the PKCι/PRMT5/NF-κB signaling axis.v/v). A linear gradient of acetonitrile from 4% to 40% in water in the presence of 0.1% formic acid over a period of 45 min was used at a flow rate of 300 nL/min. The spectra were acquired by data dependent methods, consisting of a full scan (m/z 400–2000) and then tandems on the five most abundant precursor ions. The previously selected precursor ions were scanned once during 30 s and then were excluded for 30 s. The obtained data were analyzed by Mascot software  against customized PRMT5 protein database with the setting of 10 ppm for precursor ions and 0.8 Da for product ions. Carbamidomethylation of cysteine was set as fixed modification, while oxidation of methionine, phosphorylation of serine, threonine, and tyrosine were set as variable modifications. The tandem mass spectra of candidate-modified peptides were further interpreted manually.Coomassie-stained SDS-PAGE gel band containing Flag-PRMT5 protein was subjected to in-gel tryptic digestion. Flag-PRMT5 gel pieces were subjected to destaining and reduction of cysteine residues using 50% acetonitrile in 100 mM ammonium bicarbonate and 100% acetonitrile followed by treatment, followed by 20 mM DTT at room temperature for 60 min. Alkylation with 55 mM iodoacetamide for 30 min was performed in the dark. The solution was removed, and the gel pieces were washed with 100 mM ammonium bicarbonate and dehydrated in acetonitrile. Gel pieces were then dried in a SpeedVac centrifuge, and proteolytically digested by rehydration overnight at 37 °C in 50 mM ammonium bicarbonate containing sequencing grade modified trypsin . Extracted peptides were treated with 50% acetonitrile in 5% formic acid, dried, and reconstituted in 0.1% formic acid for mass spectrometry analysis. Analysis of proteolytic digests was performed by using an LTQ Orbitrap XL linear ion-trap mass spectrometer , coupled with an Ultimate 3000 HPLC system . The digests were injected onto a reverse-phase C18 column  equilibrated with 0.1% formic acid/4% acetonitrile (The HEK293C6 (HEK293) cell line was previously described . CRC celCells were cultured to about 90–95% confluence before treatment with 10 ng/mL of IL-1β. Whole cell samples were collected and lysed using Radio Immunoprecipitation Assay buffer , 50 mM Tris-HCl pH 8.0, and protease inhibitors). Whole cell lysates were then separated by SDS/PAGE gels, and further assessed by Western blotting. Different antibodies were used to detect the target proteins of interest, obtained from the following commercial sources: anti-PRMT5 , anti-FLAG , and anti-p65 ; anti-PKCι . For cell fractionation experiments, cytoplasmic and nuclear fractions were subject to SDS-PAGE and probed with anti-LaminB1 ; anti-α-tubulin .NF-κB luciferase assays were conducted by transiently transfecting the κB-luciferase construct p5XIP10 κB into parental control or FLAG-PRMT5 stable cell lines after which luciferase activity was quantified 48 h later using the Luciferase Assay System with Reporter Lysis Buffer kit . The κB-luciferase plasmid p5XIP10 κB contains five tandem copies of the NF-κB DNA binding site derived from the IP10 gene (an established target gene of NF-κB) upstream of a luciferase reporter gene. Luciferase activity was measured using a Synergy H1 Multi-Mode Reader .v/v), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 20 µM aprotinin, 1 mM phenylmethanesulfonyl fluoride, and 1 mM pepstatin A). FLAG-PRMT5 proteins were then immunoprecipitated with anti-FLAG-M2 beads , using immunoprecipitation methods previously described , high-salt wash buffer , LiCl wash buffer , and TE buffer (10 mM Tris HCl pH 8.0 and 1 mM EDTA). Protein-DNA complexes were eluted from antibodies with elution buffer containing 1% SDS and 0.1 M NaHCO3, incubated in the presence of 192 mM NaCl for 4 h at 65 °C, and digested with proteinase K for 1 h at 45 °C. DNA was recovered using the Qiagen quick DNA purification kit and IL8 gene-specific ChIP primers  were used in the PCR analyses.Cells were left untreated or stimulated with 10 ng/mL of IL-1β for 1 and 4 h, and cross-linked with 1% formaldehyde for 10 min at room temperature (R.T.). The cross-linking was stopped by adding glycine and cells were then washed with cold× phosphate-buffered saline  as described in co-immunoprecipitation methods above. The enzyme prep was diluted in assay buffer  prior to use. SAM  and unmethylated histone H4R3  were used as the methyl group donor and PRMT5 enzyme substrate, respectively. Acceptor beads diluted in 1× Epigenetics buffer  were added at a final concentration of 20 μg/mL to the reaction mixture and the plate was incubated at R.T. for 1 h. Donor beads diluted in 1× Epigenetics buffer  were then added at a final concentration of 20 μg/mL and the plate was further incubated at R.T. for 30 min. All reactions were performed in triplicate and repeated three independent times. The plates were read using an EnVisionp values for the enrichment test were calculated using Fisher’s exact test, right-tailed. Log10 (p) was visualized to the left of the p value. p < 0.05 was considered to be statistically significant.Groups of genes regulated by S15A were analyzed by the IPA software 8.7 . The setting and filter were as follows: reference set: Ingenuity Knowledge Base ; Relationship to include: Direct and Indirect; Includes Endogenous Chemicals; Filter Summary: Consider only molecules where species _ Human OR Rat OR Mouse. The g. The remaining cell pellet was further lysed in Complete Lysis Buffer, incubated on ice for 30 min and centrifuged for 10 min at 14,000× g to collect the nuclear fraction. The cytoplasmic and nuclear proteins were then subjected to Western blot analyses.Fractionation experiments were conducted according to the manufacturer’s instructions for the nuclear extract kit . Briefly, cells were grown to about 80% confluence, washed with ice-cold 1× PBS/Phosphatase inhibitors, collected and pelleted for 5 min at 500 rpm. Cell pellets were gently resuspended in 1× hypotonic buffer and incubated for 15 min on ice. The cytoplasmic fraction was collected as the lysate following centrifuging for 30 s at 14,000× t-test was used when comparing two means between groups as specified. All statistics were carried out for triplicate experiments and a p < 0.05 was considered statistically significant.Statistical analyses were performed using Prism 6 software . Data represent the mean ±S.D. or ±SEM as indicated. A two-tailed Student’s