Both loss- and gain-of-function of Wnt/β-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/β-catenin signaling in the superficial zone (SFZ) of articular cartilage.Prg4-CreERT2;Ctnnb1fl/fl and Prg4-CreERT2;Ctnnb1-ex3fl/wt mice for loss- and gain-of-function, respectively, of Wnt/β-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/β-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/β-catenin signaling in SFZ cells.Wnt/β-catenin signaling activity was analyzed using TOPGAL mice. We generated Creb1, a potent transcription factor for Prg4.Wnt/β-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific β-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific β-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by β-catenin knockout, while it was upregulated by β-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/β-catenin signaling activation increased mRNA levels of We demonstrated that Wnt/β-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage. Osteoarthritis (OA), which involves articular cartilage degradation, is the most prevalent joint disorder. A large number of studies using genetically modified mice have revealed signaling pathways or molecules responsible for OA, such as a disintegrin-like metallopeptidase with a thrombospondin type 1 motif 5, hedgehog signaling, syndecan-4, matrix metalloproteinase-13, hypoxia-inducible factor 2-alpha, nuclear factor kappa-light-chain-enhancer of activated B cells, Notch signaling, and Wnt signaling . However2+ pathway  vector (Promega). We performed the luciferase assay using ATDC5 cells and a Dual-Luciferase Reporter Assay System (Promega) and reported the data as the ratio of firefly to Renilla activities.Proximal regions around the transcription start site of t test for comparison of two groups, one-way ANOVA followed by Tukey’s correction for comparison of multiple groups, and the Mann-Whitney U test for the OARSI score, as appropriate. P values less than 0.05 were considered significant.To assess the statistical significance of experimental data, we used a two-tailed unpaired Student’s Prg4-CreERT2 mice by mating them with Ai14 mice for further experiments. In frozen sections of Prg4-CreERT2;Ai14 knee joint 1 week after tamoxifen induction, fluorescent signal was detected in the SFZ , a representative matrix gene in mature chondrocytes, was upregulated  did not enhance transactivation of any Prg4 reporter  Prg4-CreERT2;Ctnnb1fl/fl, Ctnnb1fl/fl, (b) Prg4-CreERT2;Ctnnb1-ex3fl/wt, and Ctnnb1-ex3fl/wtmice at 8 weeks post-surgery. Tamoxifen induction was performed at 7 weeks. Insets indicate regions of enlarged images below. Scale bars, 50 and 20 μm.Additional file 3: Figure S2. Validation of primary SFZ cells isolated from joints of P5 mice. a Morphology of SFZ cells and deeper zone (DZ) chondrocytes after 1 week of culture. Scale bars, 50 μm. b mRNA levels of marker genes in SFZ and DZ cells. *P < 0.05 versus vehicle (Student’s unpaired two-tailed t-test).


Astragalus membranaceus and Curcuma zedoaria, two traditional Chinese medicines, are widely used together in colorectal cancer adjuvant treatment. Many different mechanisms should be involved in the benefit effect of Astragalus membranaceus and Curcuma zedoaria. In this study, we established that the combined extract from Astragalus membranaceus and Curcuma zedoaria (HQEZ) decreased the metastasis ability in colorectal cancer cells  in vitro, and the treatment induced the downregulation of EMT signal and decreased CXCR4 expression and the level of β-catenin. Overexpression of CXCR4 and the administration of the agonist and inhibitor to β-catenin signal pathway were used to explore the mechanism of Astragalus membranaceus and Curcuma zedoaria in colorectal cancer treatment. The data demonstrated that HQEZ increased the phosphorylation of β-catenin which related to the degradation of β-catenin, and it induced the downregulation of EMT signal and CXCR4. It meant that the influence of β-catenin should be a key event in the antimetastasis effects of Astragalus membranaceus-Curcuma zedoaria in colorectal cancer model. These findings revealed the potential effect and mechanism of Astragalus membranaceus-Curcuma zedoaria in colorectal cancer treatment and provided insight for optimization of the usage. Colorectal cancer, the fourth most common cancer, affects more than one million people per year and causes more than 600000 deaths in world . Most co Solanum incanum aqueous, Astragalus membranaceus, Curcuma zedoaria, and Rubus corchorifolius, have been proved to inhibit the growth and/or metastasis of different types of cancers [ Astragalus membranaceus (HQ) and Curcuma zedoaria (EZ) are two herbs widely used in TCM to treat different cancers [In cancer treatment, certain Chinese herbs or formulas, such as cancers , 6, 7. M cancers –10. AlthIn this research, we evaluated the influence on the metastasis and growth of colorectal cancer cells with different ratios and different concentrations of HQ and EZ, which might provide a reference for clinical use. We also researched the influence on the metastasis pathways, which might reflect the mechanisms of the treatment of HQ and EZ, and it might be useful to the usage of HQEZ. Astragalus membranaceus (HQ) and Curcuma zedoaria (EZ) were purchased from Jiangsu Province Hospital of TCM. WAY-262611 (HY-11035) and SB 216763 (HY-12012) were purchased from MedChemExpress USA. Other agents were purchased from Sigma-Aldrich.HCT116 cells are purchased from FuHeng Biology . The vector overexpressing CXCR4 (MG50621-UT) was purchased from Sino Biological.μg/ml ampicillin and 100 μg/ml streptomycin) at 37°C with 5% CO2 and 95% air.HCT116 was cultured in complete medium  containing 10% FBS , 100  Astragalus membranaceus and Curcuma zedoaria were used to be extracted with 100 ml water for 1 hour at 100°C. The solution was concentrated to 10g crude drugs/ml. After the solution was cooled, the solution was centrifuged at 12000g for 10min and the supernatant was filtered through 0.22μm filter to remove the bacteria. The filtered solution was separated and stored at -20°C. The used concentrations are represented by the weight of crude drugs per ml.3 different rates of3 cells/well HCT116 were plated into 96-well plates and treated with HQEZ 12 hours later. After 24 hours, cell viability was assessed by the CCK8 assay according to the user manual. Briefly, 10μl/well CCK8 was added to the 96-well plates and cultured for another 1 hour, then the optical density (OD) was measured at 450 nm on a multifunction microplate reader. The proliferation inhibition rate was calculated as per the following equation: (1-OD treated/ OD control)×100%.5×105 cells/well HCT116 in culture medium containing 1% FBS were performed in 24-well transwell chambers (Costar). The upper surface of polycarbonate filters with 8mm pore was coated or uncoated with 50μl Matrigel (Corning). The bottom chambers were added with culture medium containing 10% FBS. Cells were incubated for 48 hours. Migration or invasion was terminated by removing cells from upper chambers and stained with Crystal Violet.1×10β-catenin , rabbit anti-Myc , rabbit anticyclin D1 , rabbit anti-p-β-catenin , rabbit anti-ERK1/2 , rabbit anti-pERK1/2 , rabbit anti-AKT, and rabbit anti-pAKT. An internal control was performed using mouse anti-GAPDH  or mouse anti-β-actin , and appropriate horseradish peroxidase-linked secondary antibodies were used for enhanced chemiluminescence detection (Pierce). Number of cells reaching the lower surface of the filter was counted in five random fields per filter.Western bolt analysis was performed as described previously. The primary antibodies were as follows: rabbit anti-CXCR4 , rabbit anti-E-cadherin , rabbit anti-N-cadherin  and anti-Vimentin , rabbit anti-5 cells/well HCT116 were plated into 6-well plates and treated with HQEZ 12 hours later. After 24 hours, total RNA was purified by Trizol  following the manuscript's protocol. And cDNA was synthesized with M-MuLV First Strand cDNA synthesis Kit  Co., Ltd. China). We utilized qPCR Mastermix  Co., Ltd. China) and followed its protocol to perform qPCRs in triplicate. We performed the Ct method to quantify and normalize the target mRNA expression to β-actin expression. The following are the primers used: β-actin , CXCR4 , and β-catenin .5×10 p<0.05 considered statistically significant.Experiments were repeated at least three times independently. All of the data are presented as means ± standard deviation. Differences were evaluated using Student's t test, with Astragalus membranaceus and Curcuma zedoaria (HQEZ) dose-dependently inhibited the cell viability of HCT116 by CCK8 assay, and extracts from the mixture of Astragalus membranaceus and Curcuma zedoaria  showed the best effect on the inhibition of colorectal cells , and WAY-262611 also rescued the metastasis ability, EMT signal, and the expression of CXCR4 in HCT116 treated with HQEZ , an inhibitor of GSK 3β, rescued the expression of β-catenin and decreased the phosphorylation of β-catenin , is highlighted in the development and the metastasis of colorectal cancers –22. The  pathway , all of β-catenin. At first, we thought HQEZ should downregulate the Wnt/β-catenin pathway by the inhibition of CXCR4 as per previous report [β-catenin or the EMT pathway, but the enhancement of β-catenin pathway rescued CXCR4, EMT pathway, and the metastasis ability. Some researchers reported that CXCR4 is regulated by Wnt/β-catenin pathway and key factors in EMT pathway are also regulated by β-catenin related ways. Astragaloside IV, an active component in Astragalus membranaceus, and β-elemene, an active component in Curcuma zedoaria, are reported to attenuate the Wnt/β-catenin pathway by previous reporters [β-catenin rescuing the HCT116 from HQEZ. According to these results, we thought that HQEZ induced the phosphorylation of β-catenin and the following degradation of β-catenin could induce the downregulation of EMT signal and CXCR4 which were important to inhibitive effects of HQEZ on the metastasis ability of colorectal cancer.We found HQEZ decreased the expression of CXCR4 in colorectal cancer cells, and we also found the inhibition of EMT and s report . When weeporters , 27. Theβ-catenin in a relatively low concentration and therefore inhibits the metastasis of colorectal cancer cells in vitro. These results provide a potential mechanism of traditional Chinese medicine used in colorectal cancer treatment and pursue HQEZ as an adjuvant therapy used to colorectal cancer patients.In conclusion, HQEZ induces the degradation of  Astragalus membranaceus and Curcuma zedoaria on HCT116 cells in vitro. When Astragalus membranaceus and Curcuma zedoaria were used at a ratio of 2:1, it showed better effects on the inhibition of HCT116 cells. At this ratio, Astragalus membranaceus and Curcuma zedoaria also showed significantly antimetastasis effect in a relatively low concentration and this effect is related to downregulation of CXCR4 and EMT signaling. These effects owned to the degradation of β-catenin associated with the activation of GSK-3.In this study, we evaluated the effect of


Activation of IκB kinase β (IKKβ) is a central event in the NF-κB–mediated canonical pro-inflammatory pathway. Numerous studies have reported that oligomerization-mediated trans autophosphorylation of IKKβ is indispensable for its phosphorylation, leading to its activation and IKKβ-mediated phosphorylation of substrates such as IκB proteins. Moreover, IKKβ's interaction with the NF-κB essential modifier (NEMO) is necessary for IKKβ activation. Interestingly, some viruses encode virulence factors that target IKKβ to inhibit NF-κB–mediated antiviral immune responses. One of these factors is the vaccinia viral protein B14, which directly interacts with and inhibits IKKβ. Here we mapped the interaction interface on the B14 and IKKβ proteins. We observed that B14 binds to the junction of the kinase domain (KD) and scaffold and dimerization domain (SDD) of IKKβ. Molecular docking analyses identified key interface residues in both IKKβ and B14 that were further confirmed by mutational studies to promote binding of the two proteins. During trans autophosphorylation of protein kinases in the IKK complex, the activation segments of neighboring kinases need to transiently interact with each other's active sites, and we found that the B14–IKKβ interaction sterically hinders direct contact between the kinase domains of IKKβ in the IKK complex, containing IKKβ, IKKα, and NEMO in human cells. We conclude that binding of B14 to IKKβ prevents IKKβ trans autophosphorylation and activation, thereby inhibiting NF-κB signaling. Our study provides critical structural and mechanistic information for the design of potential therapeutic agents to target IKKβ activation for the management of inflammatory disorders. The inhibitor of κB kinase (IKK)XS  in its N-terminal region. These two serine residues serve as the IKKβ phosphorylation sites. The doubly phosphorylated destruction motif is then recognized by the β-TrCP–SCF ubiquitin ligase for subsequent ubiquitination and degradation. After degradation of IκBα, NF-κB is then released into the nucleus to regulate gene transcription . Because varying amounts of B14 construct were co-transfected with the same amount of WT IKKβ construct, the inhibitory effect of B14 on IKKβ autoactivation can be manifested by its loss of activity on IκBα phosphorylation. To rule out the possibility that B14 also inhibits the activated IKKβ with dual phosphorylation on Ser-177 and Ser-181 in the phosphorylation of IκBα, we conducted an in vitro kinase assay with the purified recombinant IKKβ-SSEE (constitutively active S177E/S181E mutant). B14 did not show any distinguishable inhibitory effect on IKKβ kinase activity (B), confirming that it does not inhibit the active IKKβ.Previous studies have shown that B14 inhibits IKKβ phosphorylation on Ser-177 and Ser-181 of its activation segment . B14 binds to both full-length IKKβ and a construct without the NBD (4–675), indicating that the NBD is not required for B14–IKKβ interaction. In addition, B14 binds to both the constitutively active IKKβ-SSEE and inactive IKKβ-D145N, showing that its activation state does not affect B14 association. Furthermore, Ser-to-Glu or Ser-to-Ala substitutions on Ser-177 and Ser-181 of the activation segment do not influence B14 binding, suggesting that the binding may not engage the kinase activation segment.In the structure of IKKβ, the KD, ULD, and SDD interact with each other to form an integral trimodular structural unit; however, the NBD has been shown to be an independently folded domain connected with a flexible linker to the rest of the protein . To map Fig. 2Escherichia coli (B and D). The recombinant GST-SDD constructs were not soluble, indicating that, when expressed alone separately, the SDD domain cannot be folded properly. We used the GST-IKKβ fusion proteins to pull down untagged B14 protein. All constructs pulled down B14, except GST-ULD, which clearly demonstrates that the ULD is not required for B14 binding to IKKβ. GST alone did not bind B14, indicating that the binding between B14 and other IKKβ constructs is specific (B). The NBD of IKKβ does not contribute to IKKβ binding to B14 because the GST-NBD (residues 675–756) IKKβ construct did not pull down B14 (C). Based on the results, we can conclude that B14 interacts with IKKβ on the junction between KD and SDD domains.To determine the B14-interacting domains on IKKβ, we expressed the GST fusion constructs of the KD (residues 1–307), KD-ULD (residues 1–384), ULD (residues 309–400), and ULD-SDD (residues 307–678) of human IKKβ in hia coli , B and Dspecific B. The NBdown B14 C. Based Xenopus IKKβ structure (PDB code 3QA8) in a closed conformation and a human IKKβ structure (PDB code 4E3C) in an open conformation for the modeling. We identified the same potential binding surface composed of both KD and SDD of both orthologous IKKβ structures. Five surface regions of IKKβ, encompassing residues ranging from 179–197 (M1), 235–260 (M2), 408–416 (M4), 421–426 (M5), and 577–583 (M6) are contacting B14 in the docking models (A). To verify the docking models, we have substituted all residues of the M1 and M2 segments with Gly or Ser, mutated three key interacting residues  of M3 on the GST-KD-ULD construct, and changed all residues on the M4, M5, and M6 segments to Gly or Ser on the GST-ULD-SDD construct. The GST fusion human IKKβ mutant constructs were then used to pull down the untagged B14 protein. We found that the M2 and M4 mutants have an almost 60% lower binding affinity compared with the WT constructs. The M6 mutant has a light decrease in its interaction with B14, whereas the M1, M3, and M5 mutants have no significant changes in B14 binding .Because the crystal structures of both the VACV B14 and IKKβ proteins are available, we conducted computational molecular docking to reconstruct the B14–IKKβ interaction with FRODOCK , 23. In g models A. To verC). Phe-130 of B14 has been demonstrated previously to be required for IKKβ binding and inhibition . Both the mutants T31K and Y35E demonstrated a significant decrease in binding affinity as the WT B14 to IKKβ; L126E had a slight loss of binding, whereas F130K had negligible binding to IKKβ. This is consistent with a previous study showing that the B14 F130K mutant protein not only lost its binding to IKKβ but was also unable to inhibit NF-κB signaling  in a closed conformation and a human IKKβ structure (PDB code 4E3C) in an open conformation for the modeling to indicate the evolutionarily conserved B14 binding. In these refined binding models, the small hydrophobic patch centered on Phe-130 of B14 interacts with both the M2 and M4 segments of IKKβ, which has no contact with the activation segment of the kinase domain (A–D). To verify this predicted binding interface, we created six full-length human IKKβ mutants with the substitutions L259K, Y261A, V410K, L259K/Y261A, and L259K/Y261A/V410K, and the M2M4 mutant. These mutants were expressed in human kidney embryonic 293F (HEK293F) cells with an engineered N-terminal FLAG tag. The mutant recombinant proteins were used to pull down purified GST fusion B14 protein expressed in E. coli. The IKKβ L259K/Y261A/V410K mutant was not expressed in HEK293F cells, indicating that substituting all three residues disturbs its proper folding and stability. The L259K and L259K/Y261A mutations severely weakened IKKβ binding to B14, whereas V410K or Y261A alone had a minor effect on their interaction (E). Tyr-261 is not conserved in Xenopus IKKβ (C), indicating that Tyr-261 is not critical. The M2M4 mutant has a similar binding pattern to B14 as L259K/Y261A. This shows that the three chosen residues in the M2M4 regions account for most of the binding affinity for B14. In this confirmed binding model of both the closed and open IKKβ structures, the activation segment of the kinase domain is exposed for substrate binding, which explains why B14 does not directly inhibit IKKβ kinase activity.We used the identified interface to perform a local search with a molecular docking program, ROSETTA , 27. We e domain , A–D. Toeraction E. Tyr-26pus IKKβ C, indicaBFig. S2).Our previous measurement of the recombinant full-length human NEMO and IKKβ proteins by size exclusion chromatography with multiangle light scattering has shown their dimeric status in solution. The measured molecular mass of the reconstituted IKK complex is around 2000 kDa, which corresponds to ∼12–16 NEMO and IKKβ molecules in each complex assembly . Based oFig. S2). In the Xenopus IKKβ structure, although it is an S177E/S181E constitutively active mutant, the presence of a specific kinase inhibitor renders it in an inactive kinase form, and IKKβ adopts a closed dimer conformation . It is possible that, in the preactivation IKK complex, NEMO “primes” IKKβ for activation by upstream signaling events, such as polyubiquitin chain binding or phosphorylation by an upstream kinase . B14 binds to both the constitutively active and inactive IKKβ proteins with comparable affinity (A). However, its binding to the preactivation complex stops the KD interactions, precluding trans autophosphorylation and activation. B14 can associate with the postactivation complex with little effect on the activity of the kinase (B and ) because the binding site of B14 on IKKβ has no overlap with the kinase substrate binding site and the active site  complex. In the preactivation complex, the activation segments of the neighboring KDs can contact each other for trans autophosphorylation  and the previously reported in vivo experiment in HEK293T cells (in vitro kinase inhibition assay clearly shows that B14 does not directly inhibit IκBα phosphorylation by the activated IKKβ (B). Because B14 binding attenuates IKKβ dual phosphorylation on Ser-177 and Ser-181 , which is a prelude for trans autophosphorylation and kinase activation. The B14 binding leaves some space between the activation segment and the B14 protein molecule. In principle, the activation segment of one IKKβ molecule can still have the chance to contact the active site of another for trans phosphorylation. The KD of IKKβ forms an ensemble with the SDD and ULD, and that restricts the access of the active site of one KD with the activation segment of another. In addition, the dimeric structure of IKKβ further limits the proximity of the active segment and active site of KDs, thereby restricting chances of trans autophosphorylation. It also indicates that, in the presence of the scaffold protein NEMO, the activation of IKKβ requires a well-defined structural architecture of the kinase complex that can allow optimal contact between the kinase domains.Our biochemical and computational characterization of B14–IKKβ interaction has identified a B14 binding interface consisting of both the KD and SDD of IKKβ. Although B14 inhibited IκBα phosphorylation in both our autoactivation assay A and the3T cells , our in ted IKKβ B. BecausXenopus IKKβs . Under in vivo condition, IKKβ needs to be activated in the NEMO–IKKβ complex. However, in the absence of an IKK holocomplex structure, it cannot be concluded that the observed KD interfaces in the crystal structures are relevant for IKKβ trans autophosphorylation in vivo. Our computational modeling of the B14–IKKβ interaction in all three crystal packing complexes indicates that the mode of B14 binding to IKKβ is an effective way to block the trans autophosphorylation between neighboring IKKβ dimers . This is consistent with the experimental observation that co-expression of B14 and IKKβ inhibits the IKKβ trans autoactivation induced by overexpression (A).Although IKKβ stays as a dimer in solution, different types of oligomers have been observed in the crystal structures of both human and us IKKβs , 29, 30. changes , the act changes , 29, 30.pression A.AFig. S4), the two important residues of IKKβ that are required for binding to B14, Tyr-259 and Val-410, are conserved in IKKα. However, structural analysis of IKKα has revealed a huge structural variation on the corresponding B14 binding interface in IKKα , which explains the lack of B14 binding to IKKα. However, the low resolution (4.5 Å) of the IKKα structure limits the reliability of this structural comparison to draw any conclusions . The cells were cultured in suspension and harvested 48 h post-infection. The recombinant proteins were purified by nickel affinity chromatography, anion exchange, and gel filtration chromatography. The polyhistidine tag was cleaved by the tobacco etch virus protease during protein purification.E. coli using pET28a and pGEX4T3 vectors and purified by their respective affinity tags. GST-KD, GST-KD-ULD, GST-ULD, and GST-ULD-SDD of human IKKβ were also expressed in E. coli using pGEX4T3 vectors. The tagged proteins were first purified with GSH or nickel-nitrilotriacetic acid beads, and their expression levels were assessed by SDS-PAGE.All human IκBα and VACV B14 proteins were expressed in m full-length IκBα in 20 mm HEPES (pH 7.5), 10 mm MgCl2, 20 mm β-glycerophosphate, 10 mmp-nitrophenyl phosphate, 50 mm Na3VO4, 1 mm DTT, 20 mm ATP, and 1–10 mCi)[γ-32P]ATP at 30 °C for 30 min and subjected to SDS-PAGE and autoradiography. Immunoblotting was performed using anti-FLAG (Sigma) or anti-HA (Sigma) antibodies . For the in vitro IKKβ kinase assay, purified recombinant IKKβ-S177ES/182E mutant protein was mixed with varying amounts of recombinant B14 or maltose binding protein as described above.The HA-B14 construct, Flag-IKKβ, and its mutant constructs were generated in the pcDNA3 vector using conventional PCR. HEK293T or HEK293F cells were transfected with all constructs using Lipofectamine 2000 (Invitrogen). After 24 h, cell extracts were immunoprecipitated with anti-FLAG antibodies bound to agarose beads . IKKβ kinase assays were essentially done as described previously , 34. Them Tris-HCl (pH 8.0), 100 mm NaCl, 2 mm β-mercaptoethanol, and 5 mm DTT) and eluted with 25 mm reduced GSH, and the elutions were subjected to SDS-PAGE analysis. All GST pulldown experiments were repeated at least three times for consistency.For GST pulldown, the tagged proteins were purified with GSH or nickel-nitrilotriacetic acid beads, followed by purification with gel filtration using a SuperdexG200 10/300 column . The expression levels were assessed by SDS-PAGE. Beads containing estimated equivalent quantities of the GST-tagged proteins were mixed with the purified versions of the interacting partners. The mixtures were incubated at 4 °C for 2 h with continuous rotation on a rocking platform. After centrifugation, the supernatants were removed. The beads were then washed three times with buffer (50 mm Tris-HCl (pH 8.0), 100 mm NaCl, 2 mm β-mercaptoethanol, 5 mm DTT, 2% NP40, and a protease inhibitor mixture. The protein was then mixed with anti-FLAG antibody (Sigma), purified GST-B14 protein, and protein A/G–agarose (Thermo Scientific). The resultant protein complex was eluted by 200 μg/ml FLAG peptide (Sigma) and subsequently used for Western blotting with anti-FLAG or anti-GST antibodies. The image was captured by G:Box imaging systems. All experiments were repeated at least three times.For FLAG pulldown, FLAG-tagged human IKKβ and mutant proteins expressed in HEK293F cells were lysed in buffer containing 50 m3QA8 (4E3C (2VVY (http://frodock.chaconlab.org/)https://cluspro.bu.edu/)3 to obtain more accurate complex models with these defined interfaces as restraints. The top ten models were analyzed, and the best-fitting one was chosen as an initial complex model for subsequent optimization with the Rosetta docking 2 (http://rosie.rosettacommons.org/)3 online server (Two IKKβ structures (PDB codes 3QA8  and 4E3CA8 (4E3C ) were se3C (2VVY ) was sel3C (2VVY , 23 (htte server , 27. LocQ. T., S. C., and G. X. conceptualization; Q. T. and G. X. resources; Q. T., S. C., and G. X. data curation; Q. T. software; Q. T., S. C., and G. X. formal analysis; Q. T. and S. C. validation; Q. T. and G. X. investigation; Q. T. and S. C. visualization; Q. T. and S. C. methodology; Q. T. and G. X. writing-original draft; Q. T., S. C., and G. X. writing-review and editing; G. X. supervision; G. X. funding acquisition; G. X. project administration.


Protein arginine methyltransferase 5 (PRMT5) has been implicated in the development and progression of human cancers. However, few studies reveal its role in epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells. In this study, we find that PRMT5 is up‐regulated in pancreatic cancer, and promotes proliferation, migration and invasion in pancreatic cancer cells, and promotes tumorigenesis. Silencing PRMT5 induces epithelial marker E‐cadherin expression and down‐regulates expression of mesenchymal markers including Vimentin, collagen I and β‐catenin in PaTu8988 and SW1990 cells, whereas ectopic PRMT5 re‐expression partially reverses these changes, indicating that PRMT5 promotes EMT in pancreatic cancer. More importantly, we find that PRMT5 knockdown decreases the phosphorylation level of EGFR at Y1068 and Y1172 and its downstream p‐AKT and p‐GSK3β, and then results in down‐regulation of β‐catenin. Expectedly, ectopic PRMT5 re‐expression also reverses the above changes. It is suggested that PRMT5 promotes EMT probably via EGFR/AKT/β‐catenin pathway. Taken together, our study demonstrates that PRMT5 plays oncogenic roles in the growth of pancreatic cancer cell and provides a potential candidate for pancreatic cancer treatment. This dismal outcome can be attributed to the lack of early diagnoses and effective interventions. Additionally, conventional treatment approaches such as surgery, chemotherapy and radiation have generally had little impact on the course of this aggressive cancer despite efforts over the past several years.Emerging evidence has demonstrated that epithelial‐mesenchymal transition (EMT) plays an essential role in the progression of pancreatic cancer.Protein arginine methyltransferases (PRMTs) plays critical roles in a variety of cellular processes including transcriptional regulation, chromatin regulation, signal transduction, RNA processing and DNA damage repair.In this study, we examined the roles of PRMT5 in pancreatic cancer and elucidated the underlying mechanism. Our data showed that PRMT5 promoted cell proliferation, migration and invasion in pancreatic cancer cells, and promoted tumorigenesis. Importantly, PRMT5 promoted EMT probably via EGFR/AKT/β‐catenin pathway.22.12 in humidified incubator). DMEM, FBS and trypsin were purchased from Gibco (Invitrogen).The pancreatic cancer cell lines PaTu8988 and SW1990 were obtained from ATCC (USA) and Cancer Cell Repository . Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% foetal bovine serum (FBS) at standard cell culture conditions : 5′‐CCGGGG CTCAAGCCACCAATCTATGCTCGAGCATAGATTGGTGGCTTGAGCCTTTTTG‐3′, PRMT5 shRNA (R): 5′‐AATTCAAAAAGGCTCAA GCCACCAATCTATGCTC GAGCATAGATTGGTGGCTTGAGCC‐3′. The PRMT5 shRNA sequence was inserted into the EcoR I and Age I site of the pLKO.1‐TRC plasmid and ligated into the vector (Sigma).2.36 recombinant lentivirus transduction units in the presence of 8 mg/mL polybrene (Sigma). Puromycin (1:10 000 dilutions) was added to cells until the cells in blank group died off.The packaging plasmid psPA ×2 and the envelope plasmid pMD2.G were purchased from Sigma . PLKO.1‐sh‐PRMT5 vector was cotransfected with psPA ×2 and pMD2.G into HEK293T cells using Lipofectamine2000 (Invitrogen). Viruses were harvested 48 hours after transfection, and viral titres were determined. Cells were infected with 1 × 102.45 cells/well. After 24 hours of culture, the medium was replaced by Opti‐MEM (Invitrogen) and cultured. In total, 2 μg plasmid was transfected using 6 μL Lipofectamine® 2000 Transfection Reagent. After incubation for another 48 hours, the treated cells were determined using Western blot analysis, transwell or cell counting Kit‐8 assay.The plasmids pHA‐venus and pHA‐PRMT5 were kind gifts from Professor Mo. Cells were plated in six‐well plates at a density of 4 × 102.5Whole‐cell lysates were prepared and Western blot was carried out as recently described.2.62, 37°C). Respectively in first day, second day, third day and fourth day, 10 μL CCK‐8 was added to each well, cells were incubated at 37°C for 2 hours, and the absorbance was finally determined at 490 nm.The measurement of viable cell mass was performed with Cell Counting Kit‐8 (Promega) according to manufacturer's instructions. Briefly, 3000 cells/well were seeded in a 96‐well plate, grown in an incubator  for two weeks. At the end of the incubation, the cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet. Megascopic cell colonies were counted using Image‐Pro Plus 5.0 software (Media Cybernetics). Colony formation rate = (number of colonies/number of seeded cells) × 100%. Each measurement was performed in triplicate, and the experiments were each conducted at least three times.Cells were seeded in six‐well plates at a density of 1000 cells per well and cultured in incubator , cells on the upper surface of the filter were removed with a cotton swab, while the invaded cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, photographed (×20) in five independent fields for each well and counted. Each test was repeated in triplicate.A total of 24‐well inserts (8‐μm pore size) were purchased from BD Biosciences. 5 × 102.94 PaTu8988 cells and 1 × 105 SW1990 cells were seeded in serum‐free DMEM in the upper wells, which have already been covered with a layer of BD Matrigel Basement Membrane. The cells were later processed similar to that of cell migration assay, photographed (×20) in five independent fields for each well, and counted. Each test was repeated in triplicate.Cell invasion was determined using a Boyden chamber assay. 5 × 102.106 cells/site) stably transfected with sh‐EGFP, sh‐PRMT5, pHA‐Venus and pHA‐PRMT5 were subcutaneously injected into 5‐week‐old BALB/c nude mice  to generate xenografts. There are five female mice in each group. The tumour volume was measured every week after injection and calculated using the following formula: 4/3 πlength × width × height.The protocol was approved by the Institutional Animal Care and Use Committee of Jiangsu University, Zhenjiang, China. PaTu8988 and SW1990 cells . Reverse transcription was performed using RevertAid First Strand cDNA Synthesis Kit  according to the manufacturer's specification. Real‐time PCR was performed in triplicate in 20 μL reactions with iQ SYBR Premix Ex Taq Perfect Real Time , 50 ng first strand cDNA and 0.2 μg each primer. The primers for qRT‐PCR were in Table 2.12Cells were incubated in culture medium with 10% FBS supplemented with EGFR inhibitor  over the concentration range 0, 10 μmol/L.2.13t test (two groups) or an one‐way ANOVA (multiple groups). Kaplan‐Meier survival was analysed using log‐rank analysis. P < .05 was considered statistically significant.All statistical analyses were carried out using the SPSS statistics software package. All data are presented as mean ± SD from at least three independent experiments. Comparisons between groups were analysed using the Student's 33.1http://www.proteinatlas.org). We found that PRMT5 had the positive strong expression in pancreatic cancer and negative weak expression in normal pancreas . We found that PRMT5 mRNA level was higher in pancreatic cancer tissues than that in normal pancreatic tissues  in Badea pancreas database   and evaluated the correlation of PRMT5 expression with clinicopathological features, tumour stage and patients’ outcome. PRMT5 expression in pancreas adenocarcinoma ductal was higher than that in pancreas adenocarcinoma other subtype and pancreas colloid (mucinous non‐cystic) carcinoma  and p‐EGFR (Y1172) is increased remarkably. Above all, we for the first time established the link between PRMT5 and EGFR/AKT/β‐catenin signalling in pancreatic cancer. All these findings suggest that PRMT5 is a potential biomarker for diagnostics and prognosis of pancreas cancer.The authors confirm that there are no conflicts of interest.Lu Ge and Huizhi Wang performed most experiments and wrote the manuscript; Xiao Xu performed some experiments and candled data from database; Junbo He performed some experiments; Wanxin Peng, Fengyi Du, Youli Zhang, Min Xu and Aihua Gong designed experiments and organized the manuscript. Click here for additional data file.


F-box/WD repeat-containing protein 5 (FBXW5) is a member of the FBXW subclass of F-box proteins. Despite its known function as a component of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex, the role of FBXW5 in gastric cancer tumorigenesis and metastasis has not been investigated. The present study investigates the role of FBXW5 in tumorigenesis and metastasis, as well as the regulation of key signaling pathways in gastric cancer; using in-vitro FBXW5 knockdown/overexpression cell line and in-vivo models. In-vitro knockdown of FBXW5 results in a decrease in cell proliferation and cell cycle progression, with a concomitant increase in cell apoptosis and caspase-3 activity. Furthermore, knockdown of FBXW5 also leads to a down regulation in cell migration and adhesion, characterized by a reduction in actin polymerization, focal adhesion turnover and traction forces. This study also delineates the mechanistic role of FBXW5 in oncogenic signaling as its inhibition down regulates RhoA-ROCK 1 (Rho-associated protein kinase 1) and focal adhesion kinase (FAK) signaling cascades. Overexpression of FBXW5 promotes in-vivo tumor growth, whereas its inhibition down regulates in-vivo tumor metastasis. When considered together, our study identifies the novel oncogenic role of FBXW5 in gastric cancer and draws further interest regarding its clinical utility as a potential therapeutic target. Gastric cancer is the fifth most commonly diagnosed malignancy and the third leading cause of cancer-related death worldwide . AlthougKβ [An increase in focal adhesion kinase (FAK) activity or overexpression of FAK has been reported to give rise to an aggressive tumor phenotype in gastric cancer. The activation of FAK has been widely reported to promote in-vitro cell proliferation and migration, as well as in-vivo tumorigenesis and metastasis in gastric cancer ,9,10,11.Kβ .A role of F-box protein in FAK-dependent proliferation of endothelial cells has been shown previously . DespiteFour gastric cancer cell lines, MKN1, CLS145, AGS and SNU1, were shown to have varying levels of FBXW5 mRNA and protein expressions A,B. Subsp-values = 0.01, 0.0008 and 1.74 × 10−6, respectively)  and a corresponding decrease in S phase cells    (Knockdown of FBXW5 resulted in a significant reduction in cell proliferation (ctively) C, with a = 0.01) D. Knockd = 0.03) E and a s = 0.03) F were ob = 0.03) G.The effect of FBXW5 knockdown on cell migration was determined. As compared to the non-targeting control, which remained highly migratory, FBXW5 depleted MKN1 cells exhibited reduced migratory potential A. We alsp-value = 0.006)  I.p-value = 8.23 × 10−210)     B, while  10−182) C. Subseq 10−182) D and in  10−182) E. Lastly 0.0292) F, but a  0.0037) G.The effect of FBXW5 knockdown on FAK activation was explored. Of note, FBXW5 knockdown inhibited the activation of FAK, as represented by the decrease in p-FAK (Tyr397) protein expression. Consequentially, a reduction in p-Src (Tyr 416) and p-ERK1/2 (Thr 202/Tyr 204) protein expressions was also observed even though the expression of their unphosphorylated native forms remained unchanged. A downstream effect of this was observed in c-Myc protein expression level, as shown by a marked reduction A. Converr = −0.32, p-value = 1.83 × 10−7)  , a widely studied F-box member with known tumor suppressor functions, we determined the correlation between FBXW5 and FBXW7 expressions in gastric cancer, both in-vivo and in-vitro. Here, a negative correlation between FBXW5 and FBXW7 expressions was established from the analysis of a TCGA dataset ( × 10−7) B. Consis= 0.003) C, as aga= 0.003) C did not= 0.003) B and inc= 0.003) D, respecWe then investigated if FBXW5 regulates the auto-phosphorylation of FAK through its proteasomal degradation function, since F-box members are known to play a role in ubiquitin-dependent proteosomal degradation of target proteins. Here, treatment of cells with proteasomal inhibitor MG132 did not have any effect on the decrease in p-FAK expression observed in MKN1 FBXW5 KD cells D, hence p-values = 0.004, 0.002, respectively) , as compared to those transplanted with MKN1 control cells (The role of FBXW5 in cell proliferation as suggested by our in-vitro observations was further supported by an overexpression model of FBXW5. MKN1 FBXW5 OE cells demonstrated a significant increase in in-vitro cell proliferation (ctively) A and in-ol cells A. In addol cells D.p-value = 0.0046) and MKN1 FBXW5 KO  cells with stable luciferase expression were generated using a lentiviral V5-Luciferase expression vector   F,G and t 0.0393) H, as com 0.0393) I. Immuno 0.0393) J.p-value = 6.39 × 10−4)   database (GEO Accession number: GSE13861) showed that FBXW5 expression was higher in both intestinal  and ROCK1  in advanced stage gastric cancer tissues (M1) as compared to early stage gastric cancer samples (M0) and adjacent healthy gastric tissues      revealed an elevated expression of FBXW5  D, RhoA s< 0.001) E and advF-box proteins have garnered increasing attention for their role in cancer with emerging evidence suggesting dysregulation of F-box-mediated proteolysis as a cause of various human malignancies ,26. AlbeThe current study demonstrated that knockdown of FBXW5 significantly reduced in-vitro cell proliferation. Our study further reported changes in cell cycle profiles and an increase in cell apoptosis in the absence of FBXW5. Here, the increase in G0/G1 phase cells and a decrease in S phase cells could account for the decrease in in-vitro cell proliferation. Moreover, the opposing changes observed in caspase-3 activity, p21 and survivin protein expressions observed with the knockdown of FBXW5 added further credence to the anti-apoptotic role of FBXW5. Conversely, overexpression of FBXW5 significantly promoted in-vitro cell proliferation and in-vivo tumor growth. When considered together, our findings confirmed the tumorigenic potential of FBXW5 in gastric cancer and support a previous observation in NSCLC that suggested the role of FBXW5 in promoting cell proliferation and tumor growth .Cell migration and adhesion are key processes underlying tumor metastasis, which involves the dynamic rearrangement of the actin cytoskeleton and regulation of focal adhesions turnover ,23,27. TRho proteins mediate F-actin polymerization by binding to ROCK1, which phosphorylates MLC on the serine-19 residue ,30,31 toA study previously reported that induction of RhoA-ROCK1 signaling increases focal adhesion formation, which leads to the activation of FAK, which in turns regulates the organization of stress fibers, formation and turnover of focal adhesions ,27,39. OThe oncogenic role of FBXW5 is further supported by in-silico analysis of microarray dataset GSE13861 as higher FBXW5 expression was observed in both intestinal and diffuse-type gastric cancer tissues as compared to healthy gastric tissues. In addition, in-silico analysis of another microarray dataset GSE103236 revealed differential FBXW5 and ROCK1 expressions between early and advanced stage gastric cancer tissues, whereby expressions of both FBXW5 and ROCK1 are elevated in advanced stage gastric cancer samples as compared to early stage gastric cancer samples and adjacent healthy tissues. This set of analysis further validated our in-vitro findings that showed a reduction of ROCK1 expression in FBXW5 knockdown cells. Furthermore, GSEA analyses carried out between low and high FBXW5 samples supported the pro-metastatic role of FBXW5 as high FBXW5 samples were found to be enriched in gene sets related to metastasis, RhoA signaling and advanced stage gastric cancer. Collectively, these in-silico analyses added further credence to the oncogenic and pro-metastatic role of FBXW5 as elucidated from our in-vitro and in-vivo experimental findings.v/v) heat inactivated HyCloneTM fetal bovine serum (FBS) , 1% (v/v) penicillin/streptomycin  at 37 °C, 5% CO2 in an incubator. MKN1 cells were treated with 20 µM of MG132 as per previously published protocol [Gastric cancer cell lines CLS145, MKN1 , AGS and SNU1 (purchased from American Type Culture Collection) were maintained in RPMI-1640  supplemented with 10% (protocol .® transfection reagent (Santa Cruz Biotechnology) according to the manufacturer’s protocol, followed by sorting for top 10% of green fluorescent protein (GFP) positive cells by the fluorescence-activated cell sorting (FACS) method. For lentiviral particle production, HEK293T cells were co-transfected with Lentiviral V5-Luciferase expression vector (pLenti PGK Blast V5-LUC)  using jetPRIME transfection reagent (Invitrogen). Supernatants were then harvested 48 h after transfection. For the generation of stable luciferase-tagged lines, cells were infected with viral particles, followed by 0.25 μg/mL blasticidin (Thermo Fisher Scientific) selection.To knockdown FBXW5, cells were transfected with either ON-TARGETplus Human FBXW5 siRNA–SMARTpool) or ON-TARGETplus Non-targeting Pool  using DharmaFECT transfection reagent 1 (Dharmacon) according to the manufacturer’s protocol. For the generation of stable FBXW5 overexpression lines, cells were transfected with either Myc-DDK-tagged-Human F-box and WD repeat domain containing 5 (FBXW5) cDNA clone or Myc-DDK-tagged-pCMV6-Entry vector  using jetPRIME transfection reagent  according to the manufacturer’s protocol, followed by 100 ng/mL neomycin  selection. For the generation of stable FBXW5 knockout lines, cells were transfected with either Human FBXW5 CRISPR/Cas9 KO plasmid or control CRISPR/Cas9 plasmid  using UltraCruz−ΔΔCT method.Total RNA was extracted using the RNeasy Mini kit , with the use of QIAshredder spin column for homogenization and an on-column DNase digestion. Using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), 1 µg of the total RNA was reversely transcribed. The cDNA obtained was analyzed quantitatively using PrecisionFAST qPCR Master Mix  on an ABI 7500 Fast Real-time PCR system . The primers used are listed in TM ECL Select/Prime Western Blotting Detection System; GE Healthcare), followed by exposure to chemiluminescence film . The Western blot analyses were repeated twice for each protein tested.Cells were lysed in CelLytic buffer (Sigma-Aldrich), supplemented with Pierce protease inhibitor (Thermo Fisher Scientific) and PhosSTOP phosphatase inhibitor . Protein concentrations were measured by Bradford assay . A total of 20 µg of protein was electrophoretically separated on 8% or 12% sodium dodecyl SDS-PAGE. The primary and secondary antibodies used are listed in The growth inhibitory effect of FBXW5 knockdown and the growth promoting effect of FBXW5 overexpression on MKN1 cells were measured using the BrdU proliferation assay (Roche) at 12, 24, and 48 h post siRNA transfection and at 24 and 48 h post cell seeding, respectively, according to the manufacturer’s protocol. The reaction was quantified by measurement of absorbance at 370 nm, with reference wavelength set at 492 nm, using a spectrophotometeric microplate reader . The absorbance values are directly correlated to the amount of DNA synthesis.−1; Ambion®; Thermo Fisher Scientific) and stained with propidium iodide (50 μg·mL−1)  for 30 min at 37 °C. For each sample, a total of 10,000 events were analyzed for DNA content by flow cytometry on a BDTM LSR II . The cells in sub-G1, G0/G1, S and G2/M phases were quantified using FlowJo software (version vX 0.7) .Changes in cell cycle distribution were determined by fluorescence-activated cell sorting (FACS). MKN1 cells were subjected to overnight serum starvation in serum free RPMI medium. MKN1 cells were released back into complete RPMI medium for 4 h before cell seeding. Cells were harvested at 36 h post siRNA transfection and fixed in ice-cold 70% ethanol for 30 min. Following fixation, cells were resuspended in 1 x PBS, treated with RNase (5 μg·mLTM LSR II (BD Biosciences) equipped with FlowJo software (version vX 0.7) (BD).Cell apoptosis was detected by FITC (fluorescein isothiocyanate) Annexin V Apoptosis Detection Kit I (BD Biosciences), according to manufacturer’s instructions. The cell samples were analyzed at 24 h post siRNA transfection by flow cytometry on a BD®-Multi Detection System (Promega).The effect of FBXW5 knockdown on caspase 3/7 activity was measured under serum starved condition using the Caspase-Glo 3/7 assay kit , according to the manufacturer’s protocol. Capase 3 activity was quantified at 24 h post siRNA transfection by luminescence measurement using a GloMaxTM 24-well cell migration assay kit , according to the manufacturer’s instructions. Cell migration was evaluated by images captured by an inverted fluorescence microscope  at 0, 24, and 48 h post RadiusTM Gel removal.Changes in rates of cell migration was analyzed by a Radius5 cells/well were reseeded onto 24-well plates. 3 h post cell reseeding; the 24-well plate was centrifuged twice in an inverted position at 2000 rpm for 2 min. Cells that were still adhered to the wells were fixed in 4% formaldehyde for 10 min. Following that, cells were stained with methylene blue for 10 min then rinsed thrice with 500 μL of 1× PBS with shaking for 5 min. Lastly, the number of cells that remained adhered to the plate was counted. Adhesion was defined as the percentage of plated cells that remained after centrifugation.Cell adhesion assay was performed from a protocol adapted from McClay et al., 1982 . Cells w5 cells/well) were seeded onto coverslips in a 6-well plate. At 36 h post siRNA transfection, cells were fixed in 4% formaldehyde for 15 min and washed thrice in PBS. Fixed cells were permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) for 5 min and blocked in blocking buffer , 0.3% Triton X-100 (Sigma-Aldrich) in PBS) for 1 h. Following that, cells were incubated with 1: 100 dilution of 546 Phalloidin (Thermo Fisher Scientific) for 2 h at room temperature in the dark. The nuclei were then labelled with DAPI (Thermo Fisher Scientific). Finally, fluorescence confocal images of cells were captured using a laser scanning confocal microscope (Leica TCS SP5) .MKN1 cells (1.5 × 105 cells/well) were seeded onto coverslips in a 6-well plate. After overnight incubation, cells were processed according to the above protocol.On the other hand, MKN1 control and FBXW5 OE cells (2 × 105 cells/well) were seeded onto coverslips in a 6-well plate. A nocodazole assay was performed at 24 h post siRNA transfection, in which, cells were exposed to one of the three conditions—(1) 0.025% DMSO in RPMI medium for 4 h, (2) 10 µM nocodazole in RPMI medium for 4 h or (3) 4 h 10 µM nocodazole followed by a 2 h washout with 0.025% DMSO in RPMI medium. After treatment, cells were fixed in 4% formaldehyde for 15 min, and washed thrice in PBS. Fixed cells were permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) for 5 min and blocked in blocking buffer , 0.3% TritonX-100 (Sigma-Aldrich) in PBS) for 1 h. Following that, cells were incubated with 1: 100 dilution of anti-phospho-paxillin (Tyr118)  overnight at 4 °C in the dark, followed by 2 h incubation with 1:100 dilution of Alexa Fluor 488 goat anti-Rabbit IgG (H+L) (Invitrogen) at room temperature. The nuclei were labeled with ProLongTM Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Finally, fluorescence confocal images of cells were captured using a laser scanning confocal microscope (Leica TCS SP5) (Leica).Similarly, MKN1 cells (1.5 × 10g to remove the cell debris. Cell lysate (100 µL) was ultra-centrifuged at 30,000 rpm for 1 h at 37 °C to pellet the F-actin with G-actin remaining in the supernatant. F-actin in the pellet was resuspended in 100 μL of F-actin depolymerization buffer on ice for 1 h with frequent pipetting. Equal volumes of G-actin and F-actin fractions were mixed with 5× SDS sample buffer and ran on SDS-PAGE. Western blot analysis was performed using the anti-actin primary and anti-rabbit HRP secondary antibodies provided in the kit.The amount of G-actin and F-actin in MKN1 NT and MKN1 FBXW5 KD cells was quantified using a G-actin/F-actin in-vivo assay kit  according to the manufacturer’s instructions. Cells were harvested at 36 h post siRNA transfection and lysed in pre-warmed lysis/F-actin stabilizing buffer supplemented with protease inhibitor and ATP. The cell lysate was centrifuged for 5 min at 350× 2 controls. To get reference images after the live imaging was finished, the cells were removed by adding SDS. Bead displacements (with respect to its resting state) acquired during experiments were measured with PIVlab [The traction force measurements were carried as previously described . Brieflyh PIVlab  and convh PIVlab .n = 5) were used to establish the mouse xenograft model. 1 × 106 MKN1 control or MKN1 FBXW5 overexpression (OE) cells suspended in 200 µL Matrigel (BD Biosciences) were injected subcutaneously into the left and right flanks of each mouse, respectively. Tumor growth was monitored over the course of 16 days, whereby tumor diameters were measured using Vernier calipers, and recorded three times a week. Tumor volume (TV) was calculated according to the formula: TV = (2W × L)/2 mm3, whereby ‘W’ is the width, and ‘L’ is the length of each tumor. The experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines. Mice were sacrificed if they had a tumor greater than 1.5 cm in diameter, if total tumor burden was greater than 10% of body weight if the tumor becomes ulcerated or interfered with mobility.Six weeks old NOD-SCID mice (6 MKN1 control (+Luc) or MKN1 FBXW5 KO (+Luc) cells suspended in 15 µL of Matrigel (BD Biosciences) were injected directly into the spleen of each mouse, respectively. The peritoneum and skin were then closed in a single layer with surgical thread. Tumor metastasis from the spleen to the liver was monitored twice per week via bioluminescence imaging using the IVIS® Spectrum in-vivo imaging system (PerkinElmer®) over the course of two weeks. Mice were sacrificed at two weeks post splenic injection. Bioluminescence signal was quantified using the Living Image® software (PerkinElmer®). Liver samples were harvested from each sacrificed mouse for H&E staining. The experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines.Nine weeks old NOD-SCID mice (n = 5) were used to establish the in-vivo metastasis model. Firstly, the spleen was exteriorized via a left lateral flank incision. Subsequently, 1.5 × 10https://tcga-data.nci.nih.gov/docs/publications/stad_2014/).Expression correlation analysis of FBXW5 and FBXW7 was performed using a TCGA gastric adenocarcinoma data set (n = 19), intestinal-type (n = 20) and diffuse-type (n = 31) samples, was utilized to analyze the levels of FBXW5 mRNA expression across the three types of gastric tissues. A second independent microarray dataset of gastric cancer with accession number (GSE103236) containing pairwise adjacent healthy (n = 9) and tumor tissues (n = 8) was employed to evaluate the levels of FBXW5 and ROCK1 mRNA expression across the three distinct tissues: Adjacent healthy, early tumor (n = 6), or advanced tumor (n = 3). In addition, GESA analysis was performed between high-FBXW5 and low-FBXW5 samples to explore the correlations between FBXW5 expression and genes involved in RhoA signaling, metastasis and advanced tumors.A microarray dataset for adenocarcinoma gastric cancer, downloaded from GEO with accession number (GSE13861) containing healthy , protocol number: R17-0955(A)18.In conclusion, the current study provides a detailed characterization of the oncogenic potential of FBXW5 in gastric cancer. In addition to confirming the tumorigenic role of FBXW5, our study identified a novel pro-metastatic role of FBXW5 in gastric cancer. Furthermore, we uncovered a previously unreported role of FBXW5 in regulating FAK-Src signaling. This study draws limitations in exploring the direct interacting partners of FBXW5. Nevertheless, our findings underscore a contributory role of FBXW5 to an aggressive tumor phenotype in gastric cancer and warrant further validation regarding its potential as a therapeutic strategy.


Visuomotor adaptation has been thought to occur implicitly, although recent findings suggest that it involves both explicit and implicit processes. Here, we investigated generalization between an explicit condition, in which subjects reached toward imaginary targets under a veridical visuomotor condition, and an implicit condition, in which subjects reached toward visual targets under a 30-degree counterclockwise rotation condition. In experiment 1, two groups of healthy young adults first experienced either the explicit or the implicit condition, then the other condition. The third group experienced the explicit, then the implicit condition with an instruction that the same cognitive strategy could be used in both conditions. Results showed that initial explicit learning did not facilitate subsequent implicit learning, or vice versa, in the first two groups. Subjects in the third group performed better at the beginning of the implicit condition, but still had to adapt to the rotation gradually. In experiment 2, three additional subject groups were tested. One group experienced the explicit, then an implicit condition in which the rotation direction was opposite (30-degree clockwise rotation). Generalization between the conditions was still minimal in that group. Two other groups experienced either the explicit or implicit condition, then performed reaching movements without visual feedback. Those who experienced the explicit condition did not demonstrate aftereffects, while those who experienced the implicit condition did. Collectively, these findings suggest that visuomotor adaptation primarily involves implicit processes, and that explicit processes can add up in a complementary fashion as individuals become increasingly aware of the perturbation. When onunaware of the actual task goal  and also of the fact that she is adapting to the condition .When existing motor skills are adapted, the involved neural processes can be somewhat different. For example, when one performs targeted reaching movements under a novel visuomotor condition in which the visual display of the reaching movements is rotated 30 degrees counterclockwise and she is not informed of this condition, she also attempts to improve her performance by updating her motor plan for the next trial based on the performance feedback from the previous trial. In this case, however, the task goal known to the individual often differs from that intended by the experimenter. That is, the task goal known to the individual is only to minimize the movement errors , while the actual goal, only known to the experimenter, is for the subject to adapt to the rotated visual-display condition as fast as possible. In other words, the subject is Apparently, the former example is a case of explicit learning , and the latter a case of implicit learning . However, it is currently under debate whether the latter example indeed occurs implicitly or whether it also involves explicit learning. It has been previously thought that visuomotor adaptation occurs implicitly. Mazzoni and Krakauer demonstrated that when subjects were instructed explicitly how to deal with a visuomotor rotation, they were initially successful in negating the rotation; however, they failed to sustain explicit control and the movement errors became larger . Benson Whereas the aforementioned task design allowed Taylor and colleagues to differentiate the explicit and implicit components successfully in their studies, the visuomotor adaptation task employed in their studies may be somewhat different from the typical visuomotor adaptation task employed in other previous studies. It is possible that the instruction given to the subjects  in Taylor and colleagues’ studies caused the subjects to become more aware of the visuomotor rotation, which in turn caused them to make a more conscious effort to correct their movement based on the awareness of the rotation. In other words, the authors might have added an explicit component to a visuomotor adaptation task that otherwise might primarily involve an implicit component, by having the subjects verbally report their intended aiming direction. This is in line with an argument made by Leow et al. that requiring participants to report their aiming directions during visuomotor adaptation can affect the extent or persistence of implicit learning .In the present study, we mainly attempted to determine the extent of generalization between two task conditions: one that mainly involved explicit learning, and the other that mainly involved implicit learning. In the former condition, subjects were asked to reach toward imaginary targets under a veridical visuomotor condition by using a cognitive strategy . It was assumed that this condition only involved explicit learning because the subjects were completely aware of the task goal . In the latter, subjects were asked to reach toward visual targets under a novel visuomotor rotation condition, which is the typical condition employed in many previous studies in which subjects were not informed of the visuomotor perturbation –15. ThouIn addition to the two aforementioned conditions, four other similar conditions were included and compared against each other in two separate experiments.Twenty-seven neurologically intact young adults , who were right handed and naïve to the experimental conditions employed in this study, participated in this experiment. Prior to participation, subjects signed an informed written consent, which was approved for this entire study by the Institutional Review Board of the University of Wisconsin–Milwaukee .A data acquisition system that comprised a desktop PC , a Wacom Intuos3 digitizing tablet  and an LCD monitor  was used to collect data . This syIn general, subjects performed rapid reaching movements from a start circle to a target (10 cm away from the start circle) repeatedly with the right arm. They were instructed to move the cursor to the target rapidly and as straight as possible in response to the appearance of the target, stop without correcting their movement, and bring the cursor slowly to the start circle.baseline session, all subjects were familiarized with the general reaching movement. In the training session, subjects experienced either explicit or implicit conditions. In the explicit condition, subjects reached toward imaginary targets based on the following instruction: “A target will appear on the screen. Your task is to hit an imaginary target as accurately and straight as possible, by moving the cursor in the direction that is rotated 30 degrees clockwise (CW) from the visual target.” In the implicit condition, subjects reached toward visual targets based on the following instruction: “A target will appear on the screen. Your task is to hit the target as accurately and straight as possible.” In the former condition, the cursor was shown in a veridical condition; in the latter, the cursor was rotated 30 degrees counterclockwise (CCW) about the start position. In both conditions, targets were presented in a pseudorandom order , and the cursor was always visible.The experiment consisted of three sessions (separated by approx. 3 minutes): baseline , initial learning  and subsequent learning . In the testing session, the subjects who experienced the implicit condition during the training session received the explicit condition . Among the subjects who experienced the explicit condition during the training session, a half of them received the implicit condition . The other half received the implicit condition, and also were given an instruction, prior to the beginning of this session, that the use of the instruction they received in the explicit condition would help their performance in this session as well . This group was included to determine whether the subjects would be able to learn the implicit condition better if they were told that the same instruction  could be applied in both conditions. The arrangement of these conditions for each group is shown in In the hand movement would become the same by the end of the session , the angular difference between a vector from the start circle to the target and another vector from the hand position at movement start to that at peak arm velocity. DEs obtained in the explicit and implicit conditions were adjusted in such a way that the DE in the explicit and implicit conditions were subtracted by the mean DE across the last four cycles of the baseline session. In the explicit condition, DE was zero when the cursor moved perfectly straight to the imaginary targets; in the implicit condition, DE was zero when the cursor moved perfectly straight to the visual targets.  In both conditions, positive DE values indicated that the cursor was brought to a position that was on the right side (looking from the start circle) of the target; and negative DE values indicated that the cursor was brought to a position on the left side. The DE data were subjected to a repeated-measures ANOVA with group  and cycle (1–20) as a between- and a within-subject factor, respectively, which was performed for the explicit and the implicit conditions separately. The alpha level was set at 0.05 for the ANOVAs and also for post hoc between-group comparisons (Tukey’s tests).In this experiment, subjects in the Exp-to-Imp and Exp-to-Imp/Inst groups performed reaching movements under the explicit learning condition first, then under the implicit condition, while those in the Imp-to-Exp group did so under the implicit condition first, then under the explicit condition see . Fig 4 iThe repeated-measures ANOVA for the implicit condition indicated a significant main effect of group  = 4.22, p = .027) and cycle  = 56.36, p < .001). The interaction effect between the two variables was not significant (p = .175). Post hoc analyses indicated that the difference between the Imp-to-Exp and the Exp-to-Imp groups at the first cycle of the implicit condition was not significant (p = .614), but the difference between the Imp-to-Exp and the Exp-to-Imp/Inst groups was (p = .023). This suggests that when adapting to the visuomotor rotation, the subjects in the Exp-to-Imp/Inst group were able to benefit from the instruction that was used during the explicit learning condition.trials during the implicit session for the Imp-to-Exp and Exp-to-Imp/Inst groups. A pairwise comparison between the two groups at trial 1 indicated a significant difference between the two groups (p = .014), suggesting that the subjects in the Exp-to-Imp/Inst group benefited from the instruction from the very first trial. However, the mean direction error at trial 1 was -27.7 (± 1.3 SE) and -21.7 (± 1.7 SE) degrees for the Imp-to-Exp and Exp-to-Imp/Inst groups, respectively; and the direction errors in the Exp-to-Imp/Inst group continued to decrease gradually. Given that the subjects already experienced the explicit condition and also that the difference between the two groups in terms of direction errors did not become smaller after the first trial, this result indicates that although the instruction had some beneficial effect, the effect of the prior explicit learning was minimal. , who were right handed, participated in this experiment. All subjects signed an informed consent approved by the IRB.The same apparatus used in experiment 1 was used in this experiment.baseline session , all subjects were familiarized with the general reaching movement. In the training session , subjects experienced either the explicit or implicit condition (same as those employed in experiment 1). In the testing session , two new conditions were introduced: implicit with an opposing rotation, and reaching without visual feedback. The former condition was the same as the implicit condition employed in experiment 1, except that the cursor was rotated 30 degrees CW about the start position. Adding this condition allowed us to investigate generalization between the explicit and implicit conditions in which the direction of the cursor movement (or the relationship between the target location and the cursor movement) during the late phase of the explicit condition was the same as that during the early phase of the implicit condition (hand movement was the same between the two conditions in experiment 1.) In the latter condition, subjects simply performed reaching movements without any visual feedback. This condition was included to determine whether learning under the explicit and implicit conditions would result in aftereffects.As in experiment 1, experiment 2 also consisted of three sessions: baseline, training and testing. In the ondition . . Among the subjects who experienced the explicit condition during the training session, a half of them received the implicit with an opposing rotation condition ; and the other half received the reaching without visual feedback condition .The DE data obtained from the three subject groups  tested in this experiment were investigated in the following ways:(1) A comparison between the Imp-to-Exp and the Exp-to-Imp/Opp groups (implicit condition only). The DE data from the Imp-to-Exp group tested in experiment 1 were used for this comparison. Prior to the analysis, the DE data obtained from the implicit condition of each subject group were first adjusted in such a way that DEs in the implicit condition were subtracted by the mean DE of the last 3 cycles in the baseline session within each subject. Then, the baseline-adjusted DEs of the Imp-to-Exp group were converted to absolute values, which allowed a fair comparison between the data from the two subject groups.  A comparison between the Exp-to-NoVFB and the Imp-to-NoVFB groups. The DE data from the testing session of the two subject groups were subjected to a repeated-measures ANOVA with group  and cycle (1 and 2) as a between- and a within-subject factor, respectively.The alpha level was set at 0.05 for the ANOVAs and also for post hoc comparisons.(1) A comparison between the Imp-to-Exp and the Exp-to-Imp/Opp groups.The changes in performance across cycles, using the original DE data, during the implicit condition for the Imp-to-Exp and Exp-to-Imp/Opp groups are shown in (2) A comparison between the Exp-to-NoVFB and the Imp-to-NoVFB groups.We also plotted a graph depicting the changes in performance across trials (1–12) during the training and testing sessions within the Exp-to-NoVFB, shown in The results from experiment 1 indicate that initial learning under the implicit condition did not facilitate subsequent learning under the explicit condition in the Imp-to-Exp group, as indicated by large direction errors with the opposite sign at the beginning of the explicit learning condition. If initial learning under the implicit condition had a facilitative effect, the direction errors at the beginning of the explicit condition would have been negative and closer to zero. Likewise, initial learning under the explicit condition had no influence in subsequent learning under the implicit condition in the Exp-to-Imp group either, as indicated by the lack of difference between the two groups. These findings indicate that the extent of overlap  must be minimal between the explicit and implicit conditions employed in this study.spatial errors . This interpretation is supported by our finding ; and thus, the extent of overlap in terms of explicit processes between the explicit and implicit conditions employed in this study also seems to be minimal.It should be noted that a neural imaging study has demonstrated overlapping brain activation between a mental rotation task and a visuomotor adaptation task  in the prefrontal regions  . Here, tThe results from the Exp-to-Imp/Inst group further support this argument . The first piece of evidence comes from the comparisons made among the Exp-to-Imp, Imp-to-Exp and Exp-to-Imp/Inst groups. When the subjects in the Exp-to-Imp/Inst group were told that the use of the instruction provided for the explicit condition would help them perform better during the next session, they adapted to the rotation better than those in the Imp-to-Exp group. As depicted in This interpretation is supported by a finding reported in Benson et al.’s study, in which the pattern of visuomotor adaptation was compared between a group of subjects who were given prior to adaptation an instruction as to how to deal with a perturbation (30-deg. rotation) and those who were not [Another piece of evidence comes from the comparison between the Imp-to-Exp and the Exp-to-Imp/Opp groups. This condition was tested to determine whether better generalization would occur if the direction of the cursor, as opposed to the hand, movement were the same between the explicit and implicit conditions. By rotating the visual display 30 degrees CW, the direction of the cursor movement during the late phase of the explicit condition became the same as that during the early phase of the implicit condition. Yet, the extent of generalization did not increase at all. Finally, the results from the Exp-to-NoVFB and Imp-to-NoVFB groups also provide support, by demonstrating that visuomotor learning under the implicit condition resulted in large aftereffects whereas visuomotor learning under the explicit condition did not see . This isCollectively, these findings suggest that the extent of overlap between the explicit and implicit learning conditions employed in this study is minimal; and they support the idea that “typical” visuomotor adaptation, which occurs without the subject’s awareness of the visuomotor perturbation, mainly involves implicit, but not explicit, components. Here, it should be noted that the subject’s awareness of the visuomotor perturbation can be influenced by several factors, such as the size of the rotation (e.g., 75-deg. –24 vs. 4In conclusion, the present study demonstrates that the extent of generalization between an explicit and an implicit visuomotor learning condition is minimal. This finding provides support to the traditional view that the “typical” visuomotor adaptation task primarily involves implicit learning components , 3, and