Vibrio natriegens on the resin surface after a two-month exposure to seawater than the coating formulations directly doped with non-encapsulated DCOIT. The coating formulation protection efficiency was further confirmed by twelve-month shallow field tests in the South China Sea. Replacing 2 wt.% biocide in the traditional formula with DCOIT-loaded natural environmentally friendly halloysite clay drastically improved the antifouling properties of the epoxy coating, promising scalable applications in protective marine coating. The antifouling property of epoxy resin was enhanced with silver particles synthesized on halloysite nanotubes. A natural mixture of MnO particles and halloysite could also be used as a nonbiocide additive to marine coating. The short-term White Sea water test of epoxy coating with 5% of Ag-halloysite composite of MnO-halloysite natural mixture showed no visible fouling.An antifouling epoxy resin doped with natural clay nanotubes that are loaded with biocide or silver allowed extended protection against the proliferation of marine microorganisms. Compared to the 2–3 months of protection with antifoulant dichlorooctylisothiazolone (DCOIT) directly admixed into epoxy resin, the DCOIT release time of the halloysite formulations was extended to 12 months by incorporating biocide-loaded nanoclay in the polymer matrix. The protective properties of the epoxy-halloysite nanocomposites showed much less adhesion and proliferation of marine bacteria  The annual global loss caused by marine biofouling was estimated to be $150 billion in 2016 [Biofouling starts with the accumulation of microorganisms, algae, and barnacles on vessels and equipment that are exposed to seawater, leading to higher frictional resistance, increadsed fuel consumption for ships, and shortened service life due to stimulated biocorrosion . In addi in 2016 ,4. The aBacteria colonize manmade materials immersed in seawater within hours. These early small colonizers form a biofilm: an assemblage of attached cells referred to as “slime”. Attached cells divide, rapidly giving rise to colonies that eventually coalesce to form a compact biofilm, which may achieve 1 mm in thickness, and can be a base for seaweeds and shells .Current antifouling paints utilize agents that are toxic to a variety of marine species. The tributyltin (TBT) additive, which is effective after being added directly to paint ,7, was bToxic and non-toxic coatings control fouling in different ways: toxic paint layers do not become fouled until all the active agents have been leached out; nontoxic coatings, such as silicones, rely on the “slipperiness” of their surfaces to release the microorganisms after settlement . SiliconHalloysite clay is a natural mineral, of which thousands of tons are available at a low price. Tubular halloysite is rolled-up aluminosilicate layers with 50–60 nm outer diameter, 10–15 nm lumen and 0.5–1 μm length. It is similar to kaolinite in composition, with additional water between adjacent layers in the wall . The negWe focused on antifouling agents encapsulated into clay nanotube, making an extended years-long sustained release, without a harmful bust at the beginning of exploitation. Silver nanoparticles loaded in halloysite nanotubes could also be another prospect, due to their antimicrobial activity while being non-harmful to higher organisms. Silver nanoparticles have been extensively studied due to their antiseptic properties and their inhibition of biofilms, and their clay encapsulation promises advantages in long-lasting protection ,29. CombIn this work, we report antifouling formulations based on epoxy resin doped at 22 wt.% with halloysite clay nanotubes loaded fungicides. The time of release of DCOIT from halloysite in water, non-polar solvent, and from doped epoxy composites demonstrates an essential elongation. The release time of the clay-encapsulated DCOIT was extended to 12 months, allowing for long-lasting antifoulant action, which is much more than 2–3 months of protection when non-encapsulated agents were directly admixed into epoxy.Vibrio natriegens on the epoxy composite was monitored. Vibrio natriegens, a gram-negative bacterium widely distributed in seas, lakes, and rivers, forms a primary fouling layer (biofilm) on the surfaces exposed to marine environments. The inhibition ability of microbial adhesion was further measured with the antifouling field tests by immersing coated panel in warm seawater at the Sanya Bay marine station (China) and in cold Arctic sea (Russia). The extended biofouling protection with nanoclay encapsulation containing DCOIT and Ag confirms the strategy for non-toxic, durable and effective antifouling composites. This method may be used as long-lasting coating for ships and marine structure. It also has an advantage for antifouling protection in cold Arctic waters, which is becoming much needed with marine petroleum exploration shifting to northern seas.The adhesion and proliferation of Vibrio natriegens ATCC 33899 was supplied by China General Microbiological Culture Collection Center . Cyclohexane and ethanol were obtained from Beijing Chemical Plant . All of these materials were used without further purification.Halloysite (HNT) was purified and supplied by B. Zhang, Zhengzhou University, as described in . The con−1) and kept in a vacuum at 0.1 MPa for 10 min, followed by placement in atmospheric pressure; this cycle was repeated 3 times. After washing with water, halloysite was settled in a vacuum oven at 60 °C for 6 h. The dried halloysite nanotubes loaded with DCOIT (HNTs/DCOIT) were milled into a fine powder. The loading efficiency DC was calculated using Equation (1):H-DW is the weight of DCOIT-loaded halloysite at 600 °C, HW is the weight of pristine halloysite at 600 °C.Halloysite clay powder was dispersed in DCOIT-ethanol solution  and incubated at 30 °C for 3 days. Three parallel tests were carried out for each sample. The unattached bacteria on the sample surface were removed by washing with the phosphate saline buffer. The fixation of bacteria was carried out with a conventional method by treating with 2% glutaraldehyde PBS solution for 4 h. The dehydration of bacteria was achieved by ethanol treatment at concentrations of 25, 50, 75, and 100%.The antifouling properties of epoxy resin composites with DCOIT were analyzed with monitoring of proliferation 2 coated with each epoxy resin formulation were soaked in seawater for one year.The field test of the epoxy resin composites with DCOIT was carried out in the Sanya Bay by China Shipbuilding Industry Corp.  using the antifouling panels in shallow submergence method according to ASTM D 3623-78a (2012) [−1 in a flow of nitrogen (50 mL·min−1). Fourier transform infrared spectroscopy (FTIR) was used to characterized pure DCOIT, pristine halloysite and antifoulant loaded inside the nanotubes with a TENSOR 27  instrument after dispersing in KBr, followed by pressing these powders into pellets.Scanning electron microscopy (SEM) images were taken with Hitachi S-4800  microscope at an accelerating voltage of 5 kV. Energy dispersive spectrometry (EDS) was performed on a HORIBA X-Max20 detector  attached to the SEM. Transmission electron microscopy (TEM) was performed with FEI Taicnai G2 20 STWIN  at an accelerating voltage of 200 kV. Thermogravimetric analysis (TGA) of the samples was performed with TGA-60A  in the temperature range from 30 to 600 °C at a heating rate of 10 °C·minUltraviolet-visible adsorption spectra  were recorded for monitoring fungicide release . After centrifugation, the supernatant was taken for record and fresh solvent was added. The collected supernatant absorption peak at 283 nm was used to calculate concentration of released DCOIT.As shown in The loading efficiency of DCOIT prepared with different weight ratios was calculated with the temperature decomposition weight loss . It incr−1 attributed to the C=O stretching, 1475 cm−1 due to the in-plane stretching of C=C, and 686 cm−1 related to the stretching of C–Cl were preserved. No new peaks appeared, which shows that DCOIT loading is a physical process .In the FT-IR spectrum , after eα is the release rate constant. In water, the release reached 44 ± 2 wt.% after 5 h. In cyclohexane, the release rate slowed down after 2 h and gradually reached 76 ± 2 wt.% in 7 h.UV-vis monitoring was used to study the kinetics of antifoulant release from the nanotubes, both in water and cyclohexane (as a model of organic non-polar media). It can be observed in The release from the halloysite embedded in the epoxy resin matrix was drastically slowed down, reaching hundreds of days b. These K and n are experimental constants. Values of K decreased 16 times by composing halloysite encapsulated antifoulant into the resin matrix, while n increased from 0.4 to 0.8, becoming closer to the zero-order kinetics. This almost linear and slow release is a very favorable protection characteristic. After one month, the release of biocide in epoxy/HNTs-DCOIT was only 8.8 ± 0.5%, while that in non-encapsulation epoxy/DCOIT (direct fungicide admixing) 35.0 ± 2.0% of antifoulant were lost. The nanoclay encapsulation drastically slowed down the release of the antifoulant and prolonged the biocide’s supply for the coating protection. This leads to a year-long protection potential, as will be shown below for the test samples immersed in natural seawater.Epoxy resin/HNTs-DCOIT enabled sustained biocide release from the nanotubes into the polymer matrix and its further migration to the surface led to the time-extended protection. The DCOIT release kinetic from halloysite-epoxy composite was described with Peppas’ model: Industry uses epoxy paints with the addition of silver nitrate and silver oxide as antimicrobial additives. A direct addition of the silver compounds to the paint has some disadvantages; in particular, paint acquires a characteristic yellow-brownish color due to the formation of larger silver particles. Additionally, silver nanoparticles absorb light and form plasmons, which accelerate polymer degradation. Suggested halloysite nanotube templated silver systems are better mixable with epoxy and have clay-attached Ag-nanoparticles, allowing for paint color preservation while possessing antibacterial properties. The incorporation of silver nanoparticles into the inner lumen of halloysite extends the silver stability and the lifetime of the antimicrobial effect, as shown earlier for dry polyurethane paint formulations not related to marine environments . Antimic4 according to procedure modified from [4 produces Ag-particles not covered with stabilizers that may enhance Ag+ ions release to media.We present here a method for silver-halloysite paint composite preparation for antifouling coatings with the ability to vary size of silver particles. It is based on optimization of silver ions adsorption from solution on the nanotubes and its reduction using different reduction agents. ied from . The pro+ to Ag0 was achieved through the donation of electrons from tannic acid. Within 30 min, the formation of brownish silver nanoparticles was observed. Silver particles have an average diameter of 5 ± 1 nm with the prevalence of small particles. Halloysite nanotubes act as a clay template supporting silver particles preventing their aggregation.In the second approach, we used tannic acid as a reduction agent and particle stabilizer: positively charged nanotubes modified with APTES were dispersed in solution and anionic tannic acid adsorbed on the surface due to electrostatic interaction. Silver nitrate solution was then added and a reduction of the AgEscherichia coli and gram-positive Staphyloccocus aureus bacteria [To prevent coloring the epoxy, silver 4–5 nm particles were synthesized by selective placement of silver acetate inside halloysite nanotubes (followed by thermo-reduction). This technique enabled the preservation of a white epoxy paint color after doping with Ag-HNT; bacteria . Silver 2+ ions are also efficient against biofouling and have low toxicity. A natural mixture of MnO and halloysite could be used as an additive to the epoxy coating. MnO submicron particles associated with halloysite nanotubes were kindly provided to us by Northstar Mines LLC . The EDS spectra clearly showed the presence of manganese ca. 300 nm in size at 8 wt.%. Traces of Mn ions were also distributed in the framework of the halloysite nanotubes. This mixture of clay with the micro/nano scale metal oxides is promising for the production of biocide additives to paint using such abundant natural materials. The antifouling performance of these materials was tested in the northern White Sea in July in a temperature range of 5–10 °C over 2 weeks. The test was performed in 5 L bottles with White Sea water doped with the bacteria fertilizers under moderate sun irradiation in Arctic conditions. Epoxy coatings loaded with 5% of Ag-halloysite-APTES and MnO-halloysite natural mixture showed an efficient protection and no visible fouling  at 30 °C for 3 days were taken. Many bacteria were found on the surface of the pure epoxy resin . There were very few bacteria on the surface of epoxy composites with halloysite encapsulated antifoulant  in both epoxy formulations with and without halloysite encapsulated. After 60 days of exposure to artificial seawater, the amount of bacteria proliferated on the surface of epoxy resin directly admixed with DCOIT increased significantly, and was estimated at ca. 4.0 × 10ifoulant d,f. The 2 flat panel coated with resin composited with halloysite-encapsulated DCOIT remained clean, while marine microorganism colonies were observed on the uncoated control panel. After one year, the aggregates of microorganisms on the uncoated panel were larger than those on the non-encapsulated DCOIT epoxy, and their quantity/surface density was much smaller  encased in halloysite clay serving as tubule nanocontainers for this chemical agent encapsulation and providing an extended release over one year. This nanocomposite formulation resulted in much longer protection as compared with DCOIT directly admixed into the epoxy paint.V. natriegens on the halloysite-epoxy composite surfaces displayed good and long-lasting antifouling properties. As compared with the resin directly doped with non-encapsulated (fast-leaking) DCOIT, the halloysite nanoformulation gave resulted in much fewer microorganism aggregation spots being attached to the coating after exposure to seawater for 6–12 months. One-year marine antifouling panel tests in Sanya Bay, South China Sea showed only a few microorganism colonies attached on the sample coated with 2 wt.% DCOIT encapsulated in halloysite admixed to epoxy resin, while many microorganisms aggregated onto the coating doped with non-encapsulated DCOIT.The proliferation of Such clay nanotube-fungicide-epoxy resin composites enabled long-lasting antimicrobial protection and could be used as coating materials for marine antifouling formulation. Halloysite is well dispersed not only in epoxy resin but also in most commercial paints, when tried . More tThe antibacterial property of epoxy resin can also be improved with the addition of silver nanoparticles assembled on halloysite. This method may have an advantage for antifouling protection in cold Arctic waters, which is becoming much needed with the shift of marine petroleum exploration to northern seas.


FAM83H was initially identified as a protein essential for dental enamel formation. Recent reports have shown that FAM83H is also involved in the progression of human cancers in conjunction with tumor-associated molecules, such as MYC and β-catenin. However, the role of FAM83H in sarcoma has not yet been investigated.The expression and roles of FAM83H and β-catenin were evaluated in human osteosarcomas from 34 patients and osteosarcoma cells.P <  0.001, relapse-free survival; P <  0.001). In U2OS, MG63, and KHOS/NP osteosarcoma cells, the knock-down of FAM83H decreased proliferation and invasion activity and overexpression of FAM83H increased proliferation and invasion activity. In KHOS/NP cells, knock-down of FAM83H significantly inhibited, and overexpression of FAM83H significantly increased in vivo growth of cells. In addition, the knock-down of FAM83H decreased protein expression of β-catenin, active β-catenin, cyclin D1, vimentin, and snail. Overexpression of FAM83H increased protein expression of β-catenin, active β-catenin, cyclin D1, vimentin, and snail. However, the expression of β-catenin mRNA was not significantly altered with knock-down or overexpression of FAM83H. In addition, FAM83H and β-catenin shown to directly interact via immunoprecipitation and nuclear and cytoplasmic localization of β-catenin was decreased with knock-down of FAM83H. Moreover, the ubiquitination and proteasomal degradation of β-catenin was increased with knock-down of FAM83H.The expression of nuclear FAM83H, cytoplasmic FAM83H, and β-catenin were significantly associated with each other and significantly associated with shorter survival of osteosarcoma patients by univariate analysis. In multivariate analysis, cytoplasmic expression of FAM83H was an independent indicator of shorter survival of osteosarcoma patients (overall survival; This study suggests that FAM83H is involved in the progression of osteosarcomas via a mechanism involving the stabilization of β-catenin and the promotion of proliferation and invasiveness of osteosarcomas. FAM83H was first discovered during extensive computational analysis of the human genomic sequence  and repoOsteosarcoma is the most common primary malignant tumor of the bone and commonly involves metaphysis of the long bone , 13. In In this study, 34 patients who underwent surgical resection for the primary osteosarcoma of the bone at the Chonbuk National University Hospital between January 1998 and December 2012 were evaluated. The cases included in this study consisted of the medical record, original histologic slides, and paraffin-embedded tissue blocks. All cases were reviewed according to the 2013 World Health Organization classification of tumors of soft tissue and bone . The ostThe tissue microarray (TMA) was preformed from solid areas composed mainly of tumor cells with no necrosis or degenerative changes. The diameter of each TMA core was 3.0 mm, and two cores were evaluated from each case. The TMA sections were deparaffinized, and antigen retrieval was performed by boiling the TMA tissue sections using a microwave oven for 20 min in 10 μmol/L citrate buffer (pH 6.0). Thereafter, endogenous peroxidase was blocked and incubated with Protein Block Serum-Free  to prevent nonspecific staining. The tissue sections were incubated with anti-FAM83H  and anti-β-catenin  antibodies for overnight at 4 °C. The tissue sections were incubated with dextran polymers conjugated with horseradish peroxidase in DAKO Envision system  and visualized with the enzyme substrate 3-amino-9-ethylcarbazole. The immunohistochemical staining slides were scored by two pathologists (KYJ and KMK) with consensus under a multi-viewing microscope without clinical information. The nuclear and cytoplasmic expression of FAM83H were separately evaluated, and the expression of β-catenin was evaluated by overall cellular level. Immunohistochemical expression of cytoplasmic FAM83H (Cy-FAM83H), nuclear FAM83H (Nu-FAM83H), and β-catenin were scored by assessing staining intensity and staining area. The staining intensity was graded from zero to three  and the staining area was graded from zero to five  , 24. The2.This study used one normal human osteoblast and three human osteosarcoma cell lines. Normal human osteoblast cells from the femoral head  were kindly provided by Ji Hyun Park  . U2OS . The sense and antisense sequences of FAM83H #1 duplex were 5′-CACCGCTCATCTTCAGCACGTCACATTCAAGAGATGTGACGTGCTGAAGATGAGCTTTTTTG-3′ and 5′-GATCCAAAAAAGCTCATCTTCAGCACGTCACATCTCTTGAATGTGACGTGCTGAAGATGAGC-3′, respectively. The sense and antisense sequences of FAM83H #2 duplex were 5′-CACCGCCGCCTCACTACAAAGAGTATTCAAGAGATACTCTTTGTAGAGGCGGCTTTTTTG-3′ AND 5′-GATCCAAAAAAGCCGCCTCACTACAAAGAGTATCTCTTGAATACTCTTTGTAGTGAGGCGGC-3′, respectively. The vector for wild-type FAM83H  was purchased from GeneCopoeia . The transfection was performed using JetPRIME transfection reagent .3 cells were seeded in culture plates and optical density measured at a wavelength of 560 nm after 24, 48, and 72 h. The colony-forming ability of tumor cells was evaluated by growing tumor cells for 10 days in 6-well culture plates after seeding with 1 × 103 cells. The number of colonies was determined using the densitometric software Clono-Counter [The proliferation of cells was evaluated with the 3--2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay  and colony-forming assay. For the MTT assay, 3 × 10-Counter .5 cells were seeded in a 24-transwell migration chamber with 8 μm-pore filters . The bottom chamber contained 10% FBS to promote migration of osteosarcoma cells. The invasion assay was performed by seeding 2 × 105 cells in an 8 μm-pore Matrigel Invasion Chamber . The lower chamber in the invasion assay contained DMEM culture media with 10% FBS. The chambers for the migration and invasion assays were incubated at 37 °C for 24 h. The filters in the migration and invasion assays were stained with DIFF-Quik staining solutions , and the number of migrated or invaded cells was counted in five microscopic fields (magnification × 200) per well.The migration and invasion activity of osteosarcoma cells was evaluated by migration and invasion assays. For the migration assay, 1 × 106 KHOS/NP cells transfected with control vectors, shRNA #2 for FAM83H, or the vector of wild-type FAM83H were injected into the bone marrow of the right proximal tibia. Tumor volume was calculated as length x width x height × 0.52 every week. Six weeks after tumor cell inoculation, animals were euthanized and evaluated for tumors and changes to internal organs such as lung, kidney, and liver. Animal experiments were performed with the approval of the institutional animal care and use committee of Chonbuk National University .To establish orthotopic xenografted osteosarcoma, we used six-week-old male BALB/c nude mice . Under anesthesia, 2 × 10Protein extraction was performed by using phosphatase inhibitor cocktails 2, 3  containing PRO-PREP Protein Extraction Solution . The lysates were centrifuged at 14,000 rpm for 10 min at 4 °C, and the protein concentration was measured by the Bradford method . The proteins were electrophoresed on SDS-polyacrylamide gel and electrotransferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline and 0.1% Tween 20, and incubated with primary antibodies for FAM83H , β-catenin , active β-catenin (dephosphorylated β-catenin) , cyclin D1 , p27 , vimentin , snail , lamin B1 , and actin . The membranes were incubated with corresponding secondary antibodies and detected with a luminescent image analyzer  with the ECL detection system . Immunoprecipitation was performed with a Dynabeads-Protein A kit . For immunoprecipitation, anti-FAM83H or anti-β-catenin antibodies were cross-linked to Dynabeads-Protein A for 20 min and incubated with protein lysates for 2 h at 4 °C. FAM83H- and β-catenin-immunoprecipitation complexes were washed thrice with supplied washing buffer and eluted by boiling in the elution buffer. Thereafter, the eluted proteins were immunoblotted with anti-FAM83H, anti-β-catenin, anti-ubiquitin, and anti-actin antibodies.An RNeasy Mini Kit  was used to obtain RNA and TaqMan Reverse Transcription Reagents  were used for reverse transcription of 1.5 μg RNA. Thereafter, a quantitative reverse-transcription polymerase chain reaction was performed using Applied Biosystems Prism 7900HT Sequence Detection System and Sybr Green polymerase chain reaction Master Mix . The expression level of the glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene was used to normalize all results and the experiments were performed in triplicate. Primer sequences for quantitative reverse-transcription polymerase chain reaction are listed in Table 5) were seeded in 6-cm dishes and transfected with control or FAM83H shRNA. 48 h after transfection, the cells were treated with 20 μg/ml cycloheximide  or 20 μM MG132  for 0.5 to 2 h. Western blot was performed for anti-β-catenin  and anti-actin  antibodies with the cell lysates. In addition, MG63 cells transfected with control or FAM83H shRNA for 48 h were treated with 20 μM MG132 for 4 h. Thereafter, the total lysates were immunoprecipitated with anti-β-catenin antibody and blotted with anti-ubiquitin and anti-β-catenin antibodies.MG63 cells  was used throughout and P value less than 0.05 was considered statistically significant.To determine positivity for the immunohistochemical staining for FAM83H and β-catenin, receiver operating characteristic curve analysis was preformed, and the cut-off points were determined at the points with the highest positive likelihood ratio point for the estimation of the death of osteosarcoma patients , 20, 28.P = 0.013), larger tumor size (P = 0.039), higher tumor stage (P = 0.002), and higher histologic grade (P = 0.002). Nu-FAM83H positivity was significantly associated with larger tumor size (P = 0.016), higher tumor stage (P <  0.001), and higher histologic grade (P <  0.001). In addition, there was significant association between Cy-FAM83H, Nu-FAM83H, and β-catenin expression  , tumor size , tumor stage , histologic grade , distant metastasis , Cy-FAM83H expression , Nu-FAM83H expression , and β-catenin  were significantly associated with both OS and RFS of osteosarcoma patients  and Cy-FAM83H expression  as independent indicators of poor prognostic of OS and RFS in osteosarcoma patients. Cy-FAM83H expression indicated a 15.463-fold  greater risk of death and a 15.825-fold  greater risk of relapse or death of osteosarcoma patients (Table Multivariate analysis was performed with the factors significantly associated with OS or RFS by univariate analysis: the age of patients, tumor size, tumor stage, lymph node metastasis, distant metastasis, histologic grade, Cy-FAM83H expression, Nu-FAM83H expression, and β-catenin expression. Multivariate analysis revealed presence of distant metastasis (OS; As the expression of FAM83H was significantly associated with advanced clinicopathological factors such as larger tumor size, higher tumor stage, and higher histologic grade, we evaluated the effect of the FAM83H on the proliferation and invasiveness of osteosarcoma cells. As expected, the knock-down of FAM83H with shRNA for FAM83H inhibited proliferation, and overexpression of FAM83H increased the proliferation of U2OS and MG63 osteosarcoma cells Fig. a and b. As we have shown in Fig. In this study, we investigate the roles and relationship of FAM83H and β-catenin in osteosarcomas and show that the expression of FAM83H and β-catenin are closely associated, and their expression are involved in the progression of osteosarcomas. In human osteosarcoma tissue samples, the expression of Cy-FAM83H and Nu-FAM83H were associated with advanced clinicopathologic factors such as larger tumor size, higher tumor stage, and higher histologic grade of osteosarcomas. Moreover, both Cy-FAM83H and Nu-FAM83H positivity were significantly associated with shorter OS and RFS of osteosarcoma patients by univariate analysis. Despite the limited number of cases of osteosarcomas, higher expression of Cy-FAM83H was an independent indicator of poor prognosis and shorter OS and RFS of osteosarcoma patients in multivariate analysis. In line with these results, higher expression of FAM83H was significantly associated with higher tumor stage and shorter survival of hepatocellular carcinomas and clear cell renal cell carcinoma patients , 10. In Poor prognosis in osteosarcoma patients with tumors with elevated expression of FAM83H suggests that FAM83H is involved in the progression of osteosarcomas. Supportively, in this study, in addition to the prognostic significance of Cy-FAM83H and Nu-FAM83H expression in osteosarcoma patients, overexpression of FAM83H increased proliferation of osteosarcoma cells, and knock-down of FAM83H inhibited proliferation of osteosarcoma cells both in vitro and in vivo. Moreover, overexpression of FAM83H increased expression of β-catenin, active β-catenin, and cyclin D1, and decreased expression of p27. In line with our results, knock-down of FAM83H inhibited proliferation of prostate cancer cells  and cleaAnother interesting finding of this study is that there was a significant correlation between FAM83H expression and β-catenin expression in human osteosarcoma tissue samples. Especially, the expressions of both FAM83H and β-catenin were associated with shorter survival of osteosarcoma patients. These findings suggest that both FAM83H and β-catenin are closely associated and involved in the progression of osteosarcomas. The Wnt/β-catenin signaling is important in the proliferation and progression of human cancer cells –39. MainConcerning the subcellular localization of FAM83H, FAM83H was considered a cytoplasmic membranous protein involved in the cytoskeletal arrangement , 6, 29. In conclusion, we showed that FAM38H expression is important for the proliferation and invasiveness of osteosarcoma cells and suggest that stabilizing β-catenin protein by FAM38H can be a biochemical mechanism to explain the significant association between FAM38H expression and shorter survival of osteosarcoma patients. These results highlight FAM83H as a new prognostic maker as well as a new therapeutic target of osteosarcoma.


A bio-derived dihydroxylimine hardener, Van2HMDA, for the curing of epoxy resin was prepared from vanillin (Van) and hexamethylene-1,6-diamine (HMDA) by Schiff base formation. The epoxy resin of diglycidyl ether of bisphenol A was cured with Van2HMDA in the presence of the catalyst, 2-ethyl-4-methylimidazole (EMI). The crosslinked epoxy resin showed thermal-healing properties at elevated temperatures. Moreover, the crosslinked epoxy resin can be reshaped by heating via imine metathesis of the hardener units. The imine metathesis of Van2HMDA was confirmed experimentally. Stress-relaxation properties of the epoxy resin crosslinked with Van2HMDA were investigated, and the activation energy obtained from Arrhenius plots of the relaxation times was 44 kJ/mol. The imine bonds in the epoxy polymer matrix did not undergo hydrolysis after immersing in water at room temperature for one week. However, in the presence of acid, the crosslinked polymer was easily decomposed due to the hydrolysis of imine bonds. The hydrolysis of imine bonds was used for the ecofriendly recycling of crosslinked polymer. It is inferred that thermal-healing, reshaping, and reprocessing properties can be implemented in the various crosslinked epoxy resins with the bio-derived dihydroxylimine hardener, albeit the recycled epoxy resin is of inevitably lower quality than the original material. Epoxy resin is a representative thermosetting polymer that has been widely used because of its excellent thermal and chemical stability and good mechanical properties. However, because, upon curing, epoxy resins form a highly crosslinked network structure, they cannot be reshaped or recycled at the end of their service life or after they have been damaged and may pose a risk to the environment and to human health. Therefore, extensive efforts have been devoted to designing thermosetting epoxy resins that can be reshaped or recycled and to extending the lifetimes of epoxy resins by introducing healing properties.The first strategy to introduce healing properties into epoxy resins involves introducing containers or capsules that contain a healing agent ,2,3. In In the dissociative-type mechanism, chemical bonds are first broken upon stimulus and, then, formed again under a different stimulus. The typical dissociative-type mechanism is based on a Diels–Alder reaction ,5,6,7. HIn the associative-type mechanism, dynamic covalent bonds are broken and are reformed at the same time. Because of the nature of the exchange reactions, the crosslink structures are maintained; however, the viscosity of the polymer can be controlled, enabling the epoxy thermoset to be processed like a thermoplastic ,18,19,20Concerning the environmental issue, petroleum-based materials should be replaced with bio-based materials from renewable resources. Among bio-based materials that can be incorporated into epoxy resin systems, vanillin  has been used to prepare epoxy resins ,31,32. NHexane-1,6-diamine (HMDA), vanillin, 4-hydroxylbenzaldehyde (HBA), and 2-ethyl-4-methylimidazole (EMI) as a catalyst were purchased from Sigma-Aldrich . A diglycidyl ether of bisphenol A (DGEBA)-based epoxy resin with an epoxy equivalent weight of 187 g/eq (YD-128) was provided by Kukdo Chemical . Solvents used in this work include ethanol (EtOH), methanol (MeOH), toluene (Tol), dimethylformamide (DMF), and dimethylacetamide (DMAc), which were supplied by Samchun Pure Chemical . All materials were used without further purification.1H NMR  chemical shift : 8.10 , 7.27 , 7.04 , 6.77 , 3.75 , 3.44 , 1.54 , 1.30 . 13C NMR  δ (ppm): 160.47 (–CH=N–), 148.44, 123.05, 115.72, 110.37 (C of Ar), 60.81 (–OCH3), 55.96 (–CH2–), 31.13, 27.15 (–CH2–).Van  was dissolved in 200 ml of EtOH followed by the addition of HMDA . The mixture was heated and was magnetically stirred at 80 °C for 12 h. A yellow precipitate was observed after the HMDA was dissolved in the Van solution. The precipitate, a dihydroxylimine compound (Van2HMDA), was collected by filtration, washed with cold EtOH and, then, was dried under vacuum at 70 °C for 24 h. The dried yellow powder . 1H NMR  δ (ppm): 8.11 , 7.48–7.50 , 6.74–6.76 , 3.41–3.43 , 1.51–1.53 , 1.27–1.29 . 13C NMR  δ (ppm): 160.26 (–CH=N–), 130.00, 128.04, 115.92, (C of Ar), 60.94 (–CH2–), 31.18, 27.16 (–CH2–).The model compound based on HMDA and HBA, HBA2HMDA, was prepared via a similar process and was characterized by NMR analysis. A stoichiometric mixture of epoxy resin  and Van2HMDA (10.28 g) was heated and was mechanically stirred at 140 °C in a 250 mL reactor until the resin mixture was homogeneous. The resin was cooled to 100 °C followed by the addition of the EMI catalyst (1 wt %) under stirring for an additional 1 min. Then, the resin was cast and was cured at 120 °C for 24 h and, subsequently, post-cured further at 150 °C for 2 h to obtain a 1-mm-thick film.The crosslinked epoxy polymer was recycled through the following procedure. Typically, 8 g of cured epoxy resin was added to 200 mL of 0.1 mol/L HCl standard solution. After the solution was heated and stirred at 70 °C for about 8 h, all polymer was dissolved. The solvent was removed by rotary evaporator at 65 °C. The hydrolyzed polymer was dried at 80 °C for 24 h and further 110 °C for 24 h to remove residue solvent and recover the polymer network by the condensation reaction.The FT-IR spectra were recorded with a Jasco FT/IR 4100 . The solid samples for FT-IR spectroscopy were mixed with KBr powder; then, they were ground, and pressed into thin tablets. The liquid samples were scanned after being coated directly onto KBr tablets.1H NMR and 13C NMR spectra were collected with a 600 MHz FT-NMR spectrometer . The samples (~30 mg) were dissolved in 0.75 mL of dimethyl sulfoxide d6 (DMSO-d6).The 3. The frequency of the oscillation test was 1 Hz, and the temperature dependence of the behavior was investigated in the temperature range from 20 to 200 °C at a heating rate of 3 °C/min. In addition, stress-relaxation experiments were performed on the same mechanical analyzer. To maintain the straightness of the samples, initially, they were preloaded under a force of 0.001 N. The samples were heated to the test temperature and were held there for 30 min to reach thermal equilibrium. Then, 1% strain was applied, and the stress needed to maintain constant 1% strain was recorded. The stress-relaxation moduli were obtained as a function of time.Differential scanning calorimetry  was used to characterize the imine-compound-induced curing behavior of the epoxy resin. Dynamic mechanical analysis (DMA) was carried out on a TA Instruments DMA Q800 dynamic mechanical analyzer. The single-cantilever beam was used to deform the rectangular specimens with mean dimensions of 12.5 × 1 × 17.5 mmThe imine metathesis was studied by liquid chromatography–mass spectrometry (LC/MS) . The equivalent molar ratio of imine compounds Van2HMDA and HBA2HMDA were dissolved separately in MeOH, and the two solutions were mixed and stirred at room temperature. After one hour, the solvent was removed by vacuum evaporation at room temperature for the characterization.The thermal healable of dynamic epoxy polymer was investigated by scratching test and was observed by scanning electron microscope (SEM) AIS2100C . The surface of the film was coated with gold before observation. The surface of polymer was observed before scratch and after heating at 90 °C for 5 min.Thermal stability of the epoxy polymer was analyzed by thermogravimetric analyzer . Samples (10–20 mg) were loaded in a platinum pan and heated from 30 to 800 °C under a nitrogen at a heating rate of 20 °C/min.Tensile test was carried out by Universal Test Machines . The epoxy polymer was casted in a dog-bone-shaped mold. The specimens were loaded to failure at a speed of 0.5 mm/min.GF = W2/W1 × 100, where W2 is the weight of the dry sample and W1 is the weight of the original sample.The gel fraction test was conducted by immersing crosslinked epoxy samples in different solvents at room temperature under static conditions. After a week, the solvents were removed by filtration and the samples were dried at 110 °C for 24 h. The gel fraction (GF) was calculated using the equation The imine was formed by a reversible condensation reaction between primary amine and carbonyl compounds. To enhance the yield of the reaction, benzene  were gen−1 corresponding to a C=O stretching vibration  of 78 °C for the crosslinked polymer, as determined from the enthalpy change.Epoxy groups in the resins can react with amines, hydroxyls, anhydrides, acids or thiols ,37,38,39Tg identified from the tan delta of the epoxy cured by Van2HMDA was 87 °C, which is relatively low because of the inclusion of a flexible segment of hexamethylene in the dynamic hardener. The GF test is a typical test to compare the crosslink density of thermoset polymers. The un-crosslinked polymer is dissolved in commonly used solvents. Results of the GF indicate that the epoxy resin crosslinked with dynamic polymer did not dissolve in common solvents d. In polTg. Furthermore, the stress-relaxation curves shifted to the left with increasing temperature because of the increase of the imine metathesis rate, enabling stress to relax more quickly. Relaxation times (τ*) were defined based on Maxwell’s model for viscoelastic materials as the time required to relax to 1/e of the initial stress. The dynamic-hardener-based crosslinked epoxy resin in this study showed relaxation times that ranged from 60 s at 100 °C to 10 s at 150 °C, which are much shorter than those of the crosslinked epoxy resins cured by fatty acids [τ0 is the relaxation time at infinite T, Ea is the activation energy of the imine metathesis, and R is the universal gas constant. Arrhenius plot of the relaxation time τ is shown in Ea) of 44 kJ/mol was obtained for the dynamic hardener crosslinked network of epoxy resin. This activation energy is much lower than the value of the crosslinked epoxy resins cured by fatty acids [Polymers containing dynamic bonds can relax stress and flow , which ity acids  or disulty acids ,13 reporty acids  or disulty acids ,13 reporTv) is a key transition temperature for a dynamic polymer because, at the Tv, the polymer transforms from vitrified glass to deformable solid as a consequence of the exchange reactions in the network. This transition is identified when the viscosity increases to greater than 1012 Pa·s [η = G · τ*, where G is calculated from the elastic modulus, as measured by DMA, according to the relation G = E’/2(1 + ν) , where G = E’/3 and E’ = 11.56 MPa; thus, τ* = 2.6 × 105 s. Substituting τ* into the Arrhenius’s equation, we can get the Tv. For the epoxy resin cured by Van2HMDA, the hypothetical Tv value was −38 °C, which is lower than its Tg , implying that an intrinsically fast imine metathesis occurred in a rigid polymer matrix. The value of Tv was calculated via extrapolation of stress-relaxation times; however, this transition is hypothetical because the network is not ultimately frozen by the reaction kinetics but by the lack of segmental motions associated with the Tg [The topology freezing transition temperature  of recycled polymer (63 °C) was lower than that of original polymer (78 °C) as given in Tg was lowered. However, the recycled polymer still showed the reshaping and thermal-healing properties (323 cm−1 d. The onoperties .Tg, enabling a dynamic crosslinked polymer with reshaping ability and thermal-healing ability. The dynamic epoxy polymer also shows ecofriendly recycle ability by using mild acid solution through hydrolysis of imine bonds. The recycled polymer shows lower thermal properties than that of original polymer due to lower crosslinking density. However, the recycled polymer still exhibits reshaping and thermal-healing ability.In this study, we have shown a simple, environmentally friendly route to preparing a bio-derived dynamic hardener based on Van. The successful preparation of the dynamic hardener was confirmed by FT-IR and NMR analyses. The LC/MS spectra indicated imine metathesis of the model compounds. The dihydroxylimine compound can cure epoxy resin in a manner similar to the conventional hardener. The epoxy resin cured with a dynamic hardener exhibited fast stress relaxation because of rapid imine metathesis at temperatures greater than


Background: Sorting Nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain common among all of the sorting nexin family, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27–PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells (mpkCCD) using a GST-SNX27 fusion construct as bait. We found that the C-terminal type I PDZ binding motif (DTDL) of β-catenin, an adherens junction scaffolding protein and transcriptional co-activator, interacts directly with SNX27. Using biochemical and immunofluorescent techniques, β-catenin was identified in endosomal compartments where co-localization with SNX27 was observed. Furthermore, E-cadherin, but not Axin, GSK3 or Lef-1 was located in SNX27 protein complexes. While overexpression of wild-type β-catenin protein increased TCF-LEF dependent transcriptional activity, an enhanced transcriptional activity was not observed in cells expressing β-Catenin ΔFDTDL or diminished SNX27 expression.These results imply importance of the C-terminal PDZ binding motif for the transcriptional activity of β-catenin and propose that SNX27 might be involved in the assembly of β-catenin complexes in the endosome. Many signaling pathways are regulated by endocytosis. This process plays a critical role in signaling component activation, transduction of downstream signals and signal termination. The endosomal network is composed of several intracellular compartments that begin at the plasma membrane where cargo such as a ligand-bound receptor or ion channel is internalized into the early sorting endosome . From hePX-only proteins consist of a lone PX domain with flanking regions of non-conserved sequences. SNXPX-BAR proteins contain BAR (Bin/Amphiphysin/Rvs) domains that are capable of detecting and inducing membrane curvature [PX-other proteins contain diverse domain architectures consisting of SH3, FERM or RGS  domains among others [The sorting nexin family of proteins are involved in endocytosis and endosomal trafficking . Each sourvature . SNXPX-og others ,5,7.In the latter category, sorting nexin 27 (SNX27) contains a unique Postsynaptic density protein-95, Discs-large, Zona-occludens-1 (PDZ) domain and a FERM-like/Ras-Association domain ,9.. found over 80 cell surface transmembranous proteins interact with SNX27 [Various studies have demonstrated that many of the SNX27-binding proteins, which include transmembrane receptors, ion channels and scaffolding proteins, conform to the consensus type-1 PDZ-binding motif D/E-S/T-X-L/V, where ‘X’ is any amino acid . In a coth SNX27 . These pth SNX27 .. suggest a role for SNX27 in the recycling of β2-adrenoreceptors from the early endosome to the plasma membrane [As each novel SNX27-interacting protein is identified our knowledge of the function of SNX27 that is becoming more apparent. For example, Lunn et al. have proposed that SNX27 regulates Kir3 potassium channel endocytosis and lysosomal degradation while data by Lauffer et almembrane ,14. Heremembrane .Previous work from our lab has expanded a role for SNX27 as a scaffold in the trafficking of intracellular proteins such as β-Pix/Git and ZO-2, in the regulation of cell motility and paracellular transport in epithelial cells ,17. HereThe Wnt signaling pathway is highly conserved across species and regulates many normal and pathogenic cellular processes such as embryonic development and cancer . The impIn addition to a role in Wnt signaling, β-catenin has a well-characterized role at the adherens junction where it interacts with the cytoplasmic portion of cadherin proteins enabling homotypic adhesion between adjacent cells . SimultaAs expected, the interaction of β-catenin with SNX27 is dependent on the C-terminal type-I PDZ binding motif (DTDL) of β-catenin. In the present study, we have examined the role of this novel protein–protein interaction on recruitment of β-catenin to the early endosome, to sites of cell–cell contact and transcriptional activity.A polyclonal antibody to SNX27 was generated as described previously . A monoccDNAs encoding wild-type and mutant human SNX27b were generated as described . β-Caten2 at 37°C.Mouse primary kidney cortical collecting duct cells (mpkCCD) clone 3 cells were kindly provided by Dr Mark Knepper (NHLBI/NIH). MpkCCD and 293T cells were maintained in high glucose Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum (ATCC) in humidified chambers with 5% COMpkCCD cells were transfected using Lipofectamine LTX reagent including Plus reagent (Invitrogen) using manufacturer’s recommendations. Cells were typically used in biochemical and functional assays 24 to 48 h post-transfection. Cells (293T) stably expressing SNX27 silencing and non-silencing shRNAs were generated using an inducible lentiviral shRNA system (pINDUCER10) and selected with puromycin as described in Valdes et al. . shRNA ein vitro translated β-catenin were performed as described [The purification of GST and GST–PDZ fusion proteins was performed as described previously . For GSTescribed .Wild-type (PPGDSNQLAWFDTDL) and mutant (PPGDSNQLAWFGGGG) β-catenin C-terminal peptides with an amino-terminal linker sequence (SGSG) were synthesized at the Tufts University Peptide Synthesis core . A biotin moiety was conjugated to the amino-terminal of each peptide to facilitate interaction with streptavidin. Binding assays were performed as described previously .m/z 200 to 2000. The LTQ-Orbitrap XL was operated in a data-dependent mode; i.e. MS1 in the ion trap, scan for precursor ions followed by six data-dependent MS2 scans for precursor ions above a threshold ion count of 2000 with collision energy of 35%. The raw file generated from the LTQ-Orbitrap XL was analyzed as described [To identify novel proteins bound to the PDZ domain of SNX27, samples were electrophoresed on SDS-polyacrylamide gels, and protein bands were visualized by staining with Coomassie blue. The band of interest was excised and digested with trypsin at 1:50 enzyme/substrate ratio. The dried tryptic digest was analyzed on an LTQ-Orbitrap XL (Thermo-Fisher Scientific LLC) interfaced with an Eksigent nano-LC 1D plus system  using CID fragmentation. Samples were loaded onto an Agilent Zorbax 300SB-C18 trap column at a flow rate of 5 µl/min for 10 min, and then separated on a reversed-phase PicoFrit analytical column  using a 40-min linear gradient of 2–40% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. LTQ-Orbitrap XL settings were as follows: spray voltage 1.5 kV; full MS mass range: escribed .5) were plated on square glass coverslips (484 mm2) and transfected as indicated above. Cells were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized with 0.1% Triton-X 100 for 5 min, and incubated in blocking buffer  in PBS) for 1 h at 37°C. The fixed coverslips were incubated with β-catenin and EEA1 at 1:200 dilution in blocking buffer overnight at 4°C. Coverslips were washed extensively with PBS and incubated with the indicated secondary antibodies (Alexafluor 488 nm or 597nm (Invitrogen-Molecular Probes) at 1:1000 dilutions) in blocking buffer for 1 h at 37°C. Following further washing with PBS, coverslips were mounted using FluorSave™ . Wide-field images were collected using a Personal DeltaVision system  mounted on an inverted Olympus IX71 microscope with an oil immersion PlanApo N 60×/1.42 objective lens. All images were acquired using a CoolSNAP ES2 CCD camera with 2 × 2 binning and a 512 pixels × 512 pixels imaging field. All images were deconvolved using an iterative constrained method with 10 cycles, medium noise filtering in Applied Precision’s SoftWoRx software package version 4.0.0.MpkCCD cells . The supernatant was incubated with monoclonal EEA1 or IgG control antibodies . The immunoprecipitates were collected using protein G-sepharose beads, and prepared for SDS-PAGE electrophoresis.The immunopurification of endosomes was performed using an antibody against early endosome marker, EEA1 . Briefly5) were seeded per well of a 24-well plate and transfected with 150 ng TOPFLASH or FOPFLASH reporter [5) were seeded in 3.5 cm diameter dishes and transfected with 0.67 µg of mCherry-β-catenin (wt or ΔFDTDL), HA-Tcf-4 and either TOPFLASH or FOPFLASH luciferase reporter plasmids using Fugene HD. Luciferase measurements were taken 48 h post-transfection.To examine the role of SNX27 on the Wnt-dependent transcriptional activity of β-catenin, 293T cells  was incubated with GST alone or GST-PDZ (β-Catenin contains a known type I PDZ-binding motif (DTDL), which is absent in plakoglobin (HMLA) A. We hyp GST-PDZ C. Direct GST-PDZ C. In con GST-PDZ C.To further verify that the PDZ domain on SNX27 was involved in the β-catenin interaction, a series of Myc-tagged SNX27 constructs were expressed in mpkCCD cells and isolated by immunoprecipitation . SNX27-bTo confirm that the carboxy-terminus of β-catenin mediates the SNX27 interaction, biotinylated β-catenin peptides corresponding to the wild-type (DTDL) or mutant (GGGG) β-catenin carboxy-terminus were synthesized. The peptides were immobilized with streptavidin beads, incubated with mpkCCD cell lysates and the presence of bound SNX27 detected by Western blotting E. Wild-tNumerous reports have localized SNX27 to the early endosome in a variety of cell types ,13,14, wHaving confirmed both the biochemical interaction of SNX27 with β-catenin and the endosomal localization β-catenin in mpkCCD cells, we postulated that SNX27 might be responsible for the recruitment of β-catenin to the early endosome . To testWe have previously shown a role for SNX27 as a protein scaffold . The binThe cytoplasmic unbound pool of β-catenin is tightly regulated and maintained at low levels through proteosomal degradation. However, in the presence of Wnt-signaling the pool of cytoplasmic β-catenin is elevated leading to the formation of nuclear transcriptional complexes and elevated transcription of specific Wnt target genes ,30. We aP = 0.0003, n = 3). Equal protein expression was confirmed by Western blotting for the mCherry and HA tags for β-catenin and TCF, respectively  which, in the absence of Wnt3A diminished over time at a similar rate in both control and SNX27-depleted cells A.As expected, 293T cells treated with Wnt3A led to an increase in β-catenin-TCF/LEF transcriptional activity B. HoweveProteins of the sorting nexin (SNX) superfamily are characterized by the presence of a phox-homology (PX) domain and associate with phosphatidylinositol-3-monophosphate (PtdIns3P) rich regions of the endosomal system. SNX27 is the only sorting nexin that contains a PDZ domain. Here, we identified a novel interaction between SNX27 and β-catenin. The PDZ domain of SNX27 and C-terminal residues of β-catenin mediated the interaction.+/H+ exchanger regulatory factor NHERF and membrane-associated guanylate kinase protein families suggesting that this mechanism of protein–protein interaction may regulate the distribution and function of β-catenin at different sites of the cell and in multiple cell types [While β-catenin is mainly regulated by multiple protein–protein interactions with the central ‘Arm’ repeat motifs ,31–33, all types .Numerous studies have localized SNX27 to the early endosome, yet β-catenin predominantly is found at the nucleus and at sites of cell–cell contact ,14,33,40The remaining part of the present study considered the functional consequence of SNX27 binding on β-catenin function. Given the restricted expression of SNX27 to the endosome, we proposed that SNX27 might function to sequester β-catenin alone, or in complex with known binding partners to endosomal sites to regulate the trafficking of β-catenin to the nucleus to modulate transcription or to the adherens junction to modulate cell–cell contacts.By biochemical methods, we detected E-cadherin, but not LEF-1 or GSK-3 in SNX27–β-catenin complexes in confluent mpkCCD cells. This suggested that SNX27 might specifically regulate trafficking of β-catenin and E-cadherin to or from the adherens junction, rather than participate in Wnt signaling. However, the cellular localization of both β-catenin and E-cadherin in mpkCCD cells lacking SNX27 was unchanged in cells at confluence. Furthermore, the rate of recycling of endogenous β-catenin and E-cadherin to the adherens junction in cells lacking SNX27 post-calcium switch was also unaltered (data not shown). However, we may be overlooking the fact that the intracellular trafficking of β-catenin may be highly dynamic. Hence, we attempted FRAP (fluorescence recovery after photobleaching) experiments of N-terminal tagged mCherry-β-catenin under live cell conditions in mpkCCD cells lacking SNX27 . HoweverNumerous mechanisms have been proposed by which the cytoplasmic pool of β-catenin is regulated  . There iWe postulated that this small SNX27-bound endosomal pool of β-catenin may regulate cytoplasmic levels and downstream transcriptional activity of β-catenin.To address this hypothesis, we first compared the transcriptional activity of full-length β-catenin with a SNX27-binding defective mutant (ΔFDTDL). The protein stability and relative nuclear/cytoplasmic distribution of both mutant and wild-type proteins were found to be similar (data not shown), which is in agreement with previous data from the Gottardi laboratory ,48. Howe. one of the receptors that binds Wnt, Fzd7 is a SNX27–PDZ interacting protein [There is a report of one other PDZ binding motif-containing protein that may regulate the function of β-catenin as a transcriptional co-activator. As previously reported by Sun et al protein . It is tIn summary, we present SNX27 as a novel β-catenin interacting protein which is an important player in the complex and fascinating role of β-catenin in normal and disease pathology. The observations that the SNX27-β-catenin interaction may induce a decrease in transcriptional activity suggests that inactivation of this protein-protein interaction might be beneficial in the treatment of β-catenin-dependent tumors.Click here for additional data file.Click here for additional data file.


Dolichos lablab L. (DLL-Ex) inhibited high-fat diet (HFD)-induced increases in body weight and body fat mass and ameliorated increases in body weight. In the present work, we studyed the molecular mechanism for the inhibitory effect of DLL-Ex or Chikusetsusaponin IVa (CS-IVa), as isolated from Dolichos lablab L. (DLL) seeds extract, on adipocyte differentiation. We evaluated the effect of DLL-Ex, an anti-obesity agent, and CS-IVa, an active component of DLL-Ex, on 3T3-L1 cell differentiation via Oil red O assay and Q-PCR, along with their effects on CCAAT element binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS), and fatty acid-binding protein 4 (FABP4) mRNA transcriptions. FAS and FABP4 protein expression levels after exposure to CS-IVa were also tested. The results showed that DLL-Ex and CS-IVa have potent inhibitory activity on adipocyte differentiation. Therefore, DLL and CS-IVa may be developed as a functional food material to treat obesity.Obesity, a condition where excess body fat accumulates to the extent, causes a negative effect on health. Previously, we reported the extract of  Dolichos lablab L. (DLL), also known as hyacinth bean and part of the family Fabaceae, is widely grown in Africa and southern Asia, including in India and China − . 1H-NMR  δ 6.26 , 5.40 , 4.95 , 3.34 , 3.16 , 1.28 , 1.26 , 1.07 , 0.96 , 0.90 , 0.87 , 0.81 ; 13C-NMR  δ 176.82 (C-28), 176.78 (CGlcUA-6), 144.49 (C-13), 123.23 (C-12), 107.39 (CGlcUA-1), 96.09 (CGlc-1), 89.44 (C-3), 79.67 (CGlc-5), 79.18 (CGlc-3), 78.53 (CGlcUA-4), 77.66 (CGlcUA-3), 75.77 (CGlcUA-5), 74.43 (CGlc-2), 73.94 (CGlcUA-2), 71.07 (CGlc-4), 62.51 (CGlc-6), 56.12 (C-5), 48.34 (C-9), 47.34 (C-17), 46.55 (C-19), 42.47 (C-14), 42.07 (C-18), 40.25 (C-8), 39.85 (C-4), 39.00 (C-1), 37.29 (C-10), 34.36 (C-21), 33.54 (C-7), 33.53 (C-29), 32.87 (C-22), 31.15 (C-20), 28.61 (C-23), 26.50 (C-2), 28.58 (C-15), 26.88 (C-27), 24.12 (C-16), 24.01 (C-11), 23.74 (C-30), 18.84 (C-6), 17.81 (C-26), 17.35 (C-24), 15.91 (C-25). 1H-NMR spectrum of CS-IVa exhibited seven methyl signals , and an olefin signal H-12 [5.40 ], and the 13C-NMR spectrum of isolated compound showed one oleanolic acid group . The 1H-NMR spectrum of CS-IVa showed two anomeric proton signals  and the 13C-NMR spectrum of CS-IVa also showed the existence of a glucose and a glucuronic acid group . From these results, the structure of isolated compound was identified as Chikusetsusaponin IVa (CS-IVa), indicating the molecular formula as C42H66O14, through a comparison with reference data [CS-IVa was isolated from water soluble fraction of 70% prethanol A extract . The ext 34.36 C-, 33.54 , is a carrier protein for fatty acid that is primarily expressed in adipocyte. FABP4 is similar to the FABPs family and are thought to enhance the transfer of fatty acids through binding to fatty acid ,33. SpecSaponins are potential biological activity compounds that are insisted in various natural plants. Generally, saponins contains triterpenoid saponin and steroid saponin, that triterpenoids have C30 triterpene skeleton and steroid saponins have C27 steroidal skeleton. Some of saponins, especially triterpenoid saponins, are found as saponin glycosides that attach various sugar molecules with the triterpene skeleton, such as glucose, galactose. It is reported that triterpenoid saponins showed many biological activities, such as anti-microbial, hypolipidemic, immunomodulatory, anti-tumor, anti-diabetic, hypocholesterolemia, anti-coagulant, anti-carcinogenic, hepatoprotective, hypoglycemic, neuroprotective, anti-inflammatory, and anti-oxidant activity ,42,43. SOur recent animal study revealed that DLL-Ex not only repressed hepatic steatosis but also repressed weight gain by HFD . PresentCS-IVa, which is a bioactive component of DLL-Ex (0.54% in DLL-Ex), decreased the expression of genes that are important to the adipogenesis process in 3T3-L1 adipocytes, suggesting that it is the active ingredient that suppresses the adipocyte size, number, and intracellular fat accumulation found after treatment with DLL-Ex. The biological availability concentration of CS-IVa is 16.29 mg/mL in raw seeds, or 1.85 mg/mL in DLL-Ex. These results suggest that CS-IVa and DLL-Ex might be developed into a drug for the treatment of obesity and related diseases.


The results showed that the total radiation intensity of left EX-CA1 was significantly higher than that of left control point (P <0.05) at the whole menstrual cycle, and the difference between right EX-CA1 and right control points was statistically significant before and after menstruation (P <0.05), no statistical difference during menstruation. Previous studies found that the radiation near 15μm was correlated with glucose metabolism. The results of this study showed that there were statistical differences in 10 wavelengths between left EX-CA1 and left control point from 14μm to 18μm, and there were statistical differences in 3 wavelengths on the right side (P <0.05). The left side is more prominent than the right side. The infrared radiation intensity of EX-CA1 decreased gradually with the change of cycle rhythm within menstrual cycle, but there was no statistical difference in this trend. There was no statistical difference in total radiation intensity between the right and left side of EX-CA1. Compared with the control points, the number of different wavelengths between left EX-CA1 and left control point during menstruation was significantly larger than that between right EX-CA1 and right control point (P <0.001). The results indicated that the energy of EX-CA1 was higher than control points. There was no difference in the radiation intensity between the right and left sides of EX-CA1 but there was acupoint laterality compared with nonacupoints. There was no significant rhythmic change in infrared radiation intensity of EX-CA1 during the menstrual cycle.The purpose of this study was to observe the characteristics of infrared radiation spectrum of Zigong acupoint (EX-CA1) within the menstrual cycle of healthy female. We used highly sensitive infrared radiation spectrum detection system and phase-locked amplification technology to detect and analyse the infrared radiation spectrum from 1.5 There is complex interaction between the body surface and the internal organs. The diagnosis and treatment of visceral diseases by body surface abnormalities and the treatment of visceral diseases by body surface stimulation depend on the two-way relationship between body surface and viscera. Compared with other parts of the body surface, the relationship between acupuncture points and viscera is closer. The pathological responding points of the visceral functional abnormalities are mostly located at the corresponding acupoints, and the stimulation at the acupoints generally shows better therapeutic effects than the nonacupoint. Such difference between the acupoints and other parts of the body surface is also called acupoint function specificity which is the primary evidence of the existence of acupoints. Therefore, it has become the focus and hotspot of the basic research in acupuncture and moxibustion –3.The functions of acupoints are closely related to the state of human functionality . In the Menstrual cycle (MC) is the special physiological state in female of childbearing age, and the physiological functions change greatly during menstruation. Therefore, MC is quite suitable for the acupoint-specific research under physiological conditions. Previous studies , 17 haveThe subjects were 32 female college students, with an average age of 24±2 years, an average MC of 31±3 days, and an average duration of 6±1 days per MC. Inclusion criteria were  no abnorμm to 18μm, with 0.25μm step for each band. The test points were the EX-CA1 and their lateral control points about 1 cm apart. According to the National Standard of the People's Republic of China (GBT 12346-2006) “Nomenclature and location of acupuncture points,” the EX-CA1 were located at 4 inches below the umbilicus and 3 inches left and right to the ventral midline. The detection methods and experimental conditions were of the same as those in the previous studies [The detection system of the acupoint infrared spectra was an upgraded version of the original detection system , which w studies –13. A toP<0.05 considered as statistically significant, and paired t-test was used in the comparisons of infrared radiation intensity between the acupoints and the control points. Chi-square test was used to compare the number of wavelengths with differences between acupoints and control points. Variance analysis of mixed design was used to compare the infrared radiation intensity of acupoints and control points at different time points of menstrual cycle.SPSS 21.0 software was used for statistical analysis, with μm to 14.25μm and the second wave peak appearing from 15.25μm to 16.5μm; the infrared radiation of acupoints and control points were mainly concentrated from 7.5μm to 14.25μm , except the comparison between the right EX-CA1 and right control point during menstruation (P=0.181)  .μm. Therefore, further analysis of the wavelength near the 15μm wavelength related to glucose metabolism showed that there were significant differences in multiple wavelengths between EX-A1 and control points (P<0.05). There were statistical differences in 3 wavelengths between right EX-A1 and right control points, among which 2 were before menstruation and the corresponding infrared radiation intensity of right EX-A1 was significantly higher than that of right control points (P<0.05). 1 was after menstruation and the corresponding infrared radiation intensity of right EX-A1 was significantly lower than that of right control pointsOur previous studies  have shol points . There w F  =1.718, P=0.187, time & group: F  =0.368, P=0.673. It indicates that the infrared radiation intensity of the right and left EX-CA1 does not change significantly with the menstrual cycle. . Comparing the radiation intensity of 67 wavelengths (P<0.05). The results indicated small difference between bilateral EX-CA1 at all three periods. It can be considered that the bilateral acupoints are equivalent, consistent with the previous research results [There was no statistical significance in the total radiation intensity between the left and right side of EX-CA1 at three time points in the menstrual cycle  acupoints of the healthy female have obvious acupoint specificity and indicated higher infrared radiation intensity and sugar metabolism than nonacupoint areas. The bilateral EX-CA1 of healthy female are basically equivalent; there is no difference in radiation intensity between the right and left sides of EX-CA1 but there is acupoint laterality comparing with nonacupoints. There is no significant rhythmic change in infrared radiation intensity of EX-CA1 during menstrual cycle. Right EX-CA1 is more related to the physiological changes in menstrual cycle, providing certain reference value for the selection of the acupoints in the treatment of the gynecological diseases with the acupuncture and moxibustion.


As determined in the present investigation, CdCl2, in a concentration-dependent manner, affected the viability of BMMSCs and their cytoskeletons. Exposure to 0.1 or 0.2 µM CdCl2 inhibited osteogenic differentiation of BMMSCs, which was reflected in the down-regulation of osteoblast-related genes ; in suppression of the protein expression of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2); and in decreased ALP activity and capacity for mineralization. Moreover, mRNA microarray was performed to determine the roles of these factors in BMMSCs treated with CdCl2 in comparison to control BMMSCs. As determined with the microarrays, the Wingless-type (Wnt), mothers against decapentaplegic and the C. elegans gene Sam (SMAD), and Janus kinase-Signal Transducers and Activators of Transcription (JAK-STAT) signaling pathways were involved in the effects caused by CdCl2. Moreover, during differentiation, the protein levels of Wnt3a, β-catenin, lymphoid enhancer factor 1 (LEF1), and T-cell factor 1 (TCF1) were reduced by CdCl2. The current research shows that CdCl2 suppresses the osteogenesis of BMMSCs via inhibiting the Wnt/β-catenin pathway. The results establish a previously unknown mechanism for bone injury induced by CdCl2. Cadmium is a common environmental pollutant that causes bone damage. However, the effects of cadmium on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) and its mechanism of action in this process are unclear. Here, we determined the effects of cadmium chloride (CdCl It is r2 on bone formation.As a rigid organ, bone is in a dynamic balance and is self-renewed by the activities of osteoblasts and osteoclasts . Osteocl2. Knowledge of the regulatory Wnt pathway and its role in the osteogenic differentiation of BMMSCs is limited.Various cytokines, growth factors, and signaling pathways, including the Wnt/β-catenin pathway, are essential in the regulation of osteoblast differentiation . There h2. The expression patterns of mRNAs in BMMSCs and CdCl2-treated BMMSCs (CdCl2-BMMSCs) were analyzed. For CdCl2-BMMSCs, changes of signaling pathways that may be involved in the suppression of osteogenic differentiation of BMMSCs induced by CdCl2 were addressed. Further, during osteogenic differentiation, CdCl2 inhibited the Wnt pathway. The results provide an understanding of bone dysfunction induced by CdCl2.The present study investigated the inhibition of osteogenesis in BMMSCs by CdClPrimary BMMSCs were isolated from rat bone marrow and passaged. Cells adhered to the plastic culture dishes and demonstrated a typical small spindle-shaped morphology A. BMMSCs2 cytotoxicity, BMMSCs were incubated with 0, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10, or 20 μM CdCl2 for 24 h. For low concentrations (≤1.0 μM), there were no significant differences in cell viability between the CdCl2-treated and control groups  had no effect on viability of BMMSCs, but high concentrations (≥0.5 μM) reduced the viability of BMMSCs in a concentration-dependent manner  -2,5-diphenyltetrazolium bromide (MTT)  C. Thereff BMMSCs D. These 2 was supplemented into osteogenic induction medium to determine if CdCl2 affected the osteogenesis of BMMSCs. BMMSCs were exposed to 0, 0.1, and 0.2 μM CdCl2 for various times. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and RT-PCR revealed that the osteogenic-related mRNA levels of alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), osterix (OSX), and osteopontin (OPN) in CdCl2-BMMSCs decreased after 3 days and 7 days  analysis showed that pathways related to osteogenic differentiation, including the Wnt signaling pathway, the mothers against decapentaplegic and the C. elegans gene Sam (SMAD) signaling pathway, and the Janus kinase-Signal Transducers and Activators of Transcription (JAK-STAT) signaling pathway, and pathways associated with cell motility were affected by CdCl2 , which showed that the protein expression levels increased  . For rat2, the cytoskeleton of BMMSCs were damaged, which indicated that CdCl2 may affect the osteoblast differentiation. ALP is an early marker during osteogenic differentiation of BMMSCs, and the level of ALP activity can reflect the degree of osteogenic differentiation [ALP, Runx2, OCN, OSX, and OPN were up-regulated in a time-dependent manner. In addition, we selected concentrations of Cd that did not reduce BMMSCs proliferative activity for 7 days as determined by the MTT assay. We used osteogenic media containing various concentrations of Cd for osteogenic induction for 1, 3, and 7 days. After treatment, expressions of the osteogenesis-related genes were lower in the group exposed to CdCl2 relative to the control group. Further, the inhibitory effect of CdCl2 on osteogenic differentiation of BMMSCs was reflected in lower ALP activity, lower protein levels of ALP and Runx2, and less calcification.BMMSCs derived from rats show surface markers whose characteristics indicate that they can differentiate into osteoblasts. The changes of cytoskeleton can affect the osteogenic differentiation of BMMSCs . In our ntiation ,33. Runxntiation , it can ntiation . OSX is ntiation . In addintiation ,38. In t2 damages Wnt signaling and thereby affects osteogenesis.Bone remodeling is an indispensable condition for maintaining bone balance, and bone formation is destroyed in the pathological state of bone. The imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption is an important cause of reduced bone mass and altered bone structure . The for2-BMMSCs, and performed mRNA microarray to find altered genes. The bioinformatics analysis indicated that Wnt/β-catenin signaling is involved in the suppression of osteogenesis induced by CdCl2. Therefore, we examined expression of proteins involved in the Wnt/β-catenin pathway, including Wnt3a, β-catenin, LEF1, and TCF1. The expressions of these were lower in CdCl2-BMMSCs than in controls, and the proteins in the group treated with 0.2 μM Cd were lower than those of the group treated with 0.1 μM CdCl2. Activation of the Wnt/β-catenin signaling pathway is related to the osteogenic differentiation of BMMSCs, but, in this study of BMMSCs, the expression of proteins in the Wnt/β-catenin pathway decreased after their treatment with CdCl2. CdCl2 affected activation of the Wnt/β-catenin pathway. After the treatment of Wnt3a, the protein levels of Wnt3a, β-catenin, LEF1, and TCF1 and the degrees of osteoblast differentiation of BMMSCs were rescued. Thus, inhibition of osteogenic differentiation of BMMSCs is associated with inhibition of activation of the Wnt/β-catenin pathway by CdCl2.The canonical Wnt/β-catenin pathway is triggered when the Wnts are combined with Wnt ligands. The binding of Wnt with Frizzled and LRP5/6 initiates to form a complex of proteins, which subsequently recruit the Axin complex. The protein complex, which consists of axin, glycogen synthase kinase 3 (GSK3), and the adenomatosis polyposis coli (APC) protein, stabilizes β-catenin. β-catenin entering into the nucleus activates many target genes, such as the lymphoid enhancer-binding factor/T cell factor (LEF/TCF) transcription factor family, which promotes the expression of many osteoblast-related genes ,45. In t2 incubator with changes of medium every other day. When the confluence reached 80–90%, BMMSCs were harvested by treating them with 0.25% trypsin-EDTA  and were passaged for further culture. BMMSCs, in passages 3–5, were used for experiments. All of the experiments and procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University .Primary BMMSCs were harvested from 3-week-old Sprague-Dawley (SD) rats as described previously . Brieflyl-glucose, 1% penicillin/streptomycin, and 1% HEPES.BMMSCs were induced to adipogenic differentiation in Dulbecco’s modified Eagle medium-High glucose (DMEM-HG)  supplemented with 10% FBS, 1 μmol/L dexamethasone, 100 μmol/L indomethacin , 0.5 mmol/L 3-isobutyl-1-methylxanthine , 5 μg/mL insulin , 1% BMMSCs were induced to osteogenic differentiation in DMEM-HG medium supplemented with 10% FBS, 10 nmol/L dexamethasone , 10 mmol/L β-glycerol phosphate , 50 μg/mL ascorbic acid , 1% L-glucose, 1% penicillin-streptomycin, and 1% HEPES.2, at concentrations of 0, 0.1, or 0.2 μM, was added into the osteogenic induction medium before replacing the growth medium, once BMMSCs in the culture plates reached 60% confluence.For  exposure of BMMSCs during osteogenic differentiation, CdClExpression of surface markers was assessed by flow cytometry using monoclonal antibodies for CD44 and CD90 (positive BMMSC markers) and CD45 (negative BMMSC marker) . BMMSCs at passage 3 were suspended in PBS and incubated with each antibody at a concentration of 0.5 μg/mL, along with unstained BMMSCs and isotype controls.4 cells per well) were seeded in 96-well plates. 24 h later, BMMSCs were exposed to 0, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 20 μM or to 0, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 μM CdCl2 for 24 h or for 7 days. After treatment, MTT reagent, diluted to a concentration of 0.5 mg/mL with osteogenic-inducing medium, was added to the plates, and they were incubated at 37 °C for 4 h. Next, the MTT was replaced by dimethyl sulfoxide , and preparations were incubated at 37 °C for another 15 min, and then the plates were agitated softly for 15 min. The absorbance of each well was recorded at 490 nm by an Infinite M200 Pro instrument . Each assay was repeated at least three times independently.Cell viability was assessed by 3--2,5-diphenyltetrazolium bromide (MTT)  following the manufacturer’s instructions. BMMSCs  were added into 96-well after cells were attached to the well, and then incubated for two hours in an incubator. Subsequent experiments were carried out in strict accordance with the manufacturer’s instructions. Finally, photos were taken by fluorescence microscope.For the 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay, treated BMMSCs cells were seeded into 96-well plates for EdU assay to assess the proliferation ability, and each well contained 2 × 104 /well) were cultured on coverslips (In Vitro Scientific) and treated with 0, 0.1, or 0.2 μM CdCl2 for 24 h, after which cells were fixed with 4% paraformaldehyde for 30 min. After treating of cells with 0.1% Triton X-100, their cytoskeletons were stained with anti-α-tubulin antibody  or anti-F actin antibody , together with respective fluorescence secondary antibodies (Alexa Flour 488 and Alexa Flour 546) . Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI)  for 5 min. Images were visualized and captured with a Nikon E800 confocal microscope .Cells (7 × 104 /well) were seeded in 24-well plates, and adipogenesis was induced for 14 days. Cells were then fixed with 4% formalin solution for 15 min and incubated with 0.5% Oil Red O  for 40 min. After washing the plates with 60% isopropanol, images were made at 10× magnification using an optical microscope .Cells  substrate solution, and the absorbance was read at 405 nm with an Infinite M200Pro . The ALP activity was normalized against protein concentration measured with BCA Protein Assay Kits .ALP activity was determined with Sensolyte® pNPP Alkaline Phosphatase Assay Kits  according to manufacturer’s instructions. BMMSCs were seeded in 96-well plates at a density of 1 × 104 cells/well in growth culture medium. Until the confluence reached 60%, BMMSCs were exposed to CdCl2 at concentrations of 0, 0.1, or 0.2 μM with or without the treatment of Wnt3a and induced to differentiate into osteoblasts by osteogenic induction medium. After 10 d of induction, the cells were fixed with 4% formaldehyde and 5% citrate in acetone at room temperature for 30 s. The fixed cells were washed with PBS and incubated with 0.2% naphthol AS-BI and 0.2% diazonium salt at room temperature for another 15 min. After washing the plates with PBS, images were taken at 10× magnification under an optical microscope .Leukocyte Alkaline Phosphatase Kits  were used for ALP staining according to the manufacturer’s instructions. BMMSCs were seeded in 24-well plates at a density of 7 × 104 cells/well and were exposed to CdCl2 at concentrations of 0, 0.1, or 0.2 μM with or without the treatment of Wnt3a, and induced to differentiate into osteoblasts by osteogenic induction medium for 14 days. The cells were then washed with PBS, fixed with 10% formaldehyde at room temperature for 10 min, and incubated with 40 mM alizarin red S  solution at room temperature for 20 min. After discarding the working solutions and washing the plates with PBS 4 times, images were made at 10× magnification under an optical microscope .BMMSCs were seeded in 24-well plates at a density of 7 × 10−ΔΔCt method [Total RNA was isolated from cells with TRIzol reagent  according to manufacturer’s protocol. The purity and concentration of total RNA was assessed with a NanoDrop 2000 . Reverse transcription was accomplished using Prime Script™ RT Reagent Kits with gDNA Eraser  with 1 μg of RNA according to the manufacturer’s instructions. qRT-PCR was performed with an ABI7900 Fast Real-Time System  using SYBR Premix Ex Taq™ Kits . GAPDH was used as an internal standard, and the relative expressions of genes were calculated by the 2t method . Primersw/v agarose gels. The intensities of bands were quantified with ImageJ software.Total RNA (1 μg) was treated with 10 U of RNase R  in 1× RNase R reaction buffer in a total volume of 10 μL. The mixture was incubated at 37 °C for 1 h. Total RNA (1 μg) was transcribed into cDNA by HiScript II Q RT Supermix (Vazyme Biotech). The PCR reactions were evaluated by checking the PCR products on 2% Cell were lysed with RIPA  as described previously , and pro2 at a concentration of 0.2 μM in osteogenic differentiation medium and each group included one sample that mixed by three parallel samples. Afterwards, cells were lysed, and total RNA was extracted for analysis of whole genome expression. Osteogenic differentiation medium without Cd was used as a control. The RNA samples were labeled and hybridized with 1.65 μg of Cy3-labeled cRNA using Gene Expression Hybridization Kits  in a Hybridization Oven , according to the manufacturer’s instructions. After 17 h of hybridization, slides were washed in staining dishes  with Gene Expression Wash Buffer Kits . Samples were scanned with an Agilent Microarray Scanner  with the default settings: Dye channel: Green, Scan resolution = 3 μm, PMT 100%, 20 bit. Data were extracted with Feature Extraction software 10.7 . Specific representations of heatmap and scatter plot were analyzed by bioinformatics using “pheatmap” package. The gene oncology was analyzed by bioinformatics using “ggplot2” package.BMMSCs were cultured with CdClp < 0.05, or (**) for p < 0.01.All data values were expressed as means ± standard deviations (SD). Graphpad 7.0 was applied for statistical analyses. One-way analysis of variance (ANOVA) was used for comparisons of means among multiple groups, and a multiple-range least significant difference (LSD) was used for inter-group comparisons. All statistical analyses were performed with SPSS 19.2, and the data were marked with (*) for 2 suppresses the osteogenic differentiation of BMMSCs and address the effects of CdCl2 on mRNA microarray. In this process, CdCl2 caused decreases of Wnt3a, β-catenin, LEF1, and TCF1, which are involved in the Wnt pathway, which regulates differentiation of BMMSCs. Thus, in BMMSCs exposed to CdCl2, the Wnt/β-catenin pathway may regulate inhibition of osteogenic differentiation (2-induced bone damage and have public health implications for developing strategies to reduce CdCl2 exposure and thereby to mitigate its harmful effects.In summary, our investigations show that CdClntiation . Our fin