Aims. Nuclear translocation of β-catenin is common in many cancers including esophageal squamous cell carcinoma (ESCC). As a mediator of Wnt signaling pathway, nuclear β-catenin can activate many growth-related genes including CCN1, which in turn can induce β-catenin translocation. CCN1, a matricellular protein, signals through various integrin receptors in a cell-dependent manner to regulate cell adhesion, proliferation, and survival. Its elevation has been reported in ESCC as well as other esophageal abnormalities such as Barrett's esophagus. The aim of this study is to examine the relationship between CCN1 and β-catenin in ESCC. Methods and Results. The expression and correlation between CCN1 and β-catenin in ESCC tissue were examined through immunohistochemistry and further analyzed in both normal esophageal epithelial cells and ESCC cells through microarray, functional blocking and in situ protein ligation. We found that nuclear translocation of β-catenin in ESCC cells required high level of CCN1 as knockdown of CCN1 in ESCC cells reduced β-catenin expression and translocation. Furthermore, we found that integrin α11 was highly expressed in ESCC tumor tissue and functional blocking integrin α11 diminished CCN1-induced β-catenin elevation and translocation. Conclusions. Integrin α11 mediated the effect of CCN1 on β-catenin in esophageal epithelial cells. While the incidence of most cancers is declining, esophageal cancer has been continuing its march as the fastest growing malignancy in the Western world . Nearly β-catenin. In normal epithelium, β-catenin is phosphorylated by glycogen synthase-3β and casein kinase I to keep its level low, and any loose β-catenin is degraded and recycled through ubiquitination. However, if for any reason β-catenin is unphosphorylated, it can accumulate in the cytoplasm or move into the nucleus where it becomes a mediator of Wnt signaling pathway, which could lead to transcriptional activation of several oncogenes and promote cancer [β-catenin translocation has been found in many types of cancers including ESCC.Normal esophagus is covered on the lumen side by stratified squamous epithelium in which cells are connected to each other through multiple intercellular locks, such as tight junctions, adherens junctions and desmosomes. one of the components of adherens junctions is e cancer , 3. For α and β integrin heterodimer has its own binding specificity and signaling properties, which are further diversified through alternative splicing and/or posttranslational modifications [α6β1, α2β1, αDβ2, αMβ2, αvβ3, αvβ5, and αIIbβ3 [αVβ3 in endothelial cells [αIIbβ3 in platelets [α6β1 in fibroblasts [αMβ2 in monocytes [α6β1 and heparan sulfate proteoglycans, cell migration through αvβ5, and cell proliferation through αvβ3 [κB [αvβ3.CCN1 (formerly known as Cyr61 or IGFBP10) is the first member of the CCN family , a groupications . To dateIIbβ3 –14. For al cells , throughroblasts , and thronocytes . On the  αvβ3 , it was β3 [κB  and MAPKβ3 [κB  in breasβ-catenin translocation in several cell systems including glioma [β-catenin translocation can in turn promote CCN1 expression through Wnt signaling [Overexpression of CCN1 has been found to activate g glioma , non-smag glioma , and gasg glioma , whereasignaling . Over thignaling , 22, 23,ignaling , Barrettignaling . Moreoveignaling , mediateignaling , suggestHuman esophageal squamous epithelial Het-1A cells  and esophageal squamous carcinoma OE21 cells  were maintained in BEGM plus supplements and RPMI plus 10% serum , respectively. To knock down CCN1, cells were transfected with shRNA against CCN1 in pRFP-C-RS vector , which constitutively expresses red fluorescent protein (RFP) in the transfected cells. Prior to each experiment, cells were serum-starved overnight and then treated with either vehicle (control) or recombinant human CCN1 protein  at 100 ng/mL for an indicated period of time.Total RNA was extracted from Het-1A cells using Trizol reagent  and further purified using RNeasy kit . Probe was synthesized through reverse transcription using a GEArray kit  and labeled with Biotin-16-dUTP  following the manufacturer protocol. We used GEArray Q series microarray membranes (SABiosciences) that contain 96 cDNA sequences for extracellular matrix and adhesion molecules to screen integrins. Briefly, the microarray membrane was prehybridized with the denatured salmon sperm DNA solution (Sigma-Aldrich) in a hybridization oven  at 60°C for one hour and then hybridized with the labeled probe at the same temperature overnight. The membrane was washed in 2X SSC plus 1% SDS for 2 × 15 min at 60°C and then in 0.1X SSC plus 0.5% SDS for additional 2 × 15 min. The signal was developed using a chemiluminescent kit (SABiosciences), captured on X-ray film, and quantified using ImageQuant 3.3 software .μg RNA was used as templates to generate cDNA through reverse transcription reaction in MyCycler  following standard protocol (25°C/10 min-55°C/30 min-85°C/5 min-4°C/5 min). β-actin was included as internal control. Real-time PCR was performed in iCylcer (Bio-Rad) following two-step manufacturer protocol. Data were analyzed using the ΔΔCt method based on at least 3 independent experiments. Briefly, ΔCt was calculated by subtracting the Ct value of β-actin from the Ct value for each gene of interest; then ΔΔCt was calculated by subtracting the ΔCt of the control from the ΔCt of the treatment for each gene of interest; finally the fold change was calculated using the formula: Fold Change =2t); mouse anti-phosphoserine/threonine ; mouse anti-active-β-catenin ; rabbit anti-β-catenin (Abcam); mouse anti-β-actin (Sigma-Aldrich).Protein isolation and Western blotting were done as described in our previous studies , 28. Briμg total protein was incubated with 1 μg rabbit anti-β-catenin antibody for 2 hours at 4°C on rocker prior to addition of agarose A beads (Millipore), and then the total mixture was incubated again overnight on rocker at 4°C. Beads were precipitated and washed three times with lysis buffer prior to final resuspension in 2X sample buffer. Samples were boiled for 5 minutes and then separated on 7% SDS-PAGE gels, transferred to nitrocellulose membrane, and analyzed by Western blotting as described.For immunoprecipitation, as described in our previous studies , 28, 100β-catenin antibody  for 2 hours to detect β-catenin expression and localization, and control slides were incubated with a preimmune serum to monitor the specificity of the antibody. FITC-conjugated secondary antibody  was used to detect the signal.Cells were grown in Lab-Tek chamber slides . After an appropriate treatment as indicated, cells were fixed for 10 min in cold acetone and incubated in serum-free Protein Block  for 30 min to eliminate possible nonspecific antibody binding. Then, some slides were probed with mouse anti-β-catenin (1 : 500), and goat anti-integrin α11 (1 : 50). The LSAB + kit (Dako) was used to develop a signal.Human esophageal tissue slides  were deparaffinized in Histoclear solution  and rehydrated in a series of ethanol. Antigens were retrieved by repeatedly microwave boiling in 0.01 M sodium citrate buffer (pH 6.0). Endogenous peroxidase was blocked in Dako reagent for 10 minutes. Slides were then incubated with the following primary antibodies at 4°C overnight: rabbit anti-CCN1 (1 : 200), rabbit anti-in situ using the duolink PLA-probe detection system . Briefly, cells were cultured on cover slips, fixed in cold acetone, blocked and incubated in a primary antibody as described above for cell staining, followed by 2-hour incubation in rabbit and goat PLA PLUS and PLA MINUS probes, 15-minute hybridization, 15-minute ligation, 90-minute amplification, and 60-minute detection according to the manufacturer protocol. Goat anti-integrin α11 was coupled with rabbit anti-CCN1. To maximize signal strength, samples were incubated in primary antibody overnight (4°C) and PLA probes were used at a 1/2.5 dilution. All steps after primary antibody incubation were carried out at 37°C. Nuclei were counterstained with Hoechst 33342 (blue). This system uses special secondary antibodies containing a short DNA strand (PLA probes), which can hybridize and form a circularized oligo that can be amplified and detected as a discrete fluorescent dot  when two opposite probes come in close proximity (less than 40 nm) [Protein-protein interactions were analyzed n 40 nm) .α11 at 50 μg/mL in fresh basal medium for 3 hours. Recombinant CCN1 protein (100 ng/mL) was then added to the medium, and cells were incubated again overnight. Nontreated cells and CCN1-treated cells cultured in the absence of antibody pretreatment were included as negative and positive control respectively. The effect of integrin blockage on CCN1 signaling was assessed by β-catenin nuclear translocation through immunofluorescence microscopic analysis as well as by β-catenin activation through immunoprecipitation and Western blot analysis as described above.Cells were serum starved overnight in basal medium. Where indicated, cells were then preincubated with either diluted preimmune serum (control) or the antibody against the extracellular domain of integrin P < 0.05 was considered significant.All experiments were performed at least in triplicates. All numerical data were analyzed by single-classification one-way ANOVA, and β-catenin was neatly localized to the cell membrane , β-catenin level was decreased and its distribution was more confined to the cell membrane . Out of 16 α- and 8 β-integrins examined (P < 0.01). Since integrin β5 is a known receptor of CCN1 [α11 was chosen for further investigation.Based on what we discussed previously, that is, CCN1 signals through different integrin receptors in a cell-dependent manner, we were interested to know what integrin(s) mediates CCN1-activated examined , only tw of CCN1 , 15, intα11, we used a novel protein-protein interaction technique (DUOLINK system). Nowadays, more and more studies use this technology to visualize in situ ligand-receptor binding [α11. On the other hand, blocking CCN1 with excessive antibody diminished the signal, confirming the specificity of the interaction  was increased by 109.1% (P < 0.01), which was confirmed by reprobing the blots for phosphorylated serine/threonine , which can contribute to cancer development. In addition, β-catenin translocation can also destabilize intercellular connection, facilitating cancer metastasis. CCN1 just happens to be one of Wnt signaling targets. Therefore, these two molecules can form a CCN1-β-catenin-CCN1 loop to amplify the signal, thereby to accelerate wound healing or to promote oncogenesis, depending on the context. Further studies on the relationship of these two proteins would be very rewarding.In our previous study, we found that CCN1 can induce a transient al cells  and therα11 is required for CCN1 to induce β-catenin translocation in esophageal epithelial cells. ITGA11, the gene encoding integrin α11 protein, was cloned and sequenced a decade ago by two independent groups [α11 in adult tissue and found that it was mainly expressed in muscular-rich organs such as uterus, heart, and skeletal muscle. They also identified moderate expressions of integrin α11 in the gastrointestinal tract, including the stomach, the intestine, and the colon, although the esophagus was not examined. Up to date, there are less than 20 studies on ITGA11 published, most of which focus on mesenchymal tissues, where integrin α11 mediates such processes as migration of embryonic fibroblasts [α11 in epithelial cells have never been fully investigated. In this study, we showed that ITGA11 is normally expressed at minimal levels in esophageal epithelial cells, but it can be dramatically upregulated by CCN1, a feedback mechanism that CCN1 is known to employ for some of its receptors in other tissues. Consistent with the possible role of integrin α11 as a CCN1 receptor, a direct interaction between these two proteins was demonstrated, and blocking integrin α11 function prevented CCN1 from eliciting its downstream signals in these cells. CCN1 functions through different integrin receptors in a tissue-dependent manner. Out of 26 known integrins, at least 8 have been found to mediate CCN1 actions in various cells. The context dependency of CCN1 is reflected in its receptor selection. Here we showed that integrin t groups , 43. Botroblasts , tooth eroblasts , and corroblasts . On the β-catenin in ESCC require high level of CCN1, and integrin α11, which has so far been studied mostly in mesenchymal tissues, mediates this process. Given the importance of nuclear β-catenin in cancer progression [In conclusion, our study has demonstrated that the elevation and translocation of gression , 48, pargression , 50 and gression , 52, our


HS7 is an active fraction extracted from Taiwanofungus camphoratus, which had been widely used as complementary medicine for Taiwan cancer patients in the past decades. In this study, we demonstrated the effects of HS7 on the growth inhibition, apoptosis induction, and Wnt/β-catenin signaling suppression in human colon cancer cells. HS7 significantly inhibited proliferation of HT29, HCT116, and SW480 colon cancer cells in a dose- and time-dependent manner. The apoptosis induction was evidenced by DNA fragmentation and subG1 accumulation, which was associated with increased Bax/Bcl-2 ratio, activation of caspase-3 and cleavage of PARP. By using Tcf-dependent luciferase activity assay, HS7 was found to inhibit the β-catenin/Tcf transcriptional activities. In addition, HS7 strongly suppressed the binding of Tcf complexes to its DNA-binding site shown in electrophoretic mobility shift assay. This inhibition was further confirmed by the decreased protein levels of Tcf-4 and β-catenin. The β-catenin/Tcf downstream target genes, such as survivin, c-myc, cyclin D1, MMP7, and MT1-MMP involved in apoptosis, invasion, and angiogenesis were also diminished as well. These results indicate that Taiwanofungus camphoratus may provide a benefit as integrative medicine for the treatment of colon cancer.Aberrant activation of Wnt/ As the diet and life styles of the industrialized Western countries are adopted globally, the incidence of colorectal cancer (CRC) is increasing worldwide . It becoβ-catenin signaling pathway plays an important role in both embryogenesis and tumorigenesis. Most sporadic colorectal cancer (CRC) and familial adenomatous polyposis (FAP) have mutated adenomatous polyposis coli (APC) gene. Normal APC protein could downregulate the Wnt signaling pathway through its binding to β-catenin and Axin, but most mutated APC proteins in colorectal tumors fail to inhibit Wnt signaling, leading to the overproliferation of tumor cells [β-catenin signaling pathway plays an important role in several common cancers including liver, lung, and colorectal cancers [β-catenin indicates that the binding of Wnt protein to its transmembrane-frizzled receptor could stabilize β-catenin by inhibiting the activity of the serine/threonine kinase GSK-3β [β kinase associated with β-catenin in a multiprotein complex, which is composed of the adenomatous polyposis coli tumor suppressor protein APC, axin, conductin, and protein phosphatase 2A. GSK-3β phosphorylates the NH2 terminus of β-catenin and thereby induces its degradation via the ubiquitin-proteasome pathway [β is inhibited by disheveled proteins, and, therefore, β-catenin could escape from phosphorylation. The unphosphorylated β-catenin accumulates in the cytoplasm and translocates into the nucleus. In the nucleus, β-catenin plays as a transcriptional activator in association with T-cell factor/lymphocyte enhancer factor (Tcf/Lef) family and other transcriptional cofactors [c-myc, cyclin D1, and survivin were subsequently activated [β-catenin signaling is believed to be critical to the early stages of human sporadic colorectal carcinogenesis [β-catenin signaling pathway had been proposed as a target for drug discovery [β-catenin signaling in colorectal cancer [The Wnt/or cells . Recent  cancers . The cure GSK-3β . The GSK pathway . In respofactors , by whicctivated , 9. Dysrogenesis . The Wntiscovery . Severall cancer , 13. To T. camphoratus (syn. Antrodia camphorata) is a Ganoderma-like fungus, which belongs to family of Polyporaceae, Basidiomycota, and grows in a unique host, the endemic perennial tree Cinnamomun kanehirae (Bull camphor tree) in Taiwan. In the past fifteen years, T. camphoratus has been shown to exert antioxidant [T. camphoratus exerted multiple biological activities against various cancer cells in the aspects of antiproliferation, apoptosis induction, anti-invasion, and antimetastasis [T. camphoratus by silica gel chromatography, which appears to possess significant antiproliferative activities against a panel of human cancer cell lines, including lung adenocarcinoma cells (CL1-0), prostate cancer cells (PC3), and hepatocellular carcinoma cells (Hep3B & Huh7). However, the anticancer effects of HS7 on colon cancer cells and the underlying molecular mechanisms remain unclear.ioxidant , anti-inioxidant , and immioxidant  in a vartastasis –19. ReceT. camphoratus could inhibit the proliferation of human colon cancer cells as well as the Wnt/β-catenin signaling pathway. These data may provide important insight for the use of T. camphoratus as a potential complementary therapeutic agent for colorectal cancer.The aim of this study was to address whether the active fraction HS7 isolated from β-catenin antibodies were purchased from Chemicon International, Inc. . Anti-Bcl2, anti-Bax, anti-β-catenin, and anti-survivin antibodies were purchased from Santa Cruz Biotechnology, Inc. , and anticleaved caspase-3 and anti-PARP antibodies were purchased from Cell Signaling Technology, Inc. . The plasmids TOPflash and FOPflash were purchased from Upstate Biotechnology, Inc. . NE-PER nuclear extraction reagent was purchased from Thermo Fisher Scientific Inc. . The electrophoretic-mobility shift assay (EMSA) kits were purchased from Panomics, Inc. . Propidium iodide (PI) and Sulforhodamine B (SRB) were purchased from Sigma-Aldrich Co. . Trizol reagent, fetal bovine serum (FBS), and trypsin-EDTA (T/E) were obtained from Gibco BRL Life Technologies . The QIAGEN One-tube RT-PCR system and Annexin-V/FITC Apoptosis Kits were obtained from Qiagen Inc.  and R&D Systems, Inc. , respectively. Lipofectamine was purchased from Invitrogen . An enhanced chemiluminescence (ECL) kit was purchased from Amersham Corp. . Anti-Tcf-4 and anti-active-2. The cells were regularly seeded into T-75 flasks with media changes every 3 days. For experiments, cells were grown to 70% confluence, trypsinized with 0.25% trypsin-EDTA, and then replated at a density of 105 per ml in cultured media. As an untreated solvent control, cells were treated with DMSO at a final concentration of <0.05%.The HT-29, HCT116, and SW480 human colorectal cancer cell lines were obtained from American Type Culture Collection (ATCC). All of the cell lines were maintained in RPMI-1640 culture medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin-Glutamine (100x) liquid (Gibco). Cells were cultured in an incubator with humidified air at 37°C with 5% COT. camphoratus (voucher number TC-2004-09-001) was cultivated and provided by Well Shine Biotechnology Development Co. . Briefly, air-dried ground powder of the cultivated T. camphoratus was extracted exhaustively with n-hexane and then separated by silica gel chromatography. Eight fractions (HS1 to HS8) were obtained, and the seventh (HS7) appeared to be the most active fraction on growth inhibition against a screening panel of cancer cell lines.The fruiting body-like μL of 0.4% (w/v) SRB prepared in 1% (v/v) acetic acid for 10 minutes at room temperature. The plates were washed 4 times with 1% acetic acid to remove unbound dye and then air dried until no moisture was visible. The bound dye was dissolved in 20 mmol/L Tris base (100 μL/well) for 5 minutes on a shaker. Optical densities were measured on a microplate reader  at 562 nm.According to the method described by Vichai and Kirtikara , sulforh6 cells/mL) with different doses of HS7 for 48 hours, cells were harvested and washed twice with 2 mL of ice-cold phosphate-buffered saline (PBS). Cells were then incubated with 100 μL of HEPES buffer containing 2 μL of fluorescein isothiocyanate- (FITC-) conjugated annexin V and 2 μL of propidium iodide (PI) for 15 minutes. After washing the cells of excess reagents, 400 μL of binding buffer was added. The stained cells were immediately analyzed with a FACSCalibur flow cytometer. Approximately 10,000 counts were made for each sample. The percentage distributions of apoptotic cells were calculated by CellQuest software .Apoptosis was determined by using a commercially available annexin V apoptosis detection kit and flow cytometry. Following treatment of the colon cancer cells /Triton X-100 (0.1% v/v) staining solution with RNase A (200 μg/mL) in the dark and then analyzed by a flow cytometer. Approximately 10,000 counts were made for each sample. The percentage distributions of apoptotic cells were calculated by CellQuest software.Propidium iodide (PI) staining and flow cytometry were used to determine the cell cycle distribution. Cells were treated with different doses of HS7 for 48 hours at 37°C. The treated cells were washed with 5 mL PBS and then harvested using 0.25% Trypsin EDTA and suspended in RPMI at a concentration of 1 × 10μg of DNA was loaded into each well. The agarose gels were then run at 50 V for 90 minutes in Tris-borate/EDTA electrophoresis buffer (TBE). The bands were visualized under ultraviolet (UV) light and photographed.The HT29 cells treated with different doses of HS7 were harvested, washed twice with PBS, and then lysed in 100 mL of lysis buffer for 3 hours at 56°C. The cells were treated with RNase A for another hour at 37°C. The DNA was extracted by phenol: chloroform: isoamyl alcohol  and analyzed by 1.8% agarose gel electrophoresis. Approximately 2 μg of TOPflash or FOPflash plasmids. To normalize transfection efficiency, cells were cotransfected with 0.2 μg of the internal control reporter encoding Renilla reniformis luciferase driven under the TK promoter. After transfection, cells were incubated in medium with or without HS7 (2.5–25 μg/mL) for 48 hours and then lysed with reporter lysis buffer at harvest. Luciferase activity was determined by using the Dual-Luciferase Assay System kit according to the manufacturer's protocol. The experiments were performed in triplicate, and the results were reported as folds of induction compared with control group after normalization of transfection efficiency.HT-29 cells were plated in 6-well plates, grown to 80%–90% confluence, and transiently transfected with the plasmids of TOPflash and FOPflash, respectively. TOPflash has 3 copies of the Tcf/Lef binding sites in the upstream of a thymidine kinase (TK) promoter and the firefly luciferase gene. FOPflash has mutated copies of Tcf/Lef sites and is used as control for measuring nonspecific activation of the reporter. All transfections were performed with Lipofectamine and 1.8 μg/mL of HS7 for 48 hours. Afterwards, cells were fixed in 2% paraformaldehyde for 10 minutes at room temperature, permeabilized with 0.1% Triton X-100 in 0.01 M PBS pH 7.4 containing 0.2% bovine serum albumin, air dried, and rehydrated in PBS. Cells were then incubated with a rabbit polyclonal antibody against β-catenin (1 : 500) diluted in PBS containing 3% bovine serum albumin for 2 hours at room temperature. Negative controls were performed by omitting the primary antibody. After washing with PBS twice for 10 minutes, an antirabbit IgG PE-conjugated secondary antibody (1 : 500) diluted in PBS was added and incubated for 1 hour at room temperature. Cells were then washed by PBS and then mounted using Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) to counterstain DNA. Cells were observed using a Zeiss Axiophot fluorescence microscope. Microphotographs were acquired using an AxioCam MRc digital video camera and an Axiovision Zeiss software .For immunofluorescence analysis, cells were plated in 6-well chamber slides for 24 hours before treatment with 10 To obtain nuclear extracts lysates, the HS7-treated and -untreated cells were washed twice with ice-cold 1 × PBS and lysed using NE-PER nuclear extraction reagents. The protein concentrations of all the extracts were determined by the bicinchoninic acid protein assay  with bovine serum albumin as the standard. Nuclear extracts were stored at −70°C until used.μg of nuclear extract was used for EMSA according to the manufacturer's instructions. Nuclear extracts containing equal amounts of protein for each sample were incubated with poly (dI-dC) (1 μg/μL) for 5 minutes, followed by the addition of binding buffer and biotinylated oligo (10 ng/μL). After electrophoresis, gels were transferred to nylon membranes. Transferred oligos were immobilized by UV crosslinking for 3 minutes. For detection of bound oligos, membranes were blocked using blocking buffer followed by the addition of Streptavidin-HRP, and blots were developed by ECL according to the manufacturer's instructions. Commercially available biotinylated oligonucleotide encoding the Tcf/Lef motif 5′-CCTTTGATCTTCCTTTGATCTT-3′ was used as a canonical probe .Two to five survivin, c-myc, cyclinD1, MMP7, and MT1-MMP were quantified by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, using GAPDH mRNA as an internal standard. HT29 cells (1 × 106 in 10 mL medium) were plated in 100 mm tissue culture dishes. After plating for 24 hours, HT29 cells were treated with different doses of HS7 for 48 h. At harvest, cellular RNA was extracted with a TRIzol RNA isolation kit as described in the manufacturer's manual. RNA concentration and purity were determined based on measurement of the absorbance at 260 nm and 280 nm. After adding RNase inhibitor (20 U), the total RNA was stored at −70°C. The sense and antisense primer sequences used were as follows: The expression levels of survivin: 5′-ATGGGTGCCCCGACGTTGC-3′, 5′-TCAATCCATGGCAGCCAGCTG-3′;c-Myc: 5′-CGAGCTGCTGGGAGGAGACAT-3′, 5′-AGCCGCCCACTTTTGACAGG-3′;cyclinD1: 5′-CTACAGGGGAGTTTTGTTGA-3′, 5′-GGTAGTAGGACAGGAAGTTG-3′;MMP7: 5′-CCTACAGGATCGTATCATAT-3′, 5′- GGAACAGTGCTTATCAATTC-3′;MT1-MMP: 5′-TCGGCCCAAAGCAGCAGCTTC-3′, 5′-CTTCATGGTGTCTGCATCAGC-3′;GADPH: 5′-CAAAAGGGTCATCATCTCTGC-3′, 5′-GAGGGGCCACACAGTCTTC-3′,survivin, c-Myc, cyclinD1, MMP7, MT1-MMP, and GAPDH  genes expression, using the FailSafe PCR system . The reactions took place in a volume of 50 μL, containing a final concentration of 50 mmol/L Tris-HCl, (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MnCl2, 0.2 mmol/L dNTP, 2 U of Taq DNA polymerase, and 50 pmol of primers. After initial denaturation for 2 minutes at 95°C, 26 cycles of amplification  were performed, followed by a 7-minute extension at 72°C.respectively. From each sample, 250 ng of RNA was reverse-transcribed, using 200 U of SuperScript II RNase-H reverse transcriptase, 20 U of RNase inhibitor, 0.6 mM of dNTP, and 0.5 mg/mL of oligo (dT). PCR analyses then were performed on the aliquots of the cDNA preparations to detect μL aliquot from each PCR reaction was electrophoresed in a 1.8% agarose gel containing 0.2 mg/mL ethidium bromide. The expression level of survivin was normalized by the corresponding GAPDH signal from the same gel and expressed as the ratio of survivin/GAPDH.A 10 μL of cold lysis buffer to the cell pellets on ice for 30 minutes. This was followed by centrifugation at 10,000× g for 30 minutes at 4°C. Western blotting was performed according to the method of Yeh and Yen [β-catenin (1 : 1000), Tcf-4 (1 : 1000), survivin (1 : 1000), and β-actin (1 : 5000). After incubation with primary antibodies, the membrane was washed with TBST 3 times. The membrane was then incubated with horseradish peroxidase-labeled secondary antibody for 45 minutes at room temperature and washed with TBST 3 times. Final detection was performed with enhanced chemiluminescence (ECL) Western blotting reagents.At harvest, the HS7-treated and -untreated cells were washed twice with PBS (pH 7.0), and the total proteins were extracted by adding 200  and Yen . The memt-test as appropriate. “∗” indicates that the values are significantly different from the control .Each experiment was performed in triplicate and repeated 3 times. The results were expressed as means ± SD. The significance of differences between HS7-treated and vehicle-treated control groups was analyzed by μg/mL. In contrast, the IC50 for HT-116 and SW-480 cells were 15.1 and 19.4 μg/mL, respectively. As shown in μg/mL for 48 h, the amount of annexin V-FITC-positive cells were 68.7 ± 1.1%, 39.6 ± 1.2%, and 33.5 ± 1.7% in HT29, HCT116, and SW480 cells, respectively, compared to control (less than 0.2%). In addition, it was noted that there was a remarkable dose-dependent accumulation of subG1 peak in HS7-treated HT29 cells when compared with the untreated group  to investigate whether the inhibitory effect of HS7 on the transcriptional activity of t manner . At the ecreased , which wβ-catenin-mediated transcription accompanied with decreasing the nuclear β-catenin translocation, we investigated the β-catenin subcellular localization in HS7-treated HT29 cells by indirect immunofluorescence staining. As shown in β-catenin was present in all cellular localizations and preferentially accumulated in the nucleus in the control cells, which was confirmed by DAPI staining , and membrane type I MMP (MT1-MMP) are the downstream target genes of β-catenin/Tcf pathway, we examined whether HS7 could downregulate the expressions of those genes in HT29 cells. At the dose of 2.5–10 μg/mL, HS7 was able to inhibit survivin mRNA expression in a dose-dependent manner  in colon cancer cells. The nuclear staining of β-catenin was markedly reduced as compared with that in control group. Therefore, this inhibition of β-catenin-induced transcription might result in the apoptosis of colon cancer cells. Several genes activated by dysregulated poptosis . More repoptosis . In our β-catenin signaling pathway [β-catenin/Tcf-responsive genes [β-catenin localization and β-catenin/Tcf-regulated genes [β-catenin signaling pathway in colon cancer cells [Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), being used as chemopreventive agents against CRC, had been found to inhibit the Wnt/ pathway . Both asve genes . Sulindaed genes . Celecoxer cells . Howeverer cells . Thus, sβ-catenin pathway of colon cancer cells, HS7 may serve as an alternative CRC preventing dietary supplement in addition to NSAIDs. Similar to HS7, other natural dietary substances such as epigallocatechin-3-gallate (EGCG), curcumin, and resveratrol had also been demonstrated to exert activities on Wnt/β-catenin pathway inhibition [Regarding the profound suppressing effects on the Wnt/hibition –35. Combβ-catenin signaling also appears to regulate cancer cell invasion, metastasis, and angiogenesis [β-catenin/Tcf-regulated genes, such as MMP7 and MT1-MMP, were involved in the regulation of cell growth, invasion, and angiogenesis of colorectal cancer [MMP-7 has been shown to be overexpressed in 75% of human colorectal cancers and correlate with stages of disease and/or prognosis [MT1-MMP is overexpressed in different tumors, including colon cancer and correlates with the invasiveness and metastasis [T. camphoratus suppressed the active MMP-9 levels correlated with the inhibition of transwell motility of T24 bladder cancer cells [β-catenin signaling and its downstream MMP7 and MT1-MMP mRNAs expression, it indicates the potential use of HS7 as anti-invasion and antiangiogenesis agent against colon cancer cells. Wnt/ogenesis . Holcombogenesis . Recentll cancer , 39. In rognosis . Similartastasis . Furtherer cells . Regardiβ-catenin signaling is also highlighted in modulating stemness, proliferation, and differentiation in several adult stem cell niches, such as hair follicles, mammary gland, intestinal crypt, and hematopoietic tissues [β-catenin signaling also plays an important role in cancer stem cells [β-catenin promotes the self-renewal of hepatic stem/progenitor cells and leads to tumorigenesis in the liver [β-catenin signaling and thereby suppressing the tumorigenicity of SP cells on NOD/SCID mice (data not shown), indicating that HS7 may have effects on the elimination of cancer stem cells.The importance of Wnt/ tissues . In addiem cells . Expresshe liver . Recentlβ-catenin signaling pathway, further investigations to explore its effects on other signaling pathways as well as the aspects of antiangiogenesis, anti-invasion, and elimination of cancer stem cells are strongly recommended. The effects of HS7 shown in this study may provide important insight for the use of T. camphoratus as a potential complementary and integrated medicine for the treatment of colorectal cancer.In addition to the remarkable effects of HS7 on apoptosis induction and inhibition of Wnt/


Compared to other organs the cells of the renal inner medulla are challenged with an environment of highly variable osmolality. Cellular adaption to changing osmolality is associated with changes in gene expression. To identify novel genes and pathways that are affected by high osmolality we performed microarray experiments using primary cultured rat inner medullary collecting duct cells cultivated at 300, 600, or 900 mosmol/kg for six days. Compared to 300 mosmol/kg more than 2000 genes showed significant changes in expression at 600 or 900 mosmol/kg. Pathway analysis revealed that the WNT/beta-catenin pathway was also affected. Western blot analysis confirmed the down regulation of beta-catenin and of phosphorylated beta-catenin protein. Immunofluorescence analysis indicated massive changes in intracellular localization of beta-catenin. At 300 mosmol/kg beta-catenin was distributed diffusively within the cells. At higher osmolalities beta-catenin was concentrated at the cell to cell contacts and in the nucleus. Similar effects were observed for phosphorylated beta-catenin. We also observed striking changes in cell morphology. While at 300 mosmol/kg the actin filaments were diffusively distributed the cultivation at 600 mosmol/kg was associated with massive reorganisation and enrichment of the actin filaments at the cell to cell contacts. Staining of the microtubules with beta-tubulin clearly showed the primary cilia in cells cultivated at 300 mosmol/kg. Surprisingly the length of the cilia was drastically reduced in cells cultivated at 600 mosmol/kg. The analysis of the underlying physiological mechanisms and the consequences for cilia related signalling at different osmolalities could help to add new aspects to cilia function.


High membranous β-catenin was associated with advanced stage, vascular invasion and tumor necrosis (p ≤ 0.01). Higher diameter, stage, node involvement, grade, vascular invasion and sarcomatoid differentiation (p ≤ 0.01) were found in patients with high cytoplasmic β-catenin. Patients with a high cytoplasmic β-catenin had a significantly reduced OS (hazard ratio (HR) 1.75) and CSS (HR 2.26), which was not independently associated with OS and CSS after adjustment in the multivariable model. Increased ccRCC aggressiveness was reflected by an altered Wnt1/β-catenin signaling. Cytoplasmic β-catenin was identified as the most promising candidate associated with unfavorable clinicopathology and impaired survival. Nevertheless, the shift of membranous β-catenin to the cytoplasm with a subsequently increased nuclear expression, as shown for other malignancies, could not be demonstrated to be present in ccRCC.In renal cell carcinoma (RCC), single members of the Wnt/β-catenin signaling cascade were recently identified to contribute to cancer progression. However, the role of Wnt1, one of the key ligands in β-catenin regulation, is currently unknown in RCC. Therefore, alterations of the Wnt1/β-catenin axis in clear cell RCC (ccRCC) were examined with regard to clinicopathology, overall survival (OS) and cancer specific survival (CSS). Corresponding ccRCCs and benign renal tissue were analyzed in 278 patients for Wnt1 and β-catenin expression by immunohistochemistry in tissue microarrays. Expression scores, including intensity and percentage of stained cells, were compared between normal kidney and ccRCCs. Data was categorized according to mean expression scores and correlated to tumor and patients’ characteristics. Survival was analyzed according to the Kaplan-Meier and log-rank test. Univariable and multivariable Cox proportional hazard regression models were used to explore the independent prognostic value of Wnt1 and β-catenin. In ccRCCs, high Wnt1 was associated with increased tumor diameter, stage and vascular invasion ( Approximately 64,770 new diagnoses of kidney cancer are made each year, resulting in over 13,570 deaths reported in the United States, with a growing trend in 2012 . Wnt/β-cHowever, the tumorigenic and prognostic significance of Wnt1 has never been elucidated in ccRCC patients. Independent of a reported Wnt ligand stimulation, cytoplasmic accumulation of β-catenin seems to be restricted to ccRCC in contrast to papillary or chromophobe RCCs . NeverthThe mean ± SD age of the 278 patients was 62.2 ± 12.5 years; 69.8% of patients were male. Patients were classified as ≤pT2 in 60.8% and ≥pT3 in 39.2%. Lymph node involvement was present in 13 patients (4.7%), and 39 (14.0%) patients had evidence of distant metastatic disease. Nuclear grading according to the Fuhrman classification was G1/2 in 234 (84.2%) patients and G3/4 in 44 (15.8%) patients .p < 0.0001). β-catenin was mainly expressed in the membrane of proximal and distal tubules, while only minor reactivity was observed in the cytoplasm. Higher, albeit not significant, membranous β-catenin immunoreactivity was observed in benign renal samples (60.6 ± 12.8%) in comparison to the corresponding ccRCC tissue . Furthermore, no significant cytoplasmic expression difference was found . Interestingly, nuclear β-catenin immunoreactivity was only present in 18 (6.5%) of ccRCC samples in ≤5% of cancer cells; nuclear β-catenin expression was absent in benign kidney. Representative immunohistochemical staining of Wnt1 and β-catenin in normal renal and ccRCC tissues is shown in Wnt1 expression was mainly present in the cytoplasm of proximal renal tubules. Higher expression of Wnt1 was found in normal kidney parenchyma (52.2 ± 22.6%) compared to ccRCC , tumor stage  and risk of vascular tumor infiltration . A high membranous β-catenin (>mean tumor score of 69.0%) was related to a higher tumor stage , a higher grading (G3/4: 24.3% vs. G1/2: 12.9%), a higher rate of vascular invasion  and a higher number of tumor necrosis . A high cytoplasmic β-catenin (>mean tumor score of 37.5%) expression was positively correlated to a larger tumor diameter , a higher tumor stage , the presence of lymph node involvement , a higher nuclear grade , the presence of vascular invasion  and sarcomatoid differentiation . Analysis showed no significant findings for nuclear β-catenin expression (data not shown). Detailed results of Wnt1, membranous and cytoplasmic β-catenin expression are shown in A higher Wnt1 expression (>mean tumor score of 40.0%) was associated with higher tumor diameter (5.8 cm ± 3.0 vs. a high Wnt1 protein expression . In contrast, a significantly longer OS and CSS was observed in patients with a low intratumoral cytoplasmic expression of β-catenin (OS: 124.9 (111.4–181.0) vs. 66.9 (45.7–88.1), p = 0.03; CSS: 160.9 (149.2–172.7) vs. 81.1 (58.8–103.5), p = 0.01)), as shown in The median follow-up was 65 months (interquartile range: 20–100). There was no significant difference in median OS and CSS times [months] for patients with a low p ≤ 0.002), as well as higher cytoplasmic β-catenin  were significantly associated with a higher risk of cancer-specific and non-cancer specific death. In addition, reduced OS was found in ccRCC patients with higher age above the median  and perinephric invasion (p = 0.008).In the Cox proportional hazard model , the unfp < 0.0001, CSS: HR: 4.30 p < 0.0001) and the evidence of distant metastasis  was. Higher age  was only associated with a shorter OS.If adjusted in the multivariable model , an incrThe members of the Wnt protein family are strongly implicated as regulators of mammary cell growth and differentiation . The firMany lines of evidence suggest that Wnt/β-catenin signaling is centrally involved in ccRCC tumorigenesis: First, the β-catenin coding gene is located on the short arm of chromosome 3, a region frequently affected by somatic alterations and associated with enhanced carcinogenesis, e.g., Von Hippel-Lindau (VHL)-mutations in renal cancers ,28. AlthFurthermore, the downregulation of another Wnt-antagonist, the Wnt-inhibitory factor 1 (WIF-1), is frequently observed in RCC, while the restoration of WIF-1 suppresses tumor growth . This phet al., again, no significant tumor-specific differences in mRNA levels of β-catenin were reported. Elevated cytoplasmic β-catenin expression was present in 91.7% of ccRCC patients and was associated with a shorter overall survival [In contrast, other investigators found positive nuclear β-catenin immunostaining in 44% of 152 RCC patients and associated nuclear immunoreactivity with lower Fuhrman grades . Recentlsurvival .Taken together, there seems to be great heterogeneity in the reported data, especially concerning localization-dependent β-catenin protein expression in comparison to normal kidney and their function in RCC carcinogenesis.p = 0.03) and CSS (p = 0.01) in cancers with high β-catenin levels in the cytoplasm. These findings reinforce the assumption that deregulated Wnt/β-catenin signaling plays an important role in the promotion of EMT and local infiltrative cancer growth. In contrast, weak (≤5%) nuclear β-catenin expression was only present in 6.5% of ccRCC cases, but not in normal tissue and not associated with any clinicopathology.While membranous and cytoplasmic β-catenin expression is frequently associated with unfavorable RCC biological and clinical behavior, nuclear localization is only inconsistently reported –7. In acA total of 278 ccRCCs and corresponding normal paraffin tissue specimens from patients who underwent nephrectomy or nephron-sparing surgery at the University of Tuebingen, Germany, between May 1993 and December 2006, were included in the analysis. Informed consent was obtained according to procedures approved by the institutional review board of the University of Tuebingen (396/2012BO1). Clinical and pathological data were collected by the treating physicians and by the tumor registry database of the University of Tuebingen. Samples from pathologically representative tumor regions excluding tumor regions consisting of necrosis, fibrosis or larger vessels and adjacent benign renal tissues were used for the construction of the Tissue Microarray (TMA). Specimens were classified according to the 7th edition of the UICC/AJCC system (2009) and to Fuhrman’s grading. All samples were independently evaluated by a second histological examination of hematoxylin- and eosin-stained slides. The TMA slides were prepared with a core size of 1.0 mm; all patient probes were assembled as triplets , as previously described .Staining was performed according to the following protocol. Three micrometer sections were transferred to slides . Tissues were deparaffinized by passing the specimens through xylene and rehydrated through serial dilutions of ethanol . The slides were further processed in the Benchmark XT automated slide stainer  using the standard antigen TBE-buffer-based antigen retrieval solution . The β-catenin antibody  was used at a dilution of 1/400 using antibody diluent . The Wnt1 antibody  was used at a dilution of 1/100 using antibody diluent. Both antibodies were evaluated in preceding experiments to assure antibody specificity. In line with previous reports that used Western blot analysis and the recombinant proteins, both antibodies demonstrated high specific protein detection in breast carcinoma used as positive control, as well as no immunoreactivity in negative controls, with the primary antibody omitted . As a devs. absent). TMA slides were evaluated in a blinded manner by two independent investigators. In the case of discordant scoring results, a consensus score was assigned.Counterstaining was performed using hematoxylin. In order to exclude unspecific staining due to biotin-cross reactivity in the tumor samples, control experiments were done using a biotin blocking solution. Staining was classified according to a semi-quantitative IHC reference scale: the relative amount (0%–100%) of cells stained together with the staining intensity (0–3+) resulted in a score from 0 to 300. Results were determined as a percentage of the maximum value. Only nuclear β-catenin expression was assessed dichotomously (present vs. high (> mean), according to the localization-specific mean expression scores of Wnt1 and of β-catenin, shown in Continuous normally distributed variables are given as the mean value ± standard deviation, continuous non-normally distributed variables as median values with interquartile ranges and categorical data as absolute or relative frequencies. Cancer staging and grading were dichotomized into adverse (UICC/AJCC stage >pT2 and Fuhrman classification >G2) and more favorable characteristics. Wnt1, membranous, cytoplasmic and nuclear β-catenin expression scores were compared between corresponding adjacent normal and ccRCC tissue using a non-parametric test (Wilcoxon). For the analysis of clinical and pathological features and the survival analysis data were categorized into subgroups of expression: low (≤ mean) This study examined for the first time the tumorigenic and prognostic significance of altered Wnt1/β-catenin protein expression in ccRCC patients. Although the link between higher protein expression of Wnt1, membranous and cytoplasmic β-catenin in ccRCC samples underlines the tumorigenic importance, in this study, no corresponding increase in protein levels were detected in comparison to adjacent normal kidney parenchyma in line with all the findings on mRNA-levels ,9.Furthermore, in univariate analysis, only high cytoplasmic β-catenin protein expression was associated with a significantly reduced survival and lost its prognostic significance after multivariable adjustment, while the traditional histopathological parameters did not. These results obtained in patients samples are partly in conflict with cell-line based molecular experiments of the Wnt/β-catenin signal network. Although here, the sequence of high cytoplasmic β-catenin with subsequent translocation into the nucleus and activation of oncogenes seems reasonable, the mechanism of nuclear entry and the dynamic shuttling between the cytoplasm and nucleus is not precisely understood. Furthermore, the dual role of the Wnt/β-catenin as regulator of cellular homeostasis, but also adhesion in renal cancer, remains to be clarified .While the value of Wnt/β-catenin in ccRCC is still controversial, Wnt antagonists already emerged as candidates for future targeted strategies. Recently, several non-specific Wnt inhibitors, including NSAIDs, retinoids, flavonoids or valproic acid, have been identified, while future therapeutic approaches aim to identify a more selective pathway inhibition by anti-Wnt antibodies or small interfering RNAs . These i


The size principle dictates the orderly recruitment of motoneurons (Mns). This principle assumes that Mns of different sizes have a similar voltage threshold, cell size being the crucial property in determining neuronal recruitment. Thus, smaller neurons have higher membrane resistance and require a lower depolarizing current to reach spike threshold. However, the cell size contribution to recruitment in Mns during postnatal development remains unknown. To investigate this subject, rat oculomotor nucleus Mns were intracellularly labeled and their electrophysiological properties recorded in a brain slice preparation. Mns were divided into 2 age groups: neonatal  and adult . The increase in size of Mns led to a decrease in input resistance with a strong linear relationship in both age groups. A well-fitted inverse correlation was also found between input resistance and rheobase in both age groups. However, input resistance versus rheobase did not correlate when data from neonatal and adult Mns were combined in a single group. This lack of correlation is due to the fact that decrease in input resistance of developing Mns did not lead to an increase in rheobase. Indeed, a diminution in rheobase was found, and it was accompanied by an unexpected decrease in voltage threshold. Additionally, the decrease in rheobase co-varied with decrease in voltage threshold in developing Mns. These data support that the size principle governs the recruitment order in neonatal Mns and is maintained in adult Mns of the oculomotor nucleus; but during postnatal development the crucial property in determining recruitment order in these Mns was not the modifications of cell size-input resistance but of voltage threshold. The control of posture and movement requires an understanding of how the activation of motor unit is organized. Henneman and colleagues published seminal papers in the sixties that provide a detailed account of motor unit properties, motoneuron recruitment properties, and how the relation between these two sets of properties could be summarized in terms of a unifying principle that they called the “size principle” The combination of intrinsic properties that might establish the hierarchy in Mns excitability  remains essentially unknown during postnatal development. In developing hypoglossal Mns a decrease in input resistance during the first two weeks after birth has been reported, together with a later cell size growth during the third postnatal week The anatomical and electrophysiological features of the oculomotor nucleus Mns have been separately analysed during postnatal development Experiments were carried out from 1 to 7-day-old (P1–P7) and from 20 to 30-day-old (P20-P30) Wistar rats (5–140 g) of both sexes. These age groups were selected because early after birth they show an increase in cell size and a decrease in input resistance, while physiological and anatomical features of oculomotor nucleus Mns remain essentially unchanged after P20 All recorded neurons were identified as Mns by their antidromic activation from the root of the third nerve and by the collision test P<0.05. Finally, the best fit to the raw data was calculated, represented as a dashed line (for P1-P7) and a continuous line (for P20-P30) in All Mns included in the analysis showed a stable resting membrane potential of at least −55 mV or below, an action potential larger than 60 mV, and fired repetitively in response to depolarization pulses of one second. Several parameters were analyzed: resting membrane potential, input resistance, rheobase, depolarization voltage and voltage threshold. Reconstructed Mns had a healthy appearance, with no evidence of impalement damage to the somal membrane, minimal extracellular dye leakage, and a good contrast with low background labeling of the tissue. The dendritic arborization was contained within the slices, because terminations corresponded to natural endings  as opposed to cut branches. Among the 55 Mns recorded and injected with neurobiotin, only 31 showed good labeling and 24 of these well-stained Mns were fully reconstructed. These Mns were grouped in neonatal  and young adult rats . Data for analysis were plotted and quantified in Origin software 7.5 . All statistical analyses were carried out on the raw data, significant differences between neonatal and adult age groups were determine by using a one-way ANOVA test. The significance level was established at 2 , while in adult Mns (n = 10) it ranged from 16,413 to 29,154 µm2 . Differences in the membrane surface between Mns of both age groups were significant  than for the adult one . The differences in slopes coursed in parallel to the differences in ranges of values for membrane surface area and input resistance between neonates and adult Mns. Finally, when size and input resistance of neonatal and adult Mns were combined in a single group and the linear relationship between these parameters was quantified, a significant negative co-variation was also found .To evaluate the size principle in oculomotor nucleus Mns, we first studied whether input resistance co-varies with motoneuron membrane surface . A signivs. 0.1 nA), depolarization voltage (19 mV vs. 8.6 mV), and voltage threshold (-43 mV vs. −53.4 mV) were lower in the adult Mn than in the neonatal one. Rheobase values ranged from 0.01 to 0.44 nA in Mns of the first postnatal week, and from 0.05 to 0.22 nA in adult Mns. The depolarization voltage ranged from 7.8 to 24.8 mV for the neonatal age group, and from 5.1 to 14.4 mV for the adult age group. The voltage threshold ranged from −55 to -38.7 mV in neonatal Mns and from -56.1 mV to -46.8 mV in adult Mns. A decrement in rheobase from 0.20±0.02 to 0.10±0.01 nA, in depolarization voltage from 18.9±1.2 to 9.2±0.8 mV and in voltage threshold from −42.7±2.1 mV to −52.7±1.4 mV occurred with maturation. The differences in these parameters were statistically significant when neonatal and adult age groups were compared (The depolarizing current (rheobase) and the voltage increment (depolarization voltage) required to evoke an action potential is shown in compared .did not produce an increase in rheobase. Indeed, the comparison of neonatal (n = 6) and adult (n = 4) Mns with similar sizes , dendritic complexity and input resistant, showed different values of rheobase and voltage threshold  and adult  Mns . These dhreshold . As obsePrevious findings demonstrate that Mns with similar cell size and input resistance have different rheobase when neonatal and adult Mns are compared. According to Ohm's law (rheobase  =  depolarization voltage/input resistance), rheobase differences should be attributed to changes in depolarization voltage with development. To test this proposal, rheobase was plotted as a function of the depolarization voltage for each age group and no co-variation was obtained either in neonatal or in adult epochs . That mehis work investigates the size principle in developing Mns from the rat oculomotor nucleus, studying how the somatodendritic shaping impacts input resistance and rheobase. Neonatal and adult Mns showed a strong linear relationship between size and input resistance, and between input resistance and rheobase. These relationships support that the size principle is established early after birth and is kept in the adult Mns. Nevertheless, the most noticeable finding reported here is that voltage threshold (an active membrane property that was not linked to cell size), and not input resistance, could play a key role in determining the recruitment order of Mns during postnatal development.versus mutant rats, it has been suggested that morphology contributed only partially to the modification in input resistance The size principle determines the orderly recruitment of motor units in a wide variety of muscles All in all, it can be suggested that cell size determines input resistance, but its contribution should be tempered depending on the postnatal age.In Mns from the oculomotor nucleus a significant well-fitted linear relationship was observed between the size and rheobase in both neonatal and adult Mns, which demonstrates that the size principle is established early after birth and it is maintained in the adult. However, size and rheobase did not correlate when the results from neonatal and adult Mns were combined in a single group. Furthermore, increase in size in these Mns with maturation did not lead to an increase in rheobase during the postnatal development. Rather, cell growth in developing Mns was accompanied by a decrease in rheobase. This finding could be due to the fact that input resistance decline with postnatal age did not produce an increase in rheobase, as reported in other pools of Mns + conductance and long-lasting Ca2+ currents. Such inward currents, activated at the subthreshold level, produce excitation and may be involved in Mn recruitment Present results shows that the depolarization voltage needed to reach spike threshold is lower in adult Mns than in neonatal Mns, which means a decrease in spike voltage threshold with development. Therefore, it is plausible that this change could lead to a decrease in the rheobase with postnatal age in these Mns. Thus, the excitability of these Mns increases with maturation despite the decrease of the membrane resistance. The diminution in the voltage threshold of oculomotor nucleus Mns with age could be due to an increase in persistent NaEarly after birth  the circuitry transmitting the signal from vestibular nuclei to the ocular Mns is mostly established


The “poke and release” strategy for the delivery of macromolecules using polymeric microneedle (MN) is of great importance because it eliminates microneedle reuse, the risks of biohazardous sharps and cross contamination, and it requires no special disposal mechanism. The main objective of this study was the determination of the stability and delivery of bovine serum albumin (BSA) that was transported across human skin via sodium alginate (SA) microneedle arrays (MNs) and SA needle free patches using two different analytical methods.The capability of two analytical methods, the bicinchoninic acid (BCA) assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), to precisely detect and quantify BSA within different types of polymeric MNs was assessed. The ex vivo protein release of BSA across dermatomed human abdominal skin from 10 w/w SA MNs was compared to that from needle-free patches using Franz diffusion cells. The developed applicator was mechanically characterized using a Texture Analyzer. The patch mold and its components were fabricated using a rapid prototyping machine.The BCA method was able to precisely detect BSA that had been loaded into SA MNs. However, the use of SDS-PAGE as the analytical method resulted in significantly different amounts of BSA recovered from differently conditioned polymeric MNs. The permeation of BSA across dermatomed human abdominal skin by SA MNs, which were composed of 100 pyramidal needles, increased by approximately 15.4 fold compared to the permeation obtained with SA needle-free patches. The ease of use of the applicator during the release studies was also demonstrated, as was its mechanical characterization. The delivery of medicinal substances across the skin has been limited because of the unique barrier property of the stratum corneum (SC) The primary objective of transdermal microneedle (MN) systems is the creation of microscopic holes and the transport of molecules into the skin deeper layers. Since, skin is a unique portal for vaccine administration and contains an abundance of immunocompetent cells The “poke and release” application does not allow prolonged delivery, but it facilitates bolus In MNs mediated vaccination studies, a stronger and rapid immune response received The handicap of drug delivery using “poke and release” strategy is diffusion of drug through the pores of polymer matrix, and the drawback of this strategy might be loosing a small amount of active substance during incorporation of drug with the polymer For many reasons, the value of the “poke and release” strategy should not be underestimated, and abovementioned obstacle can be overcome. This strategy eliminates the special disposal mechanism, reuse of MN, the risk of biohazardous sharps, and cross contamination Dissolvable carboxymethyl cellulose (CMC) Moreover, dissolvable microneedle patches have been reported to successfully deliver both small (MW<500 Da) and macro molecules (MW>500 Da) in “poke and release” approach An overlooked dissolvable polymer solution, sodium alginate (SA), has been found to elicit a more effective antibody response than alum An applicator developed by Mechanical Engineering Department at Bilkent University was utilized to place the microneedles on the skin, and poke the SC in order to produce uniform, reproducible 100 micro holes in the skin with a 10-by-10 MNs, and thereby enhancing the permeation profiles of the loaded protein.The protein contents of BSA-loaded SA MNs were analyzed using the bicinchoninic acid (BCA) method. Using the theoretical amount of 10 mg per MN device, the recovery of BSA was examined at different time periods .The usefulness of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for detection of BSA incorporated within abovementioned polymeric MNs were evaluated. The amounts of BSA obtained from differently conditioned MNs were compared. The absorbance values acquired using the BCA method and the band intensities received through SDS-PAGE were compared to determine the capabilities of these two different methods. The evaluation and comparison of stability of the stock BSA solution under various climate conditions was also examined.The major aim of this study was investigation of the BSA permeation profiles obtained with conventional SA patches and the formerly microfabricated SA MNs The individuals in this manuscript have given written informed consent (as outlined in PLOS consent form) to publish these case details. Written consents from patients were taken according to the regulations of Ethics Committee of the Institute of Health Sciences at Marmara University. The ethical committee evaluated and approved consent procedure and the study protocol on July 1, 2011 with a protocol number of 38/11, and an approval number of 9.The applicator inspired from a published paper Various springs supplied by a local company  were inserted between the piston cell lid and the piston tab. Once the spring was completely attached to both sides, the lid was locked . When thA rapid prototyping machine  with the aid of a computer program  used to manufacture the centrifuge patch rack, its lid, and the patch mold in a quick and simple manner, using acrylonitrile butadiene styrene (ABS) plastic.The ABS patch molds were constructed with a square (8.90 mm×8.90 mm) base with a depth of 3 mm and were inversely replicated for fabrication of polydimethylsiloxane (PDMS) needle-free patch molds that allow a comparison of drug delivery yielded by conventional and microneedle-based systems.The 10 w/w SA patch and its MNs were fabricated as 10 mg of BSA in a 90-mg gel-drug formulation. These formulations were molded, and dried at ambient conditions for 24 hours and were used for the following experiments.A colorimetric detection method (BCA assay) was utilized to calculate amount of BSA released in vitro from the polymeric MNs and permeated BSA. Measurements of diffused BSA across dermatomed human abdominal skin and released BSA from protein-loaded polymeric MNs were calculated according to the validated BCA assay. Throughout the BCA method, guidelines of the BCA kit manufacturer were followed The absorbance of each calibration standard at a wavelength of 562 nm was determined with placing multiwell plate on microplate reader and using a spectrophotometer .The data were analyzed using Gen5™ 2.0 Data Analysis Software (BioTek). The graphs were created in GraphPad Prism®  using concentrations of standards to generate the calibration curves .The linear relationships between net absorbance of the BSA standards at 562 nm and correlated concentrations were determined to generate the calibration curves . The limMoreover, a comparison was performed for the stability of stock BSA solutions  that were differently conditioned, such as freshly prepared, maintained for 24 hours at 4°C (in a refrigerator), and incubated for 24 hours at 40°C and 75% RH .The interaction between BSA and polymeric MNs  was examined. The amounts of BSA recovered from freshly prepared SA MNs, as well as from SA MNs stored for 7 and 30 days were examined using the BCA assay.The interaction between BSA and abovementioned polymeric MNs solutions was examined, similar to a paper For these cases, 5 ml of a 2000-µg/ml and 1000-µg/ml clear BSA solutions in PBS (pH 7.4) were separately added to 5 ml of an abovementioned single polymeric MNs solution in PBS (pH 7.4). The mixtures were gently shaken for 10 minutes at 800 rpm (IKA KS125 basic shaker). The results were statistically compared to determine whether these solutions interacted.The interactions between the protein and abovementioned polymeric MNs, as well as amounts of BSA recovered from SA MNs stored for different periods of time, were also analyzed and verified using SDS-PAGE: 4% stacking gel and 10% running gel in a mini electrophoresis cell . The purity and approximate molecular weight of BSA were also examined similar with a method of A clear gel containing visible bands was digitalized with a digital camera . After digital images were converted to gray scale mode, band intensities were measured with imaging software , and recovered BSA was schemed according to the standardized band intensity .A previously published method To quantify amounts of BSA released from the microneedles, 10 mg of BSA was loaded into a 10 w/w SA MNs that was secured onto the bottom of rolled rim tubes composed of soda glass with PE snap-on lid vials that contained 10 ml of PBS (pH 7.4). The PBS was freshly prepared, filtered through a Millipore filtration system  using a 0.45-µm filter , and autoclaved. After the vials were filled, they were immediately incubated in a water bath shaker at 37±0.5°C, with continuous agitation at 1000 rpm . Aliquots of 200 µl of PBS were removed from each vial at various time points  after the initiation of the experiment. An equal volume (200 µl) of autoclaved PBS (pH 7.4) at 37±0.5°C was immediately added to the vials throughout the experiment.The passive diffusion across dermatomed human abdominal skin of the commonly used, water-soluble protein, BSA, (MW = 66 kDa) from SA (10 w/w) polymeric patches and MNs was investigated ex vivo using Franz diffusion jacketed cells with flat grounded (ground O-ring) joints composed of clear borosilicate glass with an orifice diameter of 15 mm and a receptor volume of 7 ml . The Franz diffusion cells were mounted on a Franz cell stirrer with acrylic cell holders, synchronously stirred using magnetic Teflon stir bars at a constant speed of 600 rpm at 220 V/50 Hz , and thermostabilized at 37±1°C in a water bath.Human abdominal skin flaps were collected from volunteer subjects who were scheduled to undergo abdominal reconstructive surgery at Plastic Surgery Department, Marmara University in Istanbul. After the abdominoplasty was safely performed, skin flaps of approximately 10×10 cm were removed from the patient’s body. Immediately after collection, the skin flaps were tightly mounted onto a piece of smooth wood and were trimmed to a thickness of 0.76 mm with an electric dermatome  similar with a method of After the skin specimens were excised, the dermatomed samples were pre-equilibrated for 1 hour. In both the patch- and microneedles-based permeation studies, the SC side was dried with tissue paper and was secured to the vertical Franz diffusion cells with cyanoacrylate glue such with the SC and donor were facing each other, while the dermal was across from receptor.In the patch-based permeation studies, 10 µl of ultra-pure water was exposed to the SC for initiation of adhesion. The drug-loaded patch was then immediately exposed. During this exposure, donor compartment was attached with pinch clamps to the pre-equilibrated receptor phase. All procedures of permeation study were conducted similar to a published method The SA MNs were quickly visualized with a digital microscope prior to the insertion studies. Among SA MNs that experienced even a small tip buckling were discarded, only the robust and stable ones were utilized for the drug permeation studies.2, and weight = 0.42 g) was placed on donor chamber to serve as support material for the exposed MNs. The applicator was tailored to be inserted uniformly into the MNs and to maintain a consistent penetration throughout the diffusion studies  after initiation of the diffusion experiment. An equal volume (200 µl) of degassed fresh PBS (pH 7.4) pre-equilibrated at 37±1°C was immediately added to receptor phase with a cytology needle to maintain constant sink conditions throughout the experiment .The sampled receptor medium was filtered using a Millex®-HV Syringe-Driven Filter Unit  into HPLC vials  that contained the HPLC insert .After the last sample was collected from the receptor phase (24 h), pinch clamps were removed, and the donor compartment was securely transferred onto tissue paper. Upper surface of epidermis was exposed with a volume of 5 ml of methylene blue solution (1000 µg/ml), where microholes had been created, for 5 minutes. The excess solution was removed, and the surface was gently washed with 10 ml of PBS (pH 7.4). The skin specimens were pictured with a digital 500D camera (Canon Inc.) The diffused BSA samples withdrawn from the receptor phase were evaluated using the validated BCA method , accordip<0.05 was considered to indicate a statistically significant difference.All of data are presented as the means ± standard deviation (SD) of replicates. The statistical analysis of effects of the different storage conditions, i.e., 4°C and 40°C/75% RH, and results of the polymer/BSA interaction experiments were performed using a two-tailed, unpaired t-test, and p<0.05 was considered to indicate a statistically significant difference.The statistical comparisons of the percent BSA recovered from the SA MNs after 0, 7, and 30 days, as well as its raw absorbance, were complied with a one-way analysis of variance (ANOVA), and p<0.05 was considered to indicate a statistically significant difference. All of the statistical calculations were conducted using GraphPad Prism® software (Version 5.0a for Mac OS X).The statistical comparison of the stability of BSA stock solutions that were freshly prepared, frozen for 24 hours, and incubated 24 hours was complied with a one-way analysis of variance (ANOVA), and 2) was 0.999 .The stability analysis of three different sample batches of BSA stock solution (each sample represents the mean of eight replicates) revealed that these solutions were stable for 24 hours when refrigerated at 4°C or when incubated at 40°C and 75% RH . The % Cp>0.05 in all cases).p>0.05 in all cases).Moreover, raw absorbance and percentage recovery values for the abovementioned MNs were also not significantly different among the BSA-loaded SA MNs that were dried for 24 hours at room temperature (without storage) and those stored at 4°C for 7 and 30 days  and did not affect BSA analysis. Therefore, with exception of the 10% w/w SA MNs, the MNs examined in However, the 10% w/w SA MNs did not interact with BCA working reagent . As a result, SDS-PAGE was not chosen for further BSA content analyses.A calibration plot was generated with plotting a calibration standard against band intensity. There was no sequential increase in band intensities of 250, 500, and 750 ng BSA samples in SDS-PAGE analysis of BSA content. Six different BSA concentrations ranging from 100 to 600 ng/µl were used, and they exhibited useless linearity . For instance, the percent cumulative release of BSA after 5, 10, and 15 minutes was 25.79%, 58.33%, and 92.04%, respectively. The mean concentrations of released BSA were 257.98 µg/ml, 583.43 µg/ml, and 920.70 µg/ml after 5, 10, and 15 minutes, respectively .The mean permeated amount and the percent of BSA permeated through dermatomed human skin after 24 hours from 10-mg BSA-loaded SA MNs were approximately 220 µg/ml and 15.4%, respectively , Table 9What impact forces will MN tips perceive when activated applicator injects the MNs? It was assumed that the applicator would cause the MNs to be inserted into skin to minimum and maximum depths of 0.1 mm and 1.0 mm, respectively, and that different forces would be produced depending on compression distance.The mechanical measurements of forces generated in the short-distance and long-distance cases were observed. The results indicated that internal and external diameters of wire, number of coils, and free spring length were crucial parameters for generation of various impact forces as reported in a publication The impact forces generated by spring B (1.05 N and 1.14 N) were significantly smaller than forces required to axially or transversely break a single SA microneedle . Although some of springs  generated greater amounts of force than SA can withstand, and they broke microneedle arrays bases, caused tip bending, or damaged skin integrity during insertion of the needles into the skin (data not shown). The insertion of spring A into the skin was not examined 2 (0.977); therefore, band intensities of the BSA-loaded polymeric MNs in each gel were calculated by comparing them to band intensities of the 500-ng BSA solute (standard band). The SDS-PAGE band intensities of the BSA-loaded chitosan, HPC-H and cross-linked PVA-gelatin MNs were found to be different from those of the BSA solution alone , absorbance and concentration values  of the Bp values of 0.6606 and 0.3713 for 5 mg and 10 mg BSA loadings, respectively  that are not able to passively diffuse into the skin Exposing these MNs to cold temperatures, as well as hot and humid conditions and then analyzing them spectrally enabled analysis of stability of the BSA-loaded SA MNs. BSA recovered from freshly prepared SA MNs (day 0), as well as from MNs stored for 7 and 30 days, was also spectrally analyzed in triplicate. Spectral curves of the recovered BSA from days 0, 7, and 30 were virtually identical, which was supported by the data shown in In the present study, protein-loaded SA polymeric MNs were found to be a rapidly dissolvable microporation method. These MNs possess all of the abovementioned advantages of MNs. Therefore, to confirm their rapidly dissolvable behavior; the BSA-loaded SA MNs were examined in vitro. The release kinetics was found to behave as zero-order kinetics over a period of 24 hours. The kinetic results illustrate that these water-soluble polymeric microneedles (SA MNs) were able to dissolve within minutes and were entirely removed by the skin, thereby eliminating production of any biohazardous waste.As opposed to a former publication More importantly, the utilization of dissolvable MNs and the rapid delivery of proteins, as analyzed in this study, will no doubt generate practical, successful, short-term applications, and they are likely to be considered easy to use by patients as minimally invasive patches.Extremely precise and accurate microfabricated pyramidal MNs A drawback of the “poke and release” approach is protein present in the needle itself can be delivered; therefore some protein in the base section of the microneedle array are wasted, as observed in this study. This obstacle might be overcome by incorporating the macromolecule only to the needle section of the array, and should not happen in case of usage of small molecular weight drug.In conclusion, the results indicate that the SA patches were unable to deliver BSA, whereas the MNs delivered significant amounts of BSA across the skin within the same period of time and under the same experimental conditions. The ex vivo permeation of BSA across dermatomed human abdominal skin from an SA MN array with 100 pyramidal needles increased the permeation of this protein by approximately 15.4 fold compared to its needle-free patch.Because of the noteworthy dissolution behavior, and impact of the SA MNs especially in protein delivery, the SA MNs could be used as dissolvable matrix for rapid immunization studies. The commercialization of these polymeric microarrays would be of great value to patients. Due to possessing great potential to revolutionize the vaccination approaches and conventional patches, utilizing these MNs as drug delivery device will no doubt create a large economic impact.


Truncating mutations affect the adenomatous polyposis coli (APC) gene in most cases of colon cancer, resulting in the stabilization of β-catenin and uncontrolled cell proliferation. We show here that colon cancer cell lines express also the paralog APC-like (APCL or APC2). RNA interference revealed that it controls the level and/or the activity of β-catenin, but it is less efficient and binds less well to β-catenin than APC, thereby providing one explanation as to why the gene is not mutated in colon cancer. A further comparison indicates that APCL down-regulates the β-catenin level despite the lack of the 15R region known to be important in APC. To understand this discrepancy, we performed immunoprecipitation experiments that revealed that phosphorylated β-catenin displays a preference for binding to the 15 amino acid repeats (15R) rather than the first 20 amino acid repeat of APC. This suggests that the 15R region constitutes a gate connecting the steps of β-catenin phosphorylation and subsequent ubiquitination/degradation. Using RNA interference and domain swapping experiments, we show that APCL benefits from the 15R of truncated APC to target β-catenin for degradation, in a process likely involving heterodimerization of the two partners. Our data suggest that the functional complementation of APCL by APC constitutes a substantial facet of tumour development, because the truncating mutations of APC in colorectal tumours from familial adenomatous polyposis (FAP) patients are almost always selected for the retention of at least one 15R. The APC gene Truncating mutations of APC have the tendency to concentrate in the middle of the open reading frame, the so-called mutation cluster region (MCR) Compared to APC, human APCL (or APC2) It has been mentioned that the APCL mRNA is expressed in the colon and small intestine To investigate the possibility that endogenous APCL may regulate β-catenin activity and/or expression level, two siRNAs against APCL were selected for their ability to down-regulate specifically the expression of APCL but not APC (figure S1A). yAPC displayed a fiber-like appearance, likely reflecting localization at microtubules, as previously characterized figure S1A). When we measured the transcriptional activity of β-catenin in CaCo2 cells using the TOP/FOP luciferase reporter assay (see Methods), we found that yAPCL displays a weaker ability to downregulate β-catenin-dependent transcription than yAPC despite higher expression levels (figure S1B). In SW480 cells, yAPCL was also less active than yAPC in inhibiting the transcriptional activity of β-catenin  fusion protein figure 1figure S2), which is again in agreement with the previously described localization of APCL to the Golgi apparatus In the next step, we used yAPCL deletion constructs figure 1We compared the relative efficacies of the various yAPCL deletion constructs with those of their corresponding yAPC truncations in regulating β-catenin-dependent transcription in TOP/FOP reporter assays  of APC into yAPCL1728  (figure S3). This is in line with the results of the immunoprecipitation experiment mentioned above and previously performed in SW480 cells figure S3), in contrast to the situation observed in SW480 cells (figure S1).APCL may benefit from the 15Rs of APC through heterodimerization and it has been reported previously that under endogenous settings an antibody against APCL can co-immunoprecipitate the SW480 APC isoform truncated at position 1338, typical of colon cancer cells figure S4). The APC dots disappeared with the removal of the first dimerization domain (compare yAPC1404 and yAPC(205-1404)). In contrast, an even larger N-terminal deletion in truncated APCL still resulted in the retention of the dotty pattern (compare yAPCL1728 and yAPCL(410-1728). When we compared the localizations of yAPC777 lacking the second dimerization domain and rAPCL730 (tagged with the red fluorescent protein (RFP) at the N-terminus) which are almost of equivalent lengths, we found that the former one was diffusely distributed, whereas the latter one kept the dotty pattern. Thus, APC and APCL dots are of a different nature, which is in agreement with the reported cytoplasmic oligomerization of truncated APC To understand the discrepancy between the active yAPCL1241 and the inactive yAPC1466-15µ that are both supposed to interact with endogenous truncated APC, we compared the structural requirements leading to APC homo-complexes and APC-APCL hetero-complexes in SW480 figure 8Our experiments show for the first time that colon cancer cells express a functional APCL (or APC2) that controls the level and/or the transcriptional activity of β-catenin. They suggest that APCL uses the 15R region of APC to target β-catenin for degradation and offer a plausible explanation as to why APCL is not mutated and why the truncating mutations in APC almost always lead to the retention of a fragment of minimal length in colon tumour cells.). Thus, colon cancer cells expressing truncated APC may tolerate a wild-type APCL because it displays a leaky activity, allowing β-catenin to escape degradation in a sufficient amount to initiate tumourigenesis.Ectopic expression experiments revealed that APCL is less efficient than APC at inhibiting the transcriptional activity of β-catenin. This is linked to a weaker ability to bind β-catenin, as observed in co-immunoprecipitation experiments . Heterodimerization might be important for activity by allowing a direct transfer of phosphorylated β-catenin onto the 15Rs. Accordingly, a transient release of phosphorylated β-catenin in the cytoplasm is unlikely because the ectopically expressed APC construct inactivated by the introduction of point mutations that prevent β-catenin binding to the 15R , HeLa cells and CaCo2, SW837, LoVo, HT29, SW480 and DLD1 colorectal cancer cell lines were all obtained from the American Type Culture Collection (ATCC) and maintained in DMEM medium  supplemented with 10% fetal calf serum  and 1% penicillin and 1% streptomycin (PAA Laboratories). APC mutations in the cell lines are described in The mouse anti-APCL antibody was purchased from Biozol . Secondary antibodies coupled to either horseradish peroxidase or Cy3 were from Dianova , anti-GFP from Roche , anti-APC antibody Ali(12–28) from Abcam , anti-β-actin and anti-lamin from Santa Cruz Biotechnologies , anti-α-tubulin from AbD Serotec , rabbit anti-flag, anti-E-cadherin and anti-β-catenin from Sigma  and anti-phospho-β-catenin (ser33/37) from Pierce Biotechnology .The plasmids expressing the YFP-APC, YFP-APCL and RFP-APCL fusion proteins were constructed by standard molecular biology methods. The sequence of any of these plasmids is available upon request. pcDNAflag Plasmids were transfected into cells overnight using 5 µl polyethylenimine (1 mg/ml) per µg DNA. For transient transfection of plasmids in DLD1 or SW480 cells, 2 µg total DNA/250000 cells/35 mm dish were used. For transient transfection of plasmids in HEK293T cells, 10 µg total DNA/5000000 cells/85 mm dish were used.siAPC-C (gacgttgcgagaagttgga), siAPC729 (gaggtcatctcagaacaag), siAPCL413 (ggtgtttcctgctgaatga), siAPCL5100 (ggcgccaattcaattgtca), siHIF1α The TOPglow reporter consists of a tandem repeat of four TCF/LEF1 binding sites inserted in front of a TATA box Hypotonic cel extracts were prepared in a 25 mM TrisHCl pH7.5 buffer containing 1 mM EDTA, 1 mM DTT and 1 mM PMSF. Triton-X100 extracts were prepared in a 20 mM TrisHCL pH7.4 buffer containing 150 mM NaCl, 5 mM EDTA, 1% Triton-X100, 1 mM DTT and 1 mM PMSF.were performed in Triton-X100 extracts and the immunoprecipitates were washed in a 50 mM TrisHCl pH8 buffer containing 150 mM NaCl, 5 mM EDTA and 1% Triton-X100. Immunoprecipitations combined cell extracts from 293T cells expressing high levels of the different YFP-APC(L) fusions (due to a very high transfection efficiency), mixed with a cell extract from SW480 cells as an abundant source of β-catenin.The blots were developed using the chemiluminescence reagents Western Lightning™  and the signals were detected under a LAS-3000-Fuji camera from Raytest .was conducted as described Figure S1Comparison of yAPC and yAPCL in down-regulating the level and inhibiting the transcriptional activity of β-catenin.A, APCL abolishes β-catenin accumulation in SW480 cells. SW480 cells were transiently transfected on day 1 with expression vectors for either full length APC or full length APCL tagged with YFP at their N-terminus. Cells were fixed on day 3 and stained with an antibody against β-catenin and the Hoechst dye. The green, red and blue colours correspond to the fluorescences associated with YFP, β-catenin and the Hoechst dye, respectively. Bar, 10 µm. B, APCL is less efficient than APC in inhibiting the transcriptional activity of β-catenin. CaCo2 cells were transiently transfected on day 1 with reporter plasmids, together with either 100 ng of either an empty vector (flag) or yAPC or increasing amounts of yAPCL, as indicated. TOP/FOP reporter assays were performed on day 3 to measure the transcriptional activity of β-catenin . Shown is the mean of three independent values +/− standard deviation from a representative experiment.(PDF)Click here for additional data file.Figure S23D reconstruction of yAPCL1728 localization. Hela cells were transiently transfected with an expression vector for yAPCL1728. Cells were fixed 24 h after transfection, stained with Hoechst dye and imaged on a confocal microscope. The 3D reconstruction visualizes the localization of yAPCL1728 in cytoplasmic dots. The green and blue colours correspond to the fluorescences associated with YFP and the Hoechst dye, respectively. Bar, 7 µM.(PDF)Click here for additional data file.Figure S3Truncated APC colocalizes with full length APC and full length APCL. DLD1 cells were transiently transfected on day 1 with expression vectors for either YFP, APC truncated at position 1289 and flag-tagged at the N-terminus or full length APC and APCL tagged with YFP at their N-terminus, either alone or in the indicated combinations. Cells were fixed on day 3 and stained with an anti-flag antibody and the Hoechst dye. Note that none of the constructs is imposing its own localization to the other one in a dominant manner in the cell population. Similar results were obtained when replacing the flag tag by the red fluorescent protein (data not shown). The green, red and blue colours correspond to the fluorescences associated with YFP, the flag tag and the Hoechst dye, respectively. Bar, 10 µm.(PDF)Click here for additional data file.Figure S4APC homo-complexes are different from APCL homo-complexes and APC-APCL hetero-complexes. DLD1 cells were transiently transfected on day 1 with 1 µg of either RFP, the indicated APC and APCL constructs tagged with YFP at their N-terminus (see inus see  or the A(PDF)Click here for additional data file.


The work function changed dramatically as the aniline concentration increased, indicating that the aniline on the GOx surface conveyed n-type doping characteristics at a low coverage level. A higher aniline concentration increased the p-type doping character by increasing the azobenzene concentration on the GOx surface. A comparison of the oxidation reactivity of aniline molecules on the EG or GOx surfaces revealed the role of the oxygen carriers on the GOx surfaces in the context of catalytic oxidation.The oxidation of aniline to azobenzene was conducted in the presence of either monolayer graphene (EG) or graphene-oxide-like surface, such as GOx, under ultra-high vacuum conditions maintaining a 365-nm UV light exposure to enhance the oxidation reaction. The surface-bound products were investigated using micro Raman spectroscopy, high-resolution photoemission spectroscopy, and work function measurements. The oxygen carriers present on the GOx surfaces, but not on the EG surfaces, acted as reaction reagents to facilitate the oxidation reaction from aniline to azobenzene. Increasing the aniline concentration at 300 K confirmed that the exchange ratio from the aniline to the azobenzene was enhanced, as determined by the intensity ratio between the aniline- and azobenzene-induced N 1  Among the various systems composed only of carbon 5atoms, graphene  presents a framework for understanding of the properties of all other carbon allotropes. The properties of graphene, including a high intrinsic mobility,2, a larvia a reaction between the aniline amine groups and the oxygen groups on the GOx surface under ultra-high vacuum (UHV) conditions maintaining a 365-nm UV light exposure.In this study, we systematically compared and investigated the oxidation of aniline to form azobenzene on monolayer graphene (EG) or graphene-oxide-like (GOx) surfaces fabricated with benzoic acid. Moreover, we focus on examining the difference between EG and GOx surfaces in one substrate, simultaneously. Raman spectroscopy and high-resolution photoemission spectroscopy (HRPES) were used to characterize the surface-bound products. The carboxyl groups introduced onto the graphene surface upon oxidation by benzoic acid to GOx allowed aniline to react with the oxygen carriers. The oxidation of aniline proceed Generally, it is hard to distinguish the difference between EG and GOx surfaces in one substrate due to the large size of the HRPES beam. Hence, no previous systematic experimental studies have examined the oxidation of aniline on a GOx surface. However, this study is meaningful with regards to indicating this distinctive difference using the feature of micro Raman spectroscopy.A Si-terminated 6H-SiC(0001) substrate  was used to fabricate EG. The substrate was degassed, annealed at 1,200 K under a Si flux (1 Å/min), and graphitized at temperatures up to 1,500 K (for 2 min) to produce a monolayer of graphene (EG). The annealing temperature was monitored using an infrared pyrometer (with an emissivity of 0.9).s core-level spectroscopy (not shown here) and then immersed into a benzoic acid solution for 3 min. The surface was subsequently reintroduced into the UHV chamber.A GOx surface was fabricated by exposing the EG surface to benzoic acid . As shown in Figure + ion laser  as an excitation source; a spectrometer , and a CCD detector (Horiba Jobin Yvon Symphony) cooled to 140 K. The wavelength of the incident excitation beam was 514.5 nm. HRPES experiments were performed at the 8A2 beamline at the Pohang Accelerator Laboratory, which was equipped with an electron analyzer . The N 1 s core-level spectrum was obtained using photon energies of 460 eV. Secondary electron emission spectra (−20 V sample bias) and valence band spectra were measured at photon energies of 80 eV. The binding energies of the core-level spectra were determined with respect to the binding energies of the clean Au 4f core level and the valence band (Fermi energy) for the same photon energy. All spectra were recorded in the normal emission mode. The photoemission spectra were carefully analyzed using a standard nonlinear least-squares fitting procedure with Voigt functions[Aniline  was purified by turbo pumping to remove impurities prior to dosing onto the GOx surfaces. A direct doser, controlled by means of a variable leak valve, was used to dose the substrates. Raman spectra of the samples were collected using a home-built system equipped with an Ar–1 and 1,597.9 cm–1) were indistinguishable from the G band position of graphene. The ratio of the D and G band intensities, ID/IG, corresponded to the average value for graphene. The Raman D/G intensity ratios at both the bottom and small particle positions on the EG surface were 0.73, indicating that the surface properties at either position were typical of an EG surface[Raman spectroscopy, which is sensitive to the chemical functional groups on a surface, is a useful tool for comparing the properties of the EG and GOx surfaces. Optical microscopy images of the EG (a) and GOx (b) surfaces were acquired, and their corresponding Raman spectra at two positions (over a particle and over the bottom region) were collected, as shown in Figure  surface.–1. Figure –1) obtained from the particle position was shifted toward higher wavenumbers relative to the G bands of graphene and graphite. The ratio of the D and G band intensities, ID/IG, is inversely proportional to the average size of the sp2 domains. The Raman D/G intensity ratio for the GOx surface was found to be 0.92, similar to the results reported previously for graphene oxide[Figure ne oxide.s peak, which is an indicator of the overall molecular electronic properties. The morphological discrepancies observed between the optical images could only be explained in terms of a surface reaction, as supported by the HRPES results.A Raman spectrum similar to the spectrum of GO surface indicated that benzoic acid treatment successfully yielded a GOx surface. The EG and GOx surfaces were used in the subsequent experiments involving the oxidation of aniline, which is difficult to oxidize in general. We hypothesized that only the GOx surface would be able to oxidize aniline if the oxidation process is possible. Because the oxidation of aniline on a GOx surface could not be fully characterized by micro Raman spectroscopy alone, we obtained the core-level spectra of the N 1 s core-level spectra of aniline on the EG and GOx surfaces, obtained using HRPES at 460 eV photon energy. The N 1 s core spectra of 3,600 L aniline on EG or on GOx surfaces were obtained first. As expected, the presence of aniline resulted in low-intensity nitrogen peaks on the EG surface because the EG surface was too inert to react to the oxidation of aniline, illustrated in Figure s core-level spectrum was then obtained after preparing a sample to have 3,600 L aniline on the GOx surface. Two distinct nitrogen peaks corresponding to the aniline peak (NH2 is marked N1) and azobenzene peak (NO2 is marked N2) clearly appeared, as shown in Figure s aniline peak (N1) was found to be 0.84 at 397.8 eV, and the ratio of the azobenzene peak (N2) was 0.16 at 400.1 eV, for a 3,600-L aniline sample on the GOx surface[s peaks indicated that aniline had oxidized to azobenzene in the presence of the oxygen groups on the GOx surface, which suggested that the GOx surface acted as a reaction reagent at 300 K. The oxidation reaction efficiency under a 365-nm UV light exposure was measured as the aniline coverage was increased from 3,600 L to 14,400 L.Figure  surface,20. ThesThe plots of the coverage-dependent intensity of the aniline peaks N1) and the azobenzene peaks (N2) on the EG and GOx surfaces are displayed in Figure  and the s core-level spectra had confirmed that aniline did not react to form azobenzene on the EG surface, the work function measurements were conducted only on the GOx surface, for various aniline coverage levels. The red spectrum in Figure Δϕ = 0.22 eV) toward higher kinetic energies relative to the monolayer EG secondary electron edge. This result indicated that the oxygen carriers on the GOx surface acted as p-type dopant materials. After measuring the GOx surface work function, a 3,600 L aniline coverage was deposited at 300 K  toward lower kinetic energies relative to the pristine monolayer EG, indicating n-type doping due to aniline. The amine group in the aniline donated an electron carrier to the GOx surface, indicating that aniline acted as an electron dopant on the EG surface (n-type characteristic). The blue spectrum in Figure Δϕ = 0.08 eV) toward higher kinetic energies relative to the pristine monolayer EG. Unlike aniline, azobenzene acted as an electron acceptor (p-type characteristic). The presence of azobenzene on the GOx surface resulted in p-type doping carriers. Because aniline and azobenzene were in competition on the GOx surface, the secondary electron edge did not show a significant shift toward higher kinetic energies. Finally, the aniline coverage level was increased to 14,400 L at 300 K  to higher kinetic energies relative to the pristine monolayer EG. This surface yielded a work function that resembled the work function of the GOx surface. These results could be readily explained in terms of the aniline coverage. At higher coverage, the reaction rate increased, thereby facilitating the oxidation of aniline to azobenzene. Figure The work function was measured as the center position of the low kinetic energy cut-off for each sample, as shown in Figure Figure p-type to n-type due to charge transfer from the GOx surface to the adsorbed aniline or azobenzene. The doping characteristic changed from n-type to p-type as the oxidation reaction proceeded from aniline to azobenzene.The Raman spectra and HRPES experiments further supported the conclusion that the oxidation reaction occurred on the GOx surface. The work function of the surface was monitored as the doping characteristic changed from n-Type doping was observed at high aniline concentrations (at lower aniline deposition), whereas p-type doping was observed at high azobenzene concentrations (at higher aniline deposition) on the GOx surface. The oxygen carriers present on the GOx surface were found to act as the reaction reagents.The oxidation of aniline to azobenzene was investigated on a GOx surface prepared using benzoic acid. Micro optical images and their corresponding Raman spectra, HRPES measurements, and work function measurement were conducted from the samples prepared under a variety of conditions. The Raman images revealed the structure of the GOx surface prepared using benzoic acid. The HRPES measurements indicated that the relative concentration of aniline and azobenzene varied with the aniline surface coverages. The work functions of the samples were measured as a function of the aniline surface coverage to identify the major product of the surface reaction. EG: Monolayer graphene; GOx surface: Graphene-oxide-like surface; UHV: Ultra-high vacuum; HRPES: High-resolution photoemission spectroscopy.The authors declare that they have no competing interests.ML participated in overall experiments. KK conducted HRPES experiments, and HL who is a corresponding author participated in overall experiments. All authors read and approved the final manuscript.