In addition, a significantly higher expression of IQGAP1 and β-catenin also usually exists in human HCC tissues, especially their overexpression is clinicopathologically associated with tumor malignancy. Generally the overexpression and interactions of IQGAP1 and β-catenin contribute to HCC progression by promoting cell proliferation and migration.The IQ-domain GTPase-activating protein 1 (IQGAP1) is a multifunctional scaffold protein, which interacts with diverse proteins to regulate cell adhesion and cell migration. The abnormal expression of IQGAP1 widely exists in many cancers, but biological roles of IQGAP1 cooperation with its interacting proteins to involve in tumorigenesis remain to clarify. In this study, we have found that IQGAP1 interacts with β-catenin and regulates β-catenin expression in hepatocellular carcinoma (HCC) cells. The expression levels of IQGAP1 and β-catenin and their associations have a positive correlation with cell metastasis ability in several HCC cell lines. The up-regulation of IQGAP1 and β-catenin improves cell proliferation and migration ability of HCC cells, whereas the knockdown of IQGAP1 by small interfering RNA can decrease β-catenin expression, which results in the reduction of cell proliferation and migration ability  Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, and its prognosis depends on the tendency to metastasize , 2. AlthThe IQ-domain GTPase-activating proteins 1 (IQGAP1) is a scaffold protein that regulates distinct cellular processes including cell adhesion, cell migration, extracellular signals through interacting with numerous proteins , 4. MultIn this study, we focused the protein expression level of IQGAP1 and β-catenin in HCC cells and tissues and their associations with tumor malignancy degree, as well as their roles in cell proliferation and migration ability. Our findings have discovered that the expression of β-catenin can be affected by the IQGAP1 level, and their overexpression and interaction improve cell proliferation and migration ability in HCC progressing processes.2 and 95% air.Human HCC cell lines HuH7 and HepG2 were obtained from the American Type Culture Collection. Human HCC cell lines MHCC-97H and MHCC-97L were established in the Liver Cancer Institute of Fudan University, and theThirty-three pairs of HCC tissues (HCTs) and patients’ autologous para-cancerous liver tissues (PLTs) were surgically resected to collect in West China Hospital, Sichuan University . Informed written consents were obtained from the patients. This project was approved by the Institutional Ethics Committee of State Key Laboratory of Biotherapy, West China Hospital of Sichuan University.All tissues were immediately frozen after surgical operation in liquid nitrogen prior to experiments. The patients’ gender, age and histological type of tumor differentiation were obtained from the surgical and pathological records. And the histological type of tumor differentiation was classified into high differentiation group and low differentiation group. Each caSmall interfering RNA (siRNA) against IQGAP1 were synthesized as the following sequences : IQGAP1,The IQGAP1 cDNA (GenBank gi57242794) was cloned into pCMV6 plasmids by PCR-based cloning method, and the recombinant plasmid pFlag-IQGAP1 was obtained. The β-catenin cDNA (GenBank gi 148233337) was cloned into pEZ-M13 plasmids by PCR, and the recombinant plasmid was named pFlag-β-catenin. Cell transfections of pFlag-IQGAP1 plasmids were performed using Lipofectamine LTX reagent , and the transfections of pFlag-β-catenin plasmids were performed with the reagent Lipofectamine 2000  following the manufacturer’s protocols. The amount of transfected plasmids was 2.5μg for each well of a 6-well plate, and the IQGAP1-specific siRNA was used 100nM for one well of a 6-well plate. In the co-transfection of pFlag-β-catenin plasmids and IQGAP1-specific siRNA, 2.5μg plasmids and 100 nM siRNA was used for one well of a 6-well plate at the same time.Total RNA was extracted using Trizol reagent . The first-strand cDNA was generated using a cDNA synthesis kit . The real-time PCR was performed to measure the relative expression using the Supermix-Bio-Rad kit . The GAPDH mRNA level was taken as a comparison base for the target RNA expression. The relative RNA expression was calculated with the comparative CT method, which was normalized to the internal references. The primers were designed as follows. IQGAP1: forward primer 5′-TCC ATT ACT TAG GAA AGA GTG GAA ACT-3′ and reverse primer 5′-CAA ACA CCA AAG CTT ACA ATA TAG TAC TGC-3′; forward primer 5′-AAG CTC TTA CAC CCA CCA TCC-3′ and reverse primer 5′-GTG CAT GAT TTG CGG GAC AAA-3′ for β-catenin; forward primer 5′-CGT CTC CAC ACA TCA GCA CAA-3′ and reverse primer 5′-TCT TGG CAG CAG GAT AGT CCT T-3′ for c-myc; forward primer 5′-TGT TCG TGG CCT CTA AGA TGA AG-3′ and reverse primer 5′-AGG TTC CAC TTG AGC TTG TTC AC-3′ for Cyclin D1; forward primer 5′- GGG CCA CTT TAA AGA GCA G -3′ and reverse primer 5′- CCT TCA TAC ATC GGG AGC A -3′ for Axin2; forward primer 5′- TGG AAG GAC TCA TGA CCA CA-3′ and reverse primer 5′- TTC AGC TCA GGG ATG ACC TT-3′ for GAPDH.The promoter fragments of β-catenin (from -1018 to +241) were cloned into pGL3 Basic luciferase reporter plasmid (Promega), and the pGL3-catenin plasmid was obtained. HepG2 cells were transfected with 0.5μg or 2μg pFlag-IQGAP1 plasmids to incubate for 24 hours, then transfected with 1μg pGL3-catenin plasmids or the empty pGL3-basic plasmids to incubate for 24 hours. Cells were harvested to assess the luciferase activity according to a commercial Dual-luciferase reporter assay system . 100 ng pRL-TK (Promega) renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. All experiments were performed in triplicate from independent cell cultures.4 cells in 200ul serum-free DMEM were cultured in the upper wells for 24 hours. The migrated cells were fixed and stained with 1% crystal violet. Images were captured using an inverted microscope (Olympus), and the migrated cells were counted manually. The percentage of migrative cells on the condition of IQGAP1 overexpression or knockdown was calculated by comparison with the control transfection with the empty plasmids or non-targeting siRNA.Cell migration ability was assayed based on a transwell chamber apparatus (Millipore). After being transfected with pFlag-IQGAP1 plasmids or IQGAP1-specific siRNA for 48h, cells were seeded in the upper chamber of a transwell, and the bottom of the chambers was filled with 600μl of DMEM containing 10% FBS. 1×103 cells/well were seeded in one 96-well plate in DMEM supplemented with 10% FBS to incubate for 24h. And 20ul of MTT solution  was added to each well to incubate for 2–4 h at 37°C, the formazan crystals were dissolved with 150ul Dimethyl Sulfoxide (Sigma). Absorbance was determined at 570 nm on Multiskan MK3 (Thermo Scientific) immediately. Each assay was separately performed for three replicates and all experiments were repeated at least three times. The cell viability percentage was estimated as follows.Cell viability was measured using MTT assay. After IQGAP1 overexpression or knockdown for 48h, 5×10Cell viability (%) = absorption test/ absorption control ×100%Meanwhile, in order to discriminate if cell growth inhibition was only due to changes in cell proliferation or due to cell death, cells under IQGAP1-specific siRNA treatment were detected cell death by flow cytometry analysis using BD Bioscience Annexin V/PI staining kit . After cells were transfected with siRNA for 48h, cells were harvested to stain with 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) for 15 min at room temperature in the dark. Then cell suspensions were immediately evaluated by flow cytometry . The apoptotic cells were analyzed based on the percentage of Annexin V-positive cells, and the necrotic cells were analyzed based on the percentage of Annexin V-negative and PI-positive cells. Cell death included the apoptotic cells and necrotic cells.HepG2 cells were harvested to dissolve with a co-immunoprecipitation (co-IP) buffer containing 20mM Tris (pH7.5), 150mM NaCl, 1% Triton X-100 and protease inhibitor cocktail (05892970001 Roche). 1–2μg of anti-IQGAP1 antibodies  was incubated with 50μl slurry of protein A+G conjugated sepharose (P2012 Beyotime) to pull down protein complexes from cellular lysates (500μg per sample). And 1–2μg of mouse IgG (A7028 Beyotime) was taken as a control. After washing 4 times, the protein complexes were eluted with SDS-PAGE sample-loading buffer (P0015 Beyotime) and loaded onto PAGE gel for western blot detection.Cells were lysed with RIPA buffer . For protein subfractionation, nuclear and cytoplasmic proteins were respectively extracted from HepG2 cells transfected with pFlag-IQGAP1 plasmids by using NE-PER Nuclear and Cytoplasmic Extraction Reagents . Proteins were separated by 10% SDS-PAGE electrophoresis, and transferred from the gel onto the PVDF membrane (Amersham Biosciences) to detect protein expression level. The PVDF membrane was respectively incubate with anti-IQGAP1  and anti-β-catenin monoclonal antibody at a dilution of 1:1000 at 4°C overnight, followed by three 15-min washes in phosphate-buffered saline within 0.1% Tween-20. The membranes were then incubated with HRP-conjugated secondary antibodies at 37°C for 60 min. Detection was performed with Western blot reagent ECL (Amersham Biosciences). The GAPDH detection against its rabbit monoclonal anti-GAPDH antibodies with being diluted 1:5000 [EPR16891]  was taken as a control. The nuclear protein LMNB1, as a nuclear housekeeping control, was detected against anti-LMNB1 antibodies with a dilution 1:1000 [ag3631] . The Image J software was used to quantify the Western blot result.The HCTs, PLTs were paraffin-embedded and cut into sections with 5μm-thickness for hematoxylin-eosin and immunohistochemistry (IHC) analysis mainly according to our previous protocols . The ant-); scores of 1–3 were accepted as a weakly positive expression (+), while 4–6 scores were defined as a moderately positive expression (++), and 7–12 scores were accepted as a strongly positive expression (+++), namely overexpression (scoring > 7).The IHC scoring analysis of tissue slices was evaluated using light microscopy by two pathologists blinded for patient outcome to minimize variability. The estimated percentage of staining was determined by calculating average staining cells of 3–4 microscopic fields under 400-fold magnification. According to general evaluation standards, the prop<0.05 was considered to be statistically significant.All data were determined as mean ± standard error of the mean (SEM). Comparisons between two groups were performed by Student’s t test. Differences among multiple groups were assessed by one-way ANOVA analysis. And Spearman correlation was used to analyze the correlation of data. We have found that the endogenous IQGAP1 interacts with β-catenin in HepG2 cells by co-IP analysis . In ordeFor purpose of investigating the biological effects of the expression level of IQGAP1 and β-catenin in HCC progression, we further compared their expressions in several HCC cell lines with different metastasis abilities. As expected, IQGAP1 expression was gradually increased in HepG2, MHCC-97L and MHCC-97H cells than the normal liver cell line LO2 . EspeciaFurthermore, the associations of IQGAP1 and β-catenin expression were confirmed to play important roles in liver cancer progress from clinic immunohistochemistry analysis for liver cancer tissues as follows.in vivo were examined in 33 pairs of HCTs and patients’ autologous PLTs by immunostaining. Each tissue information and protein IHC scoring were summarized in the versus β-catenin . Out of all 33 cancer samples, IQGAP1 and β-catenin were overexpressed (strong expression) (The expression profiling of IQGAP1 and β-catenin ression)  in 36.36ression) , which wression)  with 8.3ression)  averagelression)  and weakression) , 8 casesression) , which wression) . The expression) . Our staSimilarly, except 9 cases (27.27%) showed weak expression of β-catenin, the other 19 HCTs (57.57%) was obviously detected in expression, including 8 cases (24.24%) with moderate staining and 11 cases (33.33%) with overexpression in HCTs . The expFurthermore, the association of protein expression with HCC clinic information was also analyzed. The clinicopathological characteristics of HCC samples included patients’ gender, age and tumor differentiation level. The average IHC score of IQGAP1 and β-catenin expression was 4.58 and 4.17 respectively, a moderate expression, among 24 HCC samples with a high differentiation , while aWhether the regulation of IQGAP1 over beta-catenin is at the transcriptional or post-transcriptional level? Firstly we detected the mRNA level of IQGAP1 and β-catenin upon IQGAP1 overexpression and knockdown by real-time PCR. As results, when IQGAP1 mRNA was overexpressed to 2.9 times, the mRNA level of β-catenin was up-regulated to 2.34 times . On the Next, we evaluated whether the upregulated β-catenin induced some target gene changes upon IQGAP1 overexpression. Response for regulations of β-catenin and IQGAP1, the mRNA expression level of three target genes, including c-myc, cyclin D1 and Axin2 were measured by real-time PCR . The uprin vitro and in vivo, and their overexpression levels are associated with tumor malignancy degree of HCC. Therefore we hypothesized that IQGAP1 may play a role in mediating cell proliferation and cell migration.Based on our findings, IQGAP1 and β-catenin are usually overexpressed p<0.001) in cell number (p<0.01) (p<0.01) (Response to IQGAP-overexpression  and knocl number . Meanwhi(p<0.01) . Whereas(p<0.01) .p<0.001) approximately detected by MTT, and cell migration ability was enhanced to 1.33-fold compared to the transfected with empty plasmids . On the contrary, when IQGAP1 knockdown by IQGAP1-specific siRNA in HuH7 cells, cell growth was decreased to 79%  in cell quantity, and a lower cell migration with 0.70-fold downregulation was observed  compared with mock cells transfected with non-target control oligonucleotides.Moreover we repeated the experiments in another HCC cell line HuH7, and the results also indicated high expression level of IQGAP1 promoted cell proliferation and migration ability . The conp>0.05), which indicated that the observed effects are only due to changes in proliferation rate.Flow cytometry analysis was performed to eliminate the decreased cell proliferation effects caused by cell death .. CorresIn order to investigate the functional associations of IQGAP1 with β-catenin, a rescue experiment was performed by overexpressing β-catenin in the condition of IQGAP1 knockdown. When the endogenous IQGAP1 in HepG2 was greatly inhibited by IQGAP1 siRNA  and protin vitro and in vivo promotes cell proliferation and migration ability in HCC, while their downregulation reduces cell growth and migration.In combination with the protein expression level changes of IQGAP1 and β-catenin and cell growth, we can conclude that the overexpression of IQGAP1 and β-catenin Due to the multiple binding partners of IQGAP1  based onIn eukaryotic cells, scaffold proteins play crucial roles in many important signaling pathways , 26. As We have demonstrated that the overexpression of IQGAP1 can upregulate the expression of β-catenin. In several hepatocellular cell lines, the overexpression level of IQGAP1 and its binding protein β-catenin have a positive correlation with cell metastasis potentials due to their contributions for cell proliferation and migration. And a significantly higher expression of IQGAP1 and β-catenin also usually exists in human HCC tissues; especially their overexpression is clinically correlated with tumor malignancy or differentiation degree. The aberrant accumulation of β-catenin is observed at high frequency in many cancers . This acDue to multiple interacting partners of IQGAP1 , IQGAP1 in vitro and in vivo due to their contributions for cell proliferation and migration.The overexpression of IQGAP1 and β-catenin is associated with tumor progression in HCC S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 FigCells were stained with Annexin V-FITC/PI. siNC: HepG2 cells transfected with control siRNA; si-IQ: HepG2 cells transfected with IQGAP1 siRNA.(TIF)Click here for additional data file.S2 FigIQGAP1 regulated cell proliferation and migration ability of HuH7 cells. Overexpression of IQGAP1 enhanced cell proliferation (A) and cell migration  in HuH7 cells. IQGAP1 Knockdown decreased cell proliferation (B) and migration . Control: HuH7 cells transfected with empty pCMV6 plasmids; flag-IQ: HuH7 cells transfected with pFlag-IQGAP1. siNC: HuH7 cells transfected with control siRNA; si-IQ: HuH7 cells transfected with IQGAP1 siRNA. Scale bar represents 100 μm .(TIF)Click here for additional data file.


However, little is known about the relationship between abnormal expression of β-catenin and cisplatin chemotherapy in oral squamous cell carcinoma (OSCC). The present study aimed to investigate the effect of β-catenin on OSCC cisplatin resistance and evaluated the drug susceptibility of stable cell lines with β-catenin knockin and knockdown. In this study, we found that higher expression level of β-catenin can be observed in CDDP-treated cell lines as compared with the control group. Furthermore, the expression levels of β-catenin increased in both a concentration- and time-dependent manner with the cisplatin treatment. More importantly, the nuclear translocation of β-catenin could also be observed by confocal microscope analysis. Stable cell lines with CTNNB1 knockin and knockdown were established to further investigate the potential role and mechanism of β-catenin in the chemoresistance of OSCC in vitro and in vivo. Our findings indicated that overexpression of β-catenin promoted cisplatin resistance in OSCC in vitro and in vivo. We confirmed that GSK-3β, C-myc, Bcl-2, P-gp, and MRP-1 were involved in β-catenin-mediated drug resistance. Our findings indicate that the Wnt/β-catenin signaling pathway may play important roles in cisplatin resistance in OSCC.Abnormal expression of  Head and neck cancer is the sixth most common cancer worldwide with approximately 600,000 new cases per year . Betweenβ-catenin, coded by CTNNB1, is an important adhesion molecule and a key regulator in the Wnt signaling pathway, which has many important functional roles including the maintenance of epithelial stability, growth, and differentiation [β-catenin pathway was reported to be associated with chemoresistance in epithelial ovarian cancer [β-catenin has been demonstrated to be associated with drug resistance in different types of human cancers [β-catenin in OSCC.ntiation . The Wntn cancer , 20. Fur cancers , 22. Howβ-catenin in tissue samples from patients who had received cisplatin chemotherapy compared with tissue specimens from patient without chemotherapy. We found that overexpression of β-catenin could promote P-gp, Bcl-2, MDR, and MRP-1 expressions that enhanced cisplatin resistance in OSCC cell lines. These findings provided important clues to the mechanisms involved in OSCC drug resistance. In addition, we found an increase in the expression levels of β-catenin in the OSCC cell lines treated with cisplatin compared with the controls. Therefore, we hypothesize that β-catenin may play a crucial role in the development of drug resistance in OSCC. In the present study, we investigated the role of β-catenin in the development of chemoresistance in OSCC cell lines.In our previous study, we demonstrated that P-glycoprotein (P-gp), Bcl-2, MDR, and multidrug resistance-associated proteins-1 (MRP-1) were involved in cisplatin resistance in tongue squamous cell carcinoma . We conf2 at 37°C and then subcultured by trypsinization . For the analysis of the expression levels of β-catenin, OSCC cells were treated with cisplatin (0.5 and 2 μg/mL) for 24 h. The control cells were treated with regular media.Two human OSCC cell lines (SCC-15 and SCC-25) were selected for analysis. SCC-15 and SCC-25 cells  were cultured in DMEM-Ham's F12 medium  with 400 ng/mL hydrocortisone , 1% penicillin/streptomycin , and 10% FBS . All cells were incubated in a humidified atmosphere containing 5% COWe used recombinant lentiviruses, pGLV3-GFP-CTNNB1-shRNA, pGLV3-GFP-NC, pGLV5-GFP-CTNNB1, and pGLV5-GFP-NC . The amplification primer sequence for CTNNB1: CTNNB1-F: 5′-ATGGCTA-CTCAAGCTGATTTGAT-3′, CTNNB1-R: 5′-TTACAGGTCAGTATCAAACCA-GG-3′. The siRNA primer sequences are siRNA-homo-1302, 5′-AGGTGCTAT-CTGTCTGCTCTA-3′, and NC siRNA, 5′-TTCTCCGAACGTGTCACGTTTC-3′.Stable SCC-25 cell lines with CTNNB1 knockin and knockdown were established via infection of the high titer lentiviral particles , which was cotransfected into 293T cells and purified according to the manufacturer's instructions. The supernatant containing infectious lentiviruses was harvested for transductions. The isolated transfectants were further selected by culture in 2 mg/mL Puromycin  and confirmed by proliferation assay. The transfection efficiency was detected by PCR and Western blotting. Stable expressing cells with CTNNB1 knockin and knockdown were used in the subsequent study.μg/mL cisplatin  for 24 h following the manufacturer's protocol. Cells were collected and stained with Annexin V PE/7AAD apoptosis kit  and detected by flow cytometry . Cells that were negative with propidium iodide (PI) and Annexin V are considered healthy, cells are considered apoptotic with PI negative and Annexin V positive, and cells that are positive to both PI and Annexin V are considered necrotic. The experiments were repeated at least three times.For the apoptosis assay, cells with stable CTNNB1 overexpression and knockdown were treated with 2 3 cells per well for cell the viability assay and 2.0 × 103 cells per well for the proliferation assay and were cultured accordingly. For the cell viability assay, cells were treated with cisplatin  for 24 h. Proliferation assay was performed on days 1 to 7. Cell growth was assessed using the Cell Counting Kit 8 assay  according to the manufacturer's protocol. After appropriate incubation, 10 μL of CCK-8 solution was added per well and incubated at 37°C for 1 hour. The absorbance of each well was measured at 450 nm using a microplate reader . Growth curve was drawn to assess the effect of β-catenin on proliferation. The cytotoxicity of cisplatin in the OSCC cell lines was quantified by the inhibitory concentration 50% (IC50), which was calculated as previously described [Cells with stable CTNNB1 overexpression and knockdown were seeded at a density of 5.0 × 103 cells) were seeded on a glass-bottom dish  and cultured with cisplatin (1 μg/mL) for 24, 48, and 72 h and with different concentrations of cisplatin  for 24 h. Regular media was used as the control. Primary antibodies to β-catenin  were added to the cells and incubated at 4°C overnight. After washing, FITC-conjugated goat anti-rabbit IgG  was added and incubated at room temperature in the dark for 2 h and then covered with Prolong® Gold Antifade Reagent and DAPI for 10 minutes at room temperature . Confocal images were taken with a Zeiss LSM 510 laser scanning confocal microscope .SCC-15 and SCC-25 cells  according to the manufacturer's instructions. The first-strand cDNA synthesis was performed by the use of Applied Biosystems® Veriti PCR . qPCR was performed using a LightCycler® 480 SYBR Green I Master , following thermal cycling parameters: preincubation at 95°C for 5 min, followed by 40 cycles of amplification , melting curve , and cooling (40°C for 10 s). Expression levels were analyzed using the ΔΔCq method [Total RNA was isolated from the OSCC cell lines using TriPure  according to the manufacturer's instructions. 1 q method , and genβ-catenin knockin and knockdown were lysed with RIPA buffer  and nuclear fractions of SCC-25 and SCC-15 cells were isolated by the use of Cellytic™ Nuclear™ Extraction Kit  to detect the cellular localization of β-catenin. Protein concentrations were quantified by the use of BCA Protein Assay Kit . Western blotting was performed according to standard protocols. 40~60 μg total protein extract was separated by SDS-PAGE on a 8%–10% gradient gel and then transferred onto a PVDF membrane  for 1 to 2 h. The membrane was blocked in 5% nonfat dry milk at room temperature for 1 hour and incubated overnight at 4°C with the following antibodies: anti-MRP-1 , anti-P-gp , anti-C-myc , anti-GSK-3β , anti-Bcl-2 , anti-β-catenin , anti-β-actin , and anti-histone . The secondary antibody was horseradish peroxidase-conjugated anti-mouse  and anti-rabbit . The membrane was then incubated with HRP-linked secondary antibodies (1 : 1000) at room temperature for 2 h. Membranes were analyzed by chemiluminescence using an ECL system  and bands were visualized using FluorChem Q . All experiments were repeated at least three times.SCC-25, SCC-15, and the cells with n = 24; 3-4 weeks old) from the Laboratory Animal Center of Sun Yat-sen University were used in the study. Mice were maintained in a temperature controlled on a 12-h light-dark cycle, with free access to food and water at 22°C. For the overexpression of β-catenin, a total of 1 × 107 SCC-25 cells with CTNNB1 knockin were injected in the left posterior flanks, and the right anterior flanks were used as the negative control. For the decreased expression of β-catenin, a total of 1 × 107 SCC-25 cells with CTNNB1 knockdown were injected in the left posterior flanks, and the right anterior flanks were used as the negative control. The tumor size was measured using Vernier calipers. Tumor volume was calculated by the following formula: volume = 0.5 × length × width2 . When the tumor had grown to an average volume of 100 mm3 (after about 14 days), the mice were given an injection of cisplatin (4 mg/kg) through the tail vein at 3-day intervals for five times. The tumor size was monitored every 3 days after inoculation. The animals were then sacrificed under anesthesia with an overdose of pentobarbital sodium  and individual tumor volumes were calculated. All animal procedures were approved by the Sun Yat-sen University Medical Experimental Animal Care Commission.Male BALB/c-nu nude mice  in SCC-25 cells with CTNNB1 knockdown compared to the controls. Correspondingly, cell proliferation and survival were significantly increased in SCC-25 cells with CTNNB1 knockin after treatment with cisplatin  compared to reduced expression of β-catenin in OSCC cells. β-catenin was reported to be involved in chemoresistance/radioresistance in colon cancer cells [β-catenin in OSCC cells suppressed cell proliferation and promoted apoptosis, making the cells more sensitive to cisplatin, which was also confirmed in our animal study results. β-catenin is an important adhesion molecule and a key regulator in Wnt signaling pathway. Wnt/β-catenin signaling pathway has been shown to be involved in cisplatin resistance through the regulation of β-catenin. Upregulation of β-catenin expression in SCC-25 cells led to an overexpression of GSK-3β and C-myc [β-catenin expression led to lower expression levels of GSK-3β and C-myc. Both GSK-3β and β-catenin are two key regulators in Wnt/β-catenin signaling pathway involved in the regulation of cell growth and adhesion. In the absence of Wnt signaling, β-catenin is phosphorylated by GSK-3β and targeted for degradation [β-catenin from being phosphorylated by GSK-3β, resulting in its nuclear translocation [β-catenin translocated and accumulated in the nucleus in OSCC cells treated with cisplatin. Accumulation of nuclear β-catenin and the subsequent binding to Tcf transcription factors [β-catenin. Moreover, evidence suggests that overexpression of Bcl-2 in tumor cells can result in their escape from cell apoptosis and resistance to anticancer drugs [β-catenin in SCC-25 cells led to higher expression levels of P-gp and MRP-1, whereas the reverse was true with reduced expression of β-catenin compared with the control cells. Substantial evidence suggests that P-gp [Several mechanisms of cisplatin resistance have been extensively reported , involvier cells . Conversnd C-myc , whereasradation . Activatlocation , 35. In  factors  promoted factors . The abn factors  allowing factors . Bcl-2 i factors , 40. We er drugs . Converser drugs , which what P-gp  and MRP-hat P-gp  are assohat P-gp , 46.β-catenin was associated with cisplatin resistance in OSCC cells and that reduced expression of β-catenin could confer sensitivity to cisplatin resulting in better treatment efficacy. However, the precise molecular mechanisms and clinical significance of our findings need to be further investigated. Our results demonstrated that β-catenin might play important roles in cisplatin resistance in OSCC through the regulatory mechanisms of Wnt/β-catenin signaling pathway. Therefore, a thorough understanding of molecular mechanism involving β-catenin would enable the development of novel strategies to overcome possible drug resistance.In conclusion, our findings revealed that overexpression of


Sorafenib is a kinase inhibitor used as anticancer drug against various human tumors, including advanced hepatocellular carcinoma (HCC). β-Catenin and prothymosin alpha (PTMA) are overexpressed in HCC and other tumors. Previous studies have shown that PTMA expression modulates the response of HCC cells to sorafenib. However, the underlying mechanism of PTMA activity in this context remains unclear. We show here that sorafenib inhibits both β-catenin and PTMA in a dose-dependent manner. Silencing β-catenin reduces PTMA level and sensitizes HCC cells to sorafenib. In contrast, ectopic expression of β-catenin induces PTMA expression and cell resistance to the drug. Sorafenib inhibits PTMA expression at the transcriptional level by inhibiting the β-catenin pathway. Nucleotide deletion analysis of the PTMA gene promoter reveals that a DNA segment lying 1,500–1,600 bp upstream of the PTMA transcription start site represents an AP-1-binding site that is critical for β-catenin modulation of gene transcription in response to sorafenib. In addition, chemical inhibitors that target JNK abrogate β-catenin/AP-1 binding to the endogenous PTMA gene and reduces PTMA transcription and protein expression. Silencing of β-catenin or c-Fos induces similar effects on gene regulation and these are reversed by ectopic expression of β-catenin. Mutations in the PTMA promoter at the predicted β-catenin/AP-1 binding site partly abrogate sorafenib's effects on PTMA transcription. These results indicate that PTMA is induced by the oncoprotein β-catenin and protects HCC cells against sorafenib-induced cell death. The β-catenin/JNK/PTMA axis may thus represent a novel target for chemotherapy against HCC. HCC cells transfected with the plasmids were incubated 48 hrs. Transcriptional activity was monitored using a dual luciferase reporter assay (Promega) and a luminescence reader  according to the instructions provided by the manufacturers. For each experiment, luciferase activity was normalized to Renilla luciferase.http://www.cbrc.jp/research/db/TFSEARCH.html; Tokyo, Japan). Primers were designed using Primer 3.0 . TCF4 and CREB-binding sites were described previously [[−ΔCt].Formaldehyde cross-linking and ChIP assays of tissue culture cells were performed based on a modified protocol  as descreviously , 62, 63.p value) was calculated with a two-tailed Student's t test for single comparison. The symbols *, **, and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively.Data were reported as mean values ± Standard deviation (SD). Three independent experiments were performed unless indicated otherwise. Statistical significance (


Our data showed that PC12 cell viability was inhibited by rotenone, but attenuated by GSK-3β antagonists LiCl or SB216763. The activity of Wnt/β-catenin pathway was deregulated on exposure of rotenone in a concentration-dependent manner. After the interference of β-catenin with siRNA, LiCl or SB216763 failed to protect PC12 cells from apoptosis by the rotenone toxicity. Our data confirmed that Wnt/β-catenin signaling activated by LiCl or SB216763 enhanced Nurr1 expression to 2.75 ± 0.55 and 4.06 ± 0.41 folds respectively compared with control detected by real-time PCR and the interaction of β-catenin with Nurr1 was identified by co-immunoprecipitate analysis. In conclusion, the data suggested that Wnt/β-catenin and Nurr1 are crucial factors in the survival of DA neurons, and the activation of Wnt/β-catenin pathway exerts protective effects on DA neurons partly by mean of a co-active pattern with Nurr1. This finding may shed a light on the potential treatment of Parkinson disease.Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic (DA) neurons in the substantial nigra pars compacta. Increasing evidence showed that Wnt/β-catenin pathway and the orphan nuclear receptor Nurr1 play crucial roles in the survival and functional maintenance of DA neurons in the midbrain and GSK-3β antagonists LiCl and SB216763 were used to activate Wnt/β-catenin pathway experimentally. However, the detail mechanism underlying the neuroprotection against apoptosis on DA neuron is still unclear and the interaction between Wnt/β-catenin and Nurr1 remains undisclosed. In this study, using cell biological assay we investigated the function of Wnt/β-catenin and its crosstalk with Nurr1 on the course of PC12 cell degeneration  Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic (DA) neurons in the substantial nigra pars compacta (SNc) –2. While-/- mice died after 24 hours without DA neurons in the midbrain[Wnt and Nurr1 are important factors in the survival and functional maintenance of DA neurons in midbrain –12. The  midbrain. The pol midbrain. Meanwhi midbrain and accu2 at 37°C. Cells were seeded on 96-well plates, 6-well plates or 25cm2 plastic flasks at a density of 1×105 cells/mL for 24 hours. The culture of HEK-293T cells was based on the instruction of Invitrogen.Differentiated PC12 cells, which were obtained 5 hours after the addition of NGF, were purchased from the Cell Library of the Chinese Academy of Science , maintained in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose  supplemented with 10% fetal bovine serum , 100 U/mL benzyl penicillin, and 100 mg/L streptomycin  and cultured in a humidified incubator with 5% COTo investigate Wnt/β-catenin signaling pathway, 2 selective GSK-3β inhibitors LiCl and SB216763 were used. LiCl  was added into the cultures for 72 hours followed by rotenone treatment for 24 h. SB216763  was added into the cultures 30 minutes prior to rotenone 24hours-exposure . In β-catenin gene silencing tests, differentiated PC12 cells were treated with the siRNA for 6 hours, and then the medium was replaced and added with LiCl for another 72 hours, followed by treatment with 1 μmol/L rotenone for last 24 hours. Similarly, following the siRNA protocol, differentiated PC12 cells were treated with SB216763 30 minutes prior to 24 hours of rotenone.The cell counting kit-8 (CCK-8) assay was performed to measure PC12 cells viability following rotenone, LiCl, and SB216763 treatments, respectively. Briefly, 10 μL of the CCK-8 kit reagent per 100 μL of medium was added to the wells of 96-well plate and incubated at 37°C for 2 hour. Cell viability was assessed by measuring the absorbance at 450nm with ELISA plate reader. The results were expressed as a percentage of the control group. Each treatment group was replicated in three wells.Caspase-3 activity was determined using the Caspase-3/CPP32 Fluor metric Assay Kit . For each assay, 50 μg of cell lysate was loaded and read on the fluorescence microliter  with 400 nm excitation and 505 nm emission filter. Caspase-3 activity in sample was expressed as folds comparing to control.B-catenin siRNA or control siRNA were chemically synthesized by Guangzhou Riborio CO. LTD . β-catenin siRNA: sense strand: 5’ -GCACCAUGCAGAAUACAAA dTdT -3’, Antisense strand: 5’ -UUUGUAUUCUGCAUGGUGCTdTd -3’. Cells were transfected with 100 nmol/L siRNA at 60% confluence using Lipofectamine2000  according to the transfection protocol. The cells were harvested for analysis the transcription and protein of β-catenin at 24 h or 72 h following transfection, respectively. During the transfection, 2 mmol/L LiCl or 5 μmmol/L SB216763 was added to determine whether these chemicals protect PC12 cells from rotenone toxicity under depletion of β-catenin. At that time, total RNA was collected to detect the relative mRNA level of the target genes including β-catenin and Nurr1.Total RNA was isolated using TRIzol reagent  and reverse transcribed into first-strand complementary DNA (cDNA) using reverse transcription kit . Real-time polymerase chain reaction (PCR) was performed in a fluorescent temperature cycler  using a Light Cycler FastStart DNA Master Plus SYBR Green kit (Roche Diagnostics GmbH) with the following primes. β-catenin forward, 5’-GTCTGAGGACAAGCCACAGGACTAC-3’; β-catenin reverse, 5’- AATGTCCAGTCCGAGATCA GCA-3’; Nurr1 forward, 5’- CCAATCCGGCAATGACCAG-3’; Nurr1 reverse, 5’-TGATGATCTCCAT AGAGCCAGTCAG -3’. Rat GAPDH gene was used as an internal control. After 94°C for 4 min, the experimental reaction consisted of 39 cycles of 94°C for 15 s and 61°C for 45 s. Fluorescent readings from real-time PCR reactions were quantitatively analyzed by determining the Ct difference (delta Ct) between β-catenin/Nurr1 and the internal GAPDH control. The mRNA expression levels were determined by the formation of 2-delta Ct.TM reagent Plus . At 72 h after transfection, cell lysates were prepared for co-immunoprecipitation.Full-length human Nurr1 and β-catenin genes were inserted into pShuttle-IRES-hrGFP-2 vectors . HEK-293T cells or PC12 cells were transfected with vectors carrying Nurr1, β-catenin gene, or control vector with EndoFectin3VO4, and protease inhibitor cocktail . The supernatant was pre-cleared with protein-A-Sepharose beads and incubated overnight at 4°C. After incubation, protein-A-Sepharose beads were added for 4 h to make the immune complex and washed in the washing buffer I , Washing buffer II , Washing buffer III  at 4°C. And then 500 μg of cell lysate was used to co-immunoprecipitation with rabbit anti-β-catenin  and mouse Nurr1 antibodies . Western blotting was carried out to detect Nurr1 and β-catenin respectively. Normal IgG  was used as a control.A Pierce Immunoprecipitation Kit  was used to detect the immunoprecipitation of Nurr1 with β-catenin proteins in transfected HEK-293T cells or PC12 cells. Briefly, cells were lysed in ice-cold immunoprecipitation (IP) buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% NP-40, 1 mmol/L NaImmunoblotting was performed in accordance with a standard procedure. Briefly, cells were homogenized with ice-cold lysis buffer  containing protease inhibitor cocktail tablets . BCA protein assay kit  was used to measure protein concentrations. Both 20μg proteins and in each group and 5ug protein marker were separated by 10% SDS-PAGE and transferred to PVDF membranes . Positive and negative controls also were used for the first time to detect the specificity antibodies. After the membranes were blocked in 5% skim milk, 0.05% Tween 20, and Tris-buffered saline (TBS) for 1 hour, they were incubated with primary antibodies: rabbit anti-Wnt3a , rabbit anti-β-catenin , rabbit anti-non-phospho-β-catenin , rabbit anti-Nurr1  and rabbit anti-GAPDH  overnight at 4°C. The second horseradish peroxidase (HRP)-labeled antibody  were used in the next day. The bands were visualized using enhanced chemiluminescences (ECL) method . Membranes probed for GAPDH used as an internal control. The protein bands were quantified using image analysis software  and the protein levels were expressed as percent (%) of controls.The GST fusion proteins used for GST pull down assay were produced by cloning Nurr1 into pGEX6p1 . The primers used for the clone were: AF1 for Nurr1 , DBD for Nurr1 , AF2 for Nurr1 . Nuclear proteins containing β-catenin were obtained by transfecting PC12 cells with β-catenin vectors for 24 hours and incubated with GST-fused mutants of Nurr1, AF1, DBD, AF2. And washed with Wash buffer containing 50mM Tris·HCl(pH7.3), 150mM NaCl, 3mM MgCl2, 1mM EDTA, 1mM DTT, 0.5%Triton X-100 β-catenin immobilized on glutathione-Sepharose beads prior to analysis by sodium dodecylsulfate-polyacrylamide gel electrophoresis.Soluble chromatin from PC12 cells was prepared with an acetyl-histone H4 immunoprecipitation assay kit (Upstate Biotechnology) and immunoprecipitated with antibodies against β-catenin in the presence, absence of LiCl and β-catenin siRNA. Specific primer pairs were designed to amplify the promoter region of Nurr1 . The CHIP resulting DNA samples were amplified by real time quantitative using SYBR Green master Mix .All results were presented as means ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test were used to compare differences between means in more than two groups. The level of significance was set at P < 0.05. All the statistical analyses were performed with SPSS 21.0 software for Windows .As showed in P < 0.01) in comparison to rotenone treatment alone. To further investigate Wnt/β-catenin signaling in rotenone-induced cell injury, we examined Wnt3a, β-catenin and non-phospho-β-catenin by Western blotting. After the cells were exposed to 1 μmol/L rotenone, a significant decrease in β-catenin and non-phospho-β-catenin was found in PC12 cells . Pretreatment of 2 mmol/L LiCl significantly attenuated those changed   attenuate rotenone-induced cell injury in a concentration-dependent manner . Therefo < 0.05) . CombineP > 0.05)  .Previous studies have demonstrated that both β-catenin and Nurr1 have protective effects on DA neurons. But little is known about whether there is a synergistic effect between β-catenin and Nurr1 and whether one regulates the expression of the other. So the interaction between β-catenin and Nurr1 was investigated. As our results showed, LiCl or SB216763 both significantly raised β-catenin and Nurr1 mRNA levels . SimilarP < 0.05). These data clearly showed that mechanistically β-catenin not only directly regulates the gene expression of Nurr1 but also interacts with Nurr1 protein to execute their cell survival function against rotenone.The crosstalk between β-catenin with Nurr1 was further studied based on the hypothesis above in both PC12 cells and rat midbrains. As shown in + or 6-OHDA resulted in dysfunction of Wnt/β-catenin signaling pathway both in mice and cellular models of Parkinson disease [This study demonstrated the protection effect of Wnt/β-catenin and Nurr1 on the differentiated PC12 cells against rotenone-induced DA neurotoxin. Differentiated PC12 cells are widely used as a suitable model to explore the mechanisms of degeneration of DA neuron in Parkinson’s disease  and the  disease , 35–36.Our data indicated that Wnt/β-catenin signaling pathway can be activated by GSK3β antagonists LiCl or SB216763 in PC12 cells. LiCl or SB216763 was found to increase the level of β-catenin and non-phospho-β-catenin (a form of active β-catenin) and attenuate the cell death affected by rotenone, but either of them failed to protect β-catenin siRNA transfected PC12 cells from the cell death induced by rotenone, indicating β-catenin might be a key molecule in Wnt/β-catenin pathway for PC12 cells to survive. Consistently, our previous data showed that 6-OHDA decreased β-catenin level, but activation of Wnt/β-catenin pathway by exogenous wnt1 attenuated 6-OHDA-induced neurotoxicity through restoring mitochondria and endoplasmic reticulum function . In addiThe mechanisms underlying the protection of the Wnt/β-catenin signaling pathway on DA neurons remain elusive. It involves in multiple mechanisms including the regulation of pro-survival process or anti-apoptosis ,9, plast


IPF), an insidious disease with grave prognosis, is characterized by heterogeneous fibrosis with densely fibrotic areas surrounded by nonfibrotic normal‐looking tissue, believed to reflect a temporal development. The etiology is incompletely elucidated, but aberrant wound healing is believed to be involved. Embryonic signaling pathways, including Wnt signaling, are reactivated in wound healing, and we therefore aimed to investigate Wnt signaling, and hypothesized that Wnt signaling would correspond to degree of fibrosis. Material from 10 patients with IPF were included (four diagnostic biopsies and six donated lungs) and compared to healthy controls (n = 7). We investigated markers of Wnt signaling  histologically in lung parenchyma with variable degree of fibrosis. Our results suggest that Wnt signaling is significantly altered (P < 0.05) already in normal‐looking parenchyma. The expression of Wnt3a and ICAT decreased (both P < 0.01) in IPF compared to healthy lungs, whereas β‐catenin, Wnt5a/b, DAAM1 and NLK increased . ICAT is further decreased in dense fibrosis compared to normal‐looking parenchyma in IPF (P < 0.001). On the basis of our results, we conclude that from a Wnt perspective, there is no normal parenchyma in IPF, and Wnt signaling corresponds to degree of fibrosis. In addition, β‐catenin and Wnt5a appears coupled, and decreased inhibition of β‐catenin may be involved. We suggest that the interaction between β‐catenin, ICAT, and Wnt5a/b may represent an important research area and potential target for therapeutic intervention.Idiopathic pulmonary fibrosis ( In this study, we investigated β‐catenin‐mediated signaling as well as Wnt/PCP and Wnt/Ca2+ in IPF.Idiopathic pulmonary fibrosis (IPF) is an insidious disease, with grave prognosis and few treatment options  and diagnostic open chest biopsies (n = 4). Control material (n = 7) was obtained from explanted lungs from healthy individuals fulfilling the criteria for lung donation after brain stem death but unmatched to a recipient. All protocols were reviewed and approved by the local ethic committee. Patient characteristics are further described in Table Fibrotic lung material was obtained from explanted lungs , before primary antibody was applied (omitted for controls) and incubated 1.5 h (RT). Slides were rinsed and secondary antibody, visualized using DAB, counterstained in HTX and mounted. The following antibodies were used to visualize markers of Wnt signaling: Wnt3a , Wnt5a/b , β‐catenin , Disheveled‐associated activator of morphogenesis 1/DAAM1 , Inhibitor of β‐catenin and TCF‐4/ICAT  and Nemo‐like kinase/NLK . For double stainings to visualize cell types, the following antibodies were used in combination with β‐catenin; Prosurfactant Protein C , pan‐cytokeratin  and Vimentin , as well as ICAT and NLK. Fluorescent secondary antibodies were used .Paraffin‐embedded tissue was section into 4‐2). For each marker, 6–20 images were taken of each lung section, the larger number in IPF lungs, where several different tissue types were found. Regions were defined based on histologic appearance   Fig. D, characDense: Densely fibrotic parenchyma, few structures can be identified beside scattered airways and vessels. Found in all IPF lungs . The number of positively labeled cells/nuclei were counted and related to the tissue area . Normal‐looking tissue was found in all diagnostic biopsies Fig. G–H, but β‐catenin copositivity with ProSpC  in IPF compared to controls Figs. . No diffBoth epithelial and endothelial cells were Wnt3a‐positive, as were spindle‐shaped mesenchymal cells likely to be fibroblasts/myofibroblasts.β‐catenin‐positive nuclei  in normal‐looking parenchyma in IPF (472 ± 68 nuclei/mm2), as well as in border zones (503 ± 211), dense fibrosis (589 ± 99), and inflammatory zones (929 ± 130) compared to healthy controls (257 ± 66). A very strong tendency was seen toward increased nuclear localization in dense fibrosis compared to normal‐looking parenchyma (P = 0.059) in IPF.The number of ei Figs. A, 4 was Most labeled cells were found to be epithelial or endothelial cells, but also mesenchymal cells, likely fibroblasts/myofibroblasts, were positive.2) is significantly decreased (P < 0.001), compared to normal‐looking parenchyma (1520 ± 54).Expression of ICAT Figs. A, 4 was β‐catenin‐positive cells.Importantly, ICAT‐positive cells appear to be the same types as P < 0.05) increased five to ninefold in the fibrotic lung  and dense fibrosis (1050 ± 316) compared to normal‐looking parenchyma (688 ± 124).Wnt5a is increased in fibroblasts isolated from IPF patients  in normal‐looking parenchyma (227 ± 66 cells/mm2), border zones (1320 ± 257), and dense fibrosis (320 ± 69) in IPF patients compared to healthy donors (104 ± 60 cells/mm2). DAAM1 also displayed a 1.5 to 6‐fold increased in border zones, dense fibrosis and inflammatory zones, compared to normal‐looking parenchyma in IPF (P < 0.01).To investigate Wnt/PCP signaling we studied expression and localization of DAAM1 Figs. B, 4, whi2+ signaling pathway, and inhibits β‐catenin activation. Results , border (715 ± 199), and densely fibrotic (287 ± 56) tissue from IPF patients expressed compared to healthy controls (161 ± 28 cells/mm2). In addition, NLK expression was also increased (3 times) in both border zones and inflammatory zones, compared to normal‐looking parenchyma.Nemo‐like kinase is activated by the Wnt/Cats Figs. B, 4 showβ‐catenin activation and decreased inhibition.To our knowledge, this study presents the first histological mapping different markers of Wnt signaling in IPF, providing a unique characterization areas with different tissue density that likely represents different temporal phases of IPF. We conclude that, the normal looking, nonfibrotic parenchyma is significantly affected, with altered Wnt signaling and increased The overall aim was to perform a histologic investigation of Wnt signaling in IPF, which we hypothesized would correspond to degree of fibrosis; no changes in normal‐looking parenchyma and severe alterations in fibrotic areas. The histologic approach allowed a more detailed quantification and importantly, this dimension is often overlooked as analyses of fibrotic lung often rely on tissue homogenates. The spatial pattern of fibrosis has been suggested to represent a temporal development  the specificity of the antibodies can be questioned. On the basis of the peptide sequence used to generate the Wnt3 and Wnt5 antibodies, we cannot exclude some promiscuous binding. Importantly, the homology between Wnt3/3a and Wnt5a/b is relatively low, thus cross‐labeling is unlikely. Furthermore, results from an MS study (data not shown) failed to detect Wnt3 in healthy or fibrotic lung , whereas both Wnt5a and Wnt5b were detected. Both Wnt5a and Wnt5b have been found to be upregulated in parallel in response to TGF‐β‐catenin signaling by phosphorylating LEF1 and rendering it inactive , which rarely expressed the β‐catenin inhibitors, ICAT and NLK. These inhibitors were more commonly found in vimentin‐positive cells within the tissue, likely fibroblasts and/or myofibroblasts. This suggests and intriguing pattern, with activated β‐catenin in epithelial cells lacking inhibitory signals. Accordingly, the lack of β‐catenin inhibitors in epithelial cells clearly warrants further investigations. Interestingly, very few cells were positive for epithelial markers (pan‐cytokeratin and prosurfactant protein C) in the densely fibrotic areas, whereas vimentin‐positive cells were commonly found in these areas, as well as in clusters beneath the epithelium and in smooth muscle bundles. β‐catenin‐positive cells were commonly found within these areas of vimentin‐positive cells. Within healthy epithelium, β‐catenin‐positive labeling was found in adherens junctions as previously described .β‐catenin, ICAT and Wnt5a/b as an important research area and potential target for therapeutic intervention.On the basis of our results, we conclude that the normal looking, nonfibrotic parenchyma may represent the fibrotic battlefront and suggest the interaction between None declared.


Flickr. We analyse 25 million photos uploaded to Flickr in 2013 across 244 countries and regions, and determine for each week in each country and region what proportion of the photographs are tagged with the word “protest” in 34 different languages. We find that higher proportions of “protest”-tagged photographs in a given country and region in a given week correspond to greater numbers of reports of protests in that country and region and week in the newspaper The Guardian. Our findings underline the potential value of photographs uploaded to the Internet as a source of global, cheap and rapidly available measurements of human behaviour in the real world.Recent years have witnessed waves of protests sweeping across countries and continents, in some cases resulting in political and governmental change. Much media attention has been focused on the increasing usage of social media to coordinate and provide instantly available reports on these protests. Here, we investigate whether it is possible to identify protest outbreaks through quantitative analysis of activity on the photo sharing site  Google [Yahoo [Wikipedia [Financial Times [Twitter [Flickr [Smartphones and computers are becoming an indispensible part of everyday life in many countries around the globe. Usage of these devices and the online services they connect us to is generating fast and cheap measurements of human behaviour at a global scale. Research in the growing interdisciplinary field of computational social science –7 has beGoogle –13 and Ye [Yahoo , 15, theikipedia , 17, newal Times  as well [Twitter –22 and t [Flickr –26. Stud [Flickr , 27–30 a [Flickr .Flickr can be used to identify protest outbreaks around the world.In recent years, news reports have described a number of prominent outbursts of protests in countries around the world, in some cases leading to political change. Much media attention has been focused on the increasing usage of social media to coordinate and provide instantly available reports on these protests –33. As aFlickr between 1st January 2013 and 31st December 2013. We retrieved data on image uploads to Flickr by accessing the Flickr API in January 2014, and downloading data in JSON format using R 3.0.1. The metadata we analyse comprise a wide range of information on where and when a photograph was taken, information about the photographer, as well as user chosen title, description and tags for each photograph, and the URL from which the photograph can be downloaded.We analyse a large corpus of metadata on the 24,944,764 geotagged photographs taken and uploaded to Wikipedia, and using the title of all articles on versions of Wikipedia which are not in English, but which are linked as translations of the article. The complete list of translations is provided in Flickr throughout 2013 in each of the 244 countries and regions are listed in For each geotagged photograph, we retrieve data on both the time and the place at which the photograph was taken. For each week, for each of the 242 countries and regions listed in Flickr in different countries and regions may differ. To account for this, we extract the total number of photos taken and uploaded during each week in 2013 for each of the 244 countries and regions analysed. We consider a week as starting on a Monday and ending on a Sunday. Using these counts, we normalise the weekly counts of photographs taken in each country and region in each week, by dividing the number of photographs labelled with a word signifying “protest” by the weekly count of all photos taken in the same country and region.The overall number of photos taken and uploaded to Flickr correspond to changes in the number of protest outbreaks, we require data on when and where protests have occurred. Such ground truth data can be difficult to obtain. Most studies of civil unrest therefore rely on data from newspaper reports as a proxy for ground truth [The Guardian in each week in 2013.To determine whether we can find any evidence that changes in the number of protest-tagged photographs taken and uploaded to nd truth , 34–36. The Guardian via The Guardian Developer Toolbox in January 2016. We deem an article as protest related if it is tagged with the word “protest”, and we deem an article as covering news related to one of the 244 countries and regions analysed if it is tagged with the country and region’s name. To account for differences in coverage of news in different countries and regions by The Guardian, we also determine the total number of articles published in each week and tagged with each country’s name. In total, we analyse data on 178,730 articles from The Guardian. The counts of The Guardian articles published in 2013 and tagged with each of the of the country and region names are listed in The Guardian uses a different tagging system for articles relating to the United Kingdom and the United States. For this reason, we determine the number of articles relating to the United Kingdom by counting articles tagged with the names “England”, “Scotland”, “Wales” and “Northern Ireland”, and we determine the number of articles relating to the United States by counting articles tagged “US news”.We retrieved data on articles in the online edition of Flickr user activity and protest outbreaks, we build a logistic regression panel model. The outcome variable is whether a The Guardian article is protest related or not. To control for underlying differences in the number of protests in a given country and region and week, we include country and region and week as fixed effects in our model. We underline that in this analysis, we focus on the relationship between Flickr activity and protest reports within the same week. Future analyses may wish to investigate whether photographic data can be used to predict protest activity before it occurs.In order to model the relationship between The Guardian as an approximation of the ground truth of when and where protest outbreaks occurred. For each of the 244 countries and regions analysed, for each month in 2013, we calculate the number of The Guardian articles tagged with the country and region’s name. In We use data on reports of protests in the online edition of Flickr reflect the ground truth data extracted from The Guardian. Again, for each of the 244 countries and regions listed in Flickr. In Flickr photographs and “protest” labelled articles in The Guardian, some key similarities can also be identified, such as an increase in “protest” labelled Flickr photographs in Brazil and Turkey in June 2013.We examine to which extent data on the number of photographs tagged with the word “protest” and uploaded to Flickr and reports of protests in The Guardian, we consider both datasets at weekly granularity. For each week in 2013, for each country and region, we calculate the number of geotagged photographs taken and uploaded to Flickr which are labelled with the character sequence “protest” in 34 different languages, and normalise this count by the total number of geotagged photographs taken and uploaded to Flickr in that week and country and region. To analyse the relationship between the data mined from Flickr and reports of protests in The Guardian, we build a logistic regression panel model. To account for unobserved differences in coverage between countries and regions and weeks, we include country and region and week as fixed effects.To determine whether we can find statistical evidence of a relationship between the number of “protest” labelled photographs taken and uploaded to Flickr photographs in a given week and country and region corresponds to a greater proportion of The Guardian articles about that country and region being tagged with the word “protest” . The odds ratio corresponding to an increase of 0.1 in the normalised number of “protest” tagged Flickr photos is 1.34 . This implies that if we fix the country and region and week effects, increasing the normalised number of “protest” tagged Flickr pictures by 0.1 will increase the odds of a protest related The Guardian article by 34%.Our results suggest that a greater normalised number of “protest” labelled Flickr predictor. We find that the model including data on the normalised number of “protest” labelled Flickr photographs allows us to account for more variance in the proportion of The Guardian articles tagged with the word “protest” than this simple baseline model of differences between different countries and regions and different weeks  = 84.48, p < 0.001, Likelihood Ratio Test).For comparison, we construct a simple baseline model which captures differences in protest frequency between countries and regions, and differences in protest frequencies across different weeks, by building a logistic regression panel model with country and region and week as fixed effects, leaving out the Flickr users are posting photographs labelled with a word signifying “protest” as a result of reading an article about protests in their country and region in The Guardian, or another news source. We posit however that the geotagged nature of the Flickr photographs we analyse makes it less likely that such an explanation may hold, in contrast with simple time series analyses of online behaviour on services such as Google or Twitter, where searching behaviour or tweets may reflect reactions to news articles. With this caveat in mind, our results are consistent with the hypothesis that data on photographs posted to Flickr may help us to identify protest outbreaks around the world.In line with other studies of civil unrest, our analysis uses data from newspaper reports of protests as a proxy for ground truth data on protest occurrences –36. As aFlickr may be of use in identifying protest outbreaks. We analyse 25 million photos uploaded to Flickr in 2013 across 244 countries and regions, and determine for each week in each country and region what proportion of the photographs are tagged with the word “protest” in 34 different languages. We find that higher proportions of “protest”-tagged photographs in a given country and region in a given week correspond to greater numbers of reports of protests in that country and region and week in the newspaper The Guardian. These results are in line with the striking hypothesis that data on photographs uploaded to Flickr may contain signs of protest outbreaks. Our findings underline the potential value of photographs uploaded to the Internet as a source of global, cheap and rapidly available measurements of human behaviour in the real world.We investigate whether data on photographs uploaded to the photo sharing website S1 TableList of country and region names used in analysis.(PDF)Click here for additional data file.S2 TableList of translations of the word “protest” in different languages.(PDF)Click here for additional data file.S3 TableFlickr during 2013.Total number of photographs per country and region taken and uploaded to (PDF)Click here for additional data file.S4 TableThe Guardian articles released during 2013 covering news relating to countries and regions listed.Total number of (PDF)Click here for additional data file.


Musa acuminata, AAA genome) and identified 28 genes belonging to 9 gene families associated with flavonoid biosynthesis. Expression analysis suggested spatial and temporal regulation of the identified genes in different tissues of banana. Analysis revealed enhanced expression of genes related to flavonol and proanthocyanidin (PA) biosynthesis in peel and pulp at the early developmental stages of fruit. Genes involved in anthocyanin biosynthesis were highly expressed during banana fruit ripening. In general, higher accumulation of metabolites was observed in the peel as compared to pulp tissue. A correlation between expression of genes and metabolite content was observed at the early stage of fruit development. Furthermore, this study also suggests regulation of flavonoid biosynthesis, at transcriptional level, under light and dark exposures as well as methyl jasmonate (MJ) treatment in banana.Flavonoid biosynthesis is largely regulated at the transcriptional level due to the modulated expression of genes related to the phenylpropanoid pathway in plants. Although accumulation of different flavonoids has been reported in banana, a staple fruit crop, no detailed information is available on regulation of the biosynthesis in this important plant. We carried out genome-wide analysis of banana ( Secondary plant products are beneficial for human health and utilized as anti-cancerous, anti-inflammatory, immunosuppressive, immunostimulant, anti-osteoporosis and in many other diseases including cardiovascular diseases12356Due to the selective and differential synthesis of flavonoids, biological activities differ across plant species as well as in different tissues of a plant. These differences have been correlated with the tight spatial and temporal regulation of genes involved in the biosynthesis of different flavonoids. Therefore, a number of food products consumed by the human are deficient in these phytochemicals. Accumulation of flavonoids in plants also fluctuates depending upon cellular, climatic and developmental conditions. These factors are major limitation for proper exploitation of secondary plant products in the drug as well as neutraceutical development.Out of different biosynthetic pathways leading to secondary plant product biosynthesis, the phenylpropanoid pathway  is proba56513Banana is a staple fruit crop for a major world population, especially in developing countries. Certain agronomic traits such as fruit quality and stress resistance are of great importance in this plant species. For various reasons, banana improvement through breeding exercises has been challenging. Therefore, genetic engineering based manipulations hold great promise for the crop improvement in banana. The recent release of banana genome sequence provides a useful resource for functional genomics and identification of candidate genes which can be utilized in banana improvement program for agronomically important traitsStructural genes encoding enzymes involved in various intermediate steps of the flavonoid biosynthesis pathway were identified through BLAST searches in banana genome using protein from different plant species as query sequences. Protein sequences identified from banana genome were analyzed through multiple sequence alignments with other known proteins. Analysis revealed that some of the sequences were partial with missing regions of variable lengths suggesting requirement of further refinement in the gene models. To achieve this, corresponding genomic sequences were retrieved, and gene models were modified using online tool (Fgenesh program) followed by manual analysis of the intron–exon boundaries. This analysis led to the identification of a total of 28 genes encoding enzymes involved in flavonoid biosynthesis pathway in banana . These pMaF3′5′H) as compared to that in other monocots.A phylogenetic tree was constructed to study the relationship of various enzymes of the flavonoid biosynthesis pathway from the banana with the corresponding genes from other plants . Most of the proteins from banana and other monocots grouped together and formed monocot-specific clusters in their respective clades . In certMaF3'5′H7 and MaLAR, could be mapped on different banana chromosomes. MaF3'5′H7 and MaLAR got mapped on the un_random chromosome in the banana genome  as well as in ripe fruit . The expression decreased with fruit maturation; however, again increased at the time of ripening  was carried out using HPLC coupled with Q-TRAP. HPLC analysis identified different classes of flavonoids such as flavanones (Naringenin), flavonols , flavonol glycoside , and flavan-3-ols (catechin and epicatechin) at varying level in different banana samples. Isoquercetin was identified in all the tissues analyzed except pulp (both unripe and ripe) of the fruit. Content of isoquercetin ranged from 10.28 to 486.00 μg/100 g DW in root and bract tissue, respectively. Quercetin-3-O-galactoside was detected in all the tissue except root and its content ranged from 2.09 to 321.69 μg/100 g DW in unripe pulp and unripe peel, respectively. Rutin was identified as the major flavonoid and its content ranged from 117.30 to 14310.37 μg/100 g DW in bract and unripe peel tissue, respectively. Quercetin and kaempferol was also identified as aglycone form of flavonols. The content of quercetin ranged from 11.08 to 1811.77 μg/100 g DW in ripe and unripe peel tissue, respectively. Similarly, the content of kaempferol ranged from 5.27 to 595.61 μg/100 g DW in pseudostem and unripe peel tissue, respectively .A detectable amount of naringenin, a flavanone, was also identified in most of the tissues except root sample. Its content ranged from 2.24 to 58.63 μg/100 g DW in pseudostem and unripe peel tissue, respectively. Catechin and epicatechin (flavan-3-ols) were also identified in different tissues. The maximum content of catechin and epicatechin was observed in the unripe peel tissue. The content of catechin ranged from 94.66 to 12742.37 μg/100 g DW in ripe pulp and unripe peel tissues, respectively. Similarly, the content of epicatechin ranged from 51.40 to 7816.92 μg/100 g DW in ripe pulp and unripe peel tissues, respectively . ModulatFlavonoid biosynthesis is modulated by various biotic and abiotic stressesLight is one of the most important modulators and prerequisite for the expression of genes involved in flavonoid biosynthetic pathway under normal growth conditions4243in vitro grown plants exposed with MJ at different time points. Analysis suggests that expression of genes encoding enzymes associated with flavonoid biosynthesis is responsive to MJ. Although MJ led to the enhancement in the expression of most of the genes, the modulation was differential for different genes  over flavonoid biosynthesis in banana was studied through qPCR using RNA isolated from the leaves of nt genes . In the reatment . To assereatment . MJ treaThe flavonoid biosynthesis has been an important target for genetic manipulation in plants owing to their role in pigmentation in flowers, providing insect resistance as well as their health beneficial properties145464748Our genome-wide analysis identified 28 genes belonging to 9 gene families associated with flavonoid biosynthesis in banana. Homology, exon-intron architecture and phylogenetic analyses suggested that identified genes are functional homologs of the genes associated with flavonoid biosynthesis from closely-related monocots. Various studies suggest that plant genes encoding enzymes of diverse functions might evolve from one of the two evolutionary processes. In the first case, single gene develops multifunctionality and produces various products utilizing different types of substrate31525538MaANR and MaLAR) were abundant at the early developmental stages of the fruit. However, after fruit maturation, there is a shift in flavonoid biosynthesis from PA to anthocyanin biosynthesis which acts as antimicrobials and might be involved in protection of the fruit from pathogens and fungiTo the best of our knowledge, there is no report available for the comprehensive analysis of various classes of flavonoids in different parts of the banana including different developmental stages of the fruit. We analyzed different tissues of banana such as young leaves, pseudostem, bract, root as well as peel and pulp of fruit at different developmental stages. Our analysis suggests that unripe peel of banana fruit contains the maximum amount of all the metabolites identified as compared to other tissues. Metabolites such as rutin, catechin and epicatechin are the predominant flavonoids in the banana peel. In our analysis, the highest amount of rutin (143.14 μg/g DW) was detected in the peel of the early developmental stage of the fruit. This is well correlated with expression analysis where flavonol biosynthetic genes were highly expressed at the early developmental stages of the fruit . Previou5758cv Mysore have been reported to provide resistance to black leaf streak disease whereas the other cultivar (Nanicao) lacking anthocyanidine is susceptible to black leaf streak diseaseThe metabolite profiling, carried out in this study, also suggested that peel contain the higher amount of flavonoids than pulp of the fruit. The higher content of flavonoids in the peel may confer disease resistance and protection from herbivores and insects to the fruit. The higher content of flavonoids and the corresponding biosynthetic genes in the berry skin have already been correlated with insect resistance5861Light is one of the most important modulators of flavonoid biosynthesis6465coi1), being defective in JA signalling is unable to induce anthocaynin biosynthesis in response to JAOur study also suggests that MJ has an inducing effect over flavonoid biosynthesis. Similar results were also observed for anthocyanin biosynthesis in diverse plant species including Arabidopsis68We have developed a comprehensive resource of gene families involved in flavonoid biosynthesis in banana. Transcript profiling and phytochemical analysis in various tissues, pulp and peel of different developmental stages of fruit as well as under different stresses suggest tight spatial and temporal regulation of flavonoid biosynthesis in banana. Future research involving development of transgenic banana lines with modified expression of specific genes should elucidate the function of individual gene family members. The manipulation of expression of the structural genes encoding the key enzymes of the limiting steps in the pathway appears to be an attractive approach to modify the flavonoid content in plantsMusa acuminata) was downloaded from the Banana Genome Hub database (http://banana-genome.cirad.fr/)Zea mays, Oryza sativa, Arabidopsis thaliana, Populus tremuloides and Vitis vinifera as a query sequence to search similar protein sequence in M. acuminata database. Banana proteins, resulting from each BLAST search were pooled, and redundant sequences were removed. The protein sequences, thus retrieved were aligned with that of maize, rice, Arabidopsis, Populus and Vitis. Further refinement of gene models of banana proteins was carried out by retrieving corresponding genomic sequences and by using Fegenesh program (www.omictools.com/fgenesh-s1037.html). Finally, manual inspection of intron and exon boundaries was carried out. The analysis for the presence of introns and exons was carried out by using tool available at http://gsds.cbi.pku.edu.cn/.The whole genome sequence of banana (http://www.megasoftware.net/). The chromosomal mapping of individual genes was carried out by performing BLASTx searches in a local database of Banana genome sequence.Multiple sequence alignments were performed using Clustal X programme. The phylogenetic analysis was performed using Maximum likelihood method by MEGA 6 software with 1000 bootstrap valueMusa acuminata (AAA genome), was taken as an experimental system in this study. Leaves, pseudostem, bract, fruit peel, and pulp, etc. were collected from the six month old banana plants growing in the field and stored at −80 °C, following freezing in liquid nitrogen. Fruits from three independent plants at different developmental stages were harvested to collect the samples. Fruits were collected at three weekly intervals, starting from 6 to 24 weeks and then treated with ethylene for ripening. Banana fingers from the same whorl of the hand representing similar developmental stage were treated with 100 μL/L ethylene for 24 h at 22 °C in dark and then allowed to ripen in the air as described previouslyDessert banana, In vitro grown plantlets were hardened in the growth chamber for 2 months. Young banana plants of uniform size were taken for different stress treatments. For dark exposure, plants were shifted in dark chamber with similar growth conditions as in the growth chamber as described previouslyTotal RNA was isolated from different banana tissues according to the previously described protocol−∆∆ct method. The list of different primers used in the study is given in the The quantitative real-time PCR (qPCR) expression was carried out with an ABI 7700 Sequence Detector . The transcripts were quantified by SYBR Green chemistry. The amount of cDNA was normalized by using amplification of housekeeping banana actin as an internal control. The data from real-time PCR amplification was estimated in terms of comparative fold expression following 2Dried and powdered plant material (1 g) was extracted three times successively with 10 ml of methanol: water (80:20 v/v) using an IKA analytical mill . Samples were homogenized and put in on an orbital shaker for 15 min at room temperature76All the samples were analyzed on an HPLC system  equipped with a pump, autosampler, column compartment and thermostat using Agilent Zorbax Eclipse Plus column  maintained at 40 °C. A guard column of the same chemistry was also used. The mobile phase consisted aqueous solution of 0.1% formic acid  and 0.1% formic acid in acetonitrile . The gradient was programmed as follows: 0–2 min, solution B 2%; 2–22 min, Solution B 2-98%; 22.01–25 min, Solution B 98-2%. Other chromatographic parameters included a constant flow of 0.6 ml/min, injection volume was 5 μl, autosampler temperature was set at 10 °C and a run time of 25 min including equilibration. Before analysis, all of the samples were filtered through a 0.22 μm syringe membrane filters .The detection was performed using a HPLC system coupled to a triple quadrupole system QTRAP 5500  using ESI probe both mode of ionisation. The voltage was set at 5.5 KV for positive ionisation and −4.5 KV for negative ionisation, The value of Gas1 and Gas2 was set at 50, curtain gas (CUR) was set at 30, collision assisted dissociation (CAD) was set at medium, temperature of the source (TEM) was set at 550 °C. The mass spectrometer was used in multiple reactions monitoring (MRM) mode. Identification and quantification analysis was performed using Analyst software (version 1.5.2) and MultiQuant software (version2.0.2).For all the experiments three biological and three technical replicates were used for the analysis. The data were analyzed by Student’s paired t-test, and the mean values under each treatment were compared at P ≤ 0.05–0.001.How to cite this article: Pandey, A. et al. Genome-wide Expression Analysis and Metabolite Profiling Elucidate Transcriptional Regulation of Flavonoid Biosynthesis and Modulation under Abiotic Stresses in Banana. Sci. Rep.6, 31361; doi: 10.1038/srep31361 (2016).


In the present study we explored the role of β-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear β-catenin in the CM and RPE after injury (retinectomy). β-catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear β-catenin was maintained as long as FGF2 was present. However, nuclear β-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear β-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear β-catenin. Moreover, retinectomy followed by disruption of active β-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that β-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of β-catenin is a pre-requisite for chick retina regeneration. Retina regeneration studies have been conducted in different animal models for many years, however, the molecular mechanism underlying regeneration via different cellular sources is still under rigorous investigation β-catenin is a dual function protein, playing a critical role in cell-cell adhesion as well as mediating gene transcription through Wnt signaling The transcriptional functions of β-catenin can be regulated by Wnt signaling which controls β-catenin levels in the cytoplasm by modulating phosphorylation events. In the absence of Wnt signaling, the β-catenin destruction complex continuously phosphorylates β-catenin protein at Ser 33, 37, 45 and Thr 41 to target β-catenin for degradation via an ubiquitin-dependent pathway Since β-catenin has been reported to be important for the development or maintenance of the RPE and CM, we sought to explore the role of β-catenin in these tissues during retina regeneration. Here, we report that active β-catenin is associated with cells having a low proliferative status in the CM and RPE and that blocking the transcriptional activity of β-catenin is a necessary step in retina regeneration.Fertilized Specific Pathogen Free (SPF) chicken eggs  were incubated in a humidified rotating incubator at 38°C.Retinectomies were performed in eyes of chick embryos at E4 (HH stage 22–24) using fine forceps as previously described lef1 (NM_205013) cDNA was synthetized by RT-PCR using RNA from the CM at E4 followed by PCR amplification of the partial sequence of lef1 using the primers listed in Table S1 in RCAS DN-Lef1-HA was generated using the Gateway system as previously described 2. The RCAS DN-Lef1-HA infection was confirmed by RT-PCR using specific primers listed in Table S1 in CM explants were isolated from E4 embryos as previously described in vitro electroporation RCAS DN-Lef1-HA or RCAS-GFP  were injected into the vitreous cavity or sub-retinal space for CM or RPE infection respectively in E3 (HH Stage 19–20) embryos. The embryos were collected at 3 and 7 d PR and the viral infection was confirmed by immunohistochemistry using the AMV-3C2 antibody. Electroporation was performed also in E3 embryos as described in Thirty microliters of 10 mg/ml BrdU  solution was injected over the eyes of embryos 1 hour before collection. Tissues were fixed and processed for immunohistochemistry.Tissues used for histology were fixed in Bouin's solution fixative  for 1 hour and transferred into 70% ethanol until embedded in paraffin wax. The tissues were sectioned and stained with hematoxylin and eosin. Histological results are representative of 10 or more eyes. Histological images were photographed using an Olympus BX-51 microscope .Tissues used for immunohistochemistry were fixed in 4% paraformaldehyde for 1 hour, rinsed in 1x PBS and cryoprotected in 30% sucrose, then snap frozen in optimal cutting temperature medium (O.C.T.) . The frozen tissues were sectioned at 10 µm, washed in 1x PBS and blocked in serum for 1 hour. When antibodies against transcription factors were used, a 10-minute 1% saponin  permeabilization was used followed by three washes in 1x PBS. Primary antibodies (Table S2 in http://primer3.wi.mit.edu/) and the sequences and Ensembl or GenBank IDs are provided as well as the primer efficiency for each set in Table S3 in −ΔΔCt) was used to determine relative changes in transcript levels compared to gapdh mRNA levels as previously reported Total RNA was extracted from the CM explants electroporated with RCAS constructs (see in vitro electroporation) using Nucleospin RNA II isolation kit  following the manufacture's protocol. The quality and quantity of RNA was determined using Agilent RNA 6000 nano LabChip . In general, samples with RIN >9 were used for RT-qPCR experiments. Approximately 200 ng of total RNA were used for cDNA synthesis using ImProm-II Reverse Transcription System  and random-primer hexamers according to the manufacturer's instructions. RT-qPCR reactions and conditions were performed as previously described ImagePro Plus software  was used for all quantitative analyses of the histology data. The area of regenerated neuroepithelium  was outlined and the average area for each treated eye was graphed. Exact two-sample permutation tests were used to determine statistical significance. Error bars represent SEM. The alpha level was set at 0.05.2 in regenerated neuroepithelium/retina was determined by counting 3 different areas of the regenerating neuroepthelium/retina derived from the CM and from RPE transdifferentiation in each treated eye. Three different eyes were included. Split-plot ANOVA was used to determine statistical significance between experimental and control groups. Error bars represent SEM.Quantitation of BrdU-positive cells per 10,000 µmin situ hybridization and reporter assays + NPE cells extended to the optic cup lip (OCL), the most anterior domain of the CM that has been reported to house multipotent optic cup stem/progenitor cells including ones for the retina + area is defined by collagen IX staining, a widely used marker for the anterior region of the CM known as the ciliary body, that has been shown to contain retinal stem cells +  at E4 or E7 , the posterior region of the CM, where retinal progenitors are located We then examined if nuclear β-cateninE4 or E7 . Some So+ cells were actively cycling and entering the S phase, we added BrdU to the eyes one hour before collection, and subsequently performed immunohistochemistry for BrdU and nuclear β-catenin. Many of the nuclear β-catenin+ cells in the NPE, PE and in the RPE of E4 eyes were BrdU negative (BrdU-)  . Althougwith PH3 , a label+ cells of the PE and RPE were Sox2- at 1 and 3 d PR . A clear reduction of nuclear β-catenin was detected in the NPE at 1 and 3 d PR  comparedd 3 d PR . While fd 3 d PR  comparedd 3 d PR , most oft 1 d PR . Insteadd 3 d PR .+ and Sox2- (+ (We further examined the patterns of nuclear β-catenin in the CM and RPE of eyes at 1 and 3 d PR in the presence of FGF2. Nuclear β-catenin was clearly down-regulated or absent in FGF2-induced regenerating neuroepithelium from the CM and from the RPE . At 1 d nd Sox2- . At 3 d nd Sox2- . The nonSox2- (+ . Therefomsi-1) and cyclin D1 (cD1) was detected in DN-Lef1 electroporated ciliary explants verifying the activity of the construct  and 7 d PR (p = 0.01)  . Most of = 0.01) . At 7 d  = 0.01) .The retina houses neurons arranged in three cell layers. The innermost cell layer contains retinal ganglion cells, the inner nuclear layer has bipolar, amacrine, and horizontal cells, and the outer nuclear layer contains the bodies of photoreceptors. Müller glia cells expand throughout the different cell layers of the retina β-catenin is the key mediator of the canonical Wnt signaling pathway. Nuclear targeted β-catenin binds to the transcription factor TCF/Lef1 to induce the β-catenin/TCF/Lef1 transcription activity, which is a hallmark of Wnt/β-catenin signaling activation XAV939 is an antagonist of the Wnt/β-catenin pathway. It inhibits the enzymes tankyrase 1/2 that inhibits the activity of axin 2 (a component of the β-catenin destruction complex), thus promoting axin 2 stabilization, and as a consequence β-catenin degradation The present study revealed the involvement of β-catenin dependent TCF/Lef1 transcriptional activity in the regulation of the stem/progenitor cell niche located in the CM and of the maintenance of the RPE in developing chick eyes, especially upon injury, as well as establishing for the first time its role in regulating chick retina regeneration from activation of stem/progenitor cells and from RPE transdifferentiation. We observed dynamic changes of active nuclear β-catenin protein in early stages of eye development . During early eye development, nuclear β-catenin is widespread throughout the optic vesicle and by E4 (HH stage 22–24), nuclear β-catenin positive cells were present in the CM, NE and RPE. This temporal and spatial pattern of nuclear β-catenin in developing chick eyes is consistent with the reported expression pattern of Wnt signaling components Lef1 and activation of Wnt/β-catenin signaling during chick and mouse eye development detected by Lef1/TCF reporter assays It is important to note that while canonical Wnt signaling plays a role in the specification of the CM including the iris and ciliary body in birds and mice + RPE cells were also found to be Sox2-, BrdU- and PH3- indicating the low-proliferative state of those cells. This data is consistent with previous reports that showed that the Wnt/β-catenin signaling is activated in the developing RPE and is required for RPE specification In the differentiated RPE of E4 eyes, we detected reduced nuclear β-catenin immunoreactivity compared to the anterior CM region, but the few nuclear β-cateninThe dynamic changes of nuclear β-catenin staining in the CM in response to retinectomy and during FGF-induced regeneration further suggests that nuclear β-catenin is important to keep the NPE cells  in a low proliferative state during development . Moreover, the loss of nuclear β-catenin protein in response to retinectomy is likely to release those cells from their low proliferative state, but not sufficient to induce continuous proliferation. Adding exogenous FGF2 is able to further interrupt β-catenin nuclear accumulation in those NPE cells and induce proliferation to give rise to a new neuroepithelium.As to the RPE, nuclear β-catenin was detected in differentiating RPE cells at E4; however it was decreased at E5 and older embryos. Upon retinectomy, the significant up-regulation of nuclear β-catenin in the PE and the RPE suggests that it prevents those cells from entering the cell cycle, however, in the presence of FGF2, RPE cells lose nuclear β-catenin and undergo further dedifferentiation and proliferation. Previous work has shown that a precise regulation of Wnt/β-catenin signaling is critical for normal RPE development in mouse. The inactivation of Wnt/β-catenin signaling in developing RPE results in the downregulation of RPE-specific genes Mitf and Otx2 and induction of neural retinal markers Chx10 and Rx, resulting in the conversion of RPE to neural retina. On the other hand, over-activation of Wnt/β-catenin signaling impairs RPE patterning during mouse eye development D. lacteum, P. kawakatsui and P. fluviatilisThe disruption of β-catenin nuclear distribution by XAV939 in developing chick eyes and the induction of retina regeneration by XAV939 as well as by the retroviral DN-Lef1construct in the absence of FGF2 strongly suggest that the canonical Wnt pathway is involved in both modes of retina regeneration. Canonical Wnt signaling regulates stem and progenitor cell fate and proliferation in other systems In summary, our study provides important information on the regulation of β-catenin activation to maintain retinal stem/progenitor cells and RPE cells, and its pivotal role during the process of injury and retina regeneration  coincide with Collagen IX+ area.Nuclear β-catenin (A) Collagen IX labels the CB of the developing chick eye at E7. (B) β-catenin immunostaining of a neighboring section of the same chick eye shows that nuclear β-catenin+ cells located in the NPE of the CB, overlap with the Collagen IX+ domain. Scale bar in (A) represents 100 µm and applies to (B).(TIF)Click here for additional data file.Figure S2Pax6 and Chx10 co-localization identifies retinal progenitor cells in DN-Lef1 induced neuroepithelium/retina. (A–B) The majority of cells in DN-Lef1 induced neuroepithelium from the CM (A) and RPE transdifferentiation (B) at 3 d PR co-express Pax6 and Chx10, indicating their retinal progenitor identity. (C–D) At 7 d PR, there is still a region of retinal progenitors in DN-Lef1 induced neuroepithelium/retina from the CM (C) and from the transdifferentiated RPE (D). (E–H) Pax6/Chx10 double immunostaining on FGF2-induced neuroepithelium/retina from the CM  and RPE transdifferentiation  at 3 d and 7 d PR are used for comparison. Cr = ciliary regeneration; Td = transdifferentiation; RPE: retinal pigment epithelium. Scale bar in (A) represents 100 µm and applies to all panels.(TIF)Click here for additional data file.File S1Supporting tables.Table S1, Primer sequences utilized for RCAS construction and RT-PCR detection. Table S2, Primary antibodies utilized for immunohistochemistry experiments. Table S3, Primer sequences utilized for RT-qPCR.(DOCX)Click here for additional data file.


Human T-cell leukemia virus -1 (HTLV-1) is a delta retrovirus that infects an estimated 15-25 million people worldwide. HTLV-1 is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL) and a neurodegenerative disease (HAM/TSP). While the probability of presenting any symptoms related to HTLV-1 infection is relatively low , the disease progression and prognosis of those infected individuals who develop ATL is fatal, with a median survival range of 8-10 months. Unfortunately, ATL is highly chemotherapy resistant and while many current therapies improve ATL patient survival, the patients consistently relapse. Therefore, a need exists to develop treatments that improve ATL outcome. We have recently identified PRMT5 (protein arginine methyltransferase 5) as a potential target to modulate HTLV-1 gene expression. We find that PRMT5 protein levels are elevated in T-cell leukemia/lymphoma cell lines compared to freshly isolated naïve T-cells. PRMT5 RNA levels do not correlate to PRMT5 protein levels, suggesting a possible post-transcriptional method of regulation. Furthermore, we also show that PRMT5 protein expression is slightly elevated during short-term immortalization, but gains highest expression after transformation and IL-2 independence. Utilizing shRNA vectors, we demonstrate that knockdown of endogenous PRMT5 results in a decrease in viral p19 production in a HTLV-1-transformed cell line. Finally, we observe a decrease in cell proliferation and in viral gene expression when HTLV-1-infected/-transformed cells are treated with a novel small molecule inhibitor of PRMT5. In conclusion, we find PRMT5 to be a positive regulator of HTLV-1 gene expression.