Microparticles (MPs) are increasingly recognized as important mediators of cell-cell communication in tumour growth and metastasis by facilitating angiogenesis-related processes. While the effects of the MPs on recipient cells are usually well described in the literature, the leading process remains unclear. Here we isolated MPs from ovarian cancer cells and investigated their effect on endothelial cells. First, we demonstrated that ovarian cancer MPs trigger β-catenin activation in endothelial cells, inducing the upregulation of Wnt/β-catenin target genes and an increase of angiogenic properties. We showed that this MPs mediated activation of β-catenin in ECs was Wnt/Frizzled independent; but dependent on VE-cadherin localization disruption, αVβ3 integrin activation and MMP activity. Finally, we revealed that Rac1 and AKT were responsible for β-catenin phosphorylation and translocation to the nucleus. Overall, our results indicate that MPs released from cancer cells could play a major role in neo-angiogenesis through activation of beta catenin pathway in endothelial cells. Clinical and experimental evidences indicate that tumor initiation and progression is intimately related to the complex dialogue between tumor microenvironment and malignant cells , 2. In oCancer cell derived MPs are involved in angiogenesis promotion. For instance, glioblastoma (GBM) cell-derived MPs are efficiently internalized by endothelial cell human umbilical vein (HUVEC) and primary human brain microvascular cells (HBMECs), inducing microvascular sprouting . Renal cAngiogenesis is crucial in advanced ovarian cancer where anti-angiogenic treatment targeting VEGF demonstrated an effect on progression free survival , 27. HowHere, we investigated the effect of MPs derived from ovarian cancer cell lines (OCC-MPs) on ECs. We demonstrated that ovarian cancer MPs trigger β-catenin activation in ECs increasing angiogenic properties. We showed that the activation of β-catenin in ECs by OCC-MPs was Wnt/Frizzled independent; but related to integrin and subsequent MMP activation leading to VE-cadherin disruption from the membrane. Finally we established that Rac1 and AKT were the intracellular mediators of β-catenin phosphorylation and translocation to the nucleus.To investigate β-catenin activation, we established a co-culture of eGFP-E4+ECs, with two ovarian cancer cell lines Skov3 and APOCC  to the nucleus. Therefore, we investigated whether OCC-MPs uptake by ECs affected β-catenin localization in E4+ECs. β-catenin translocation to the nucleus was observed in E4+ECs after 1h of incubation with OCC-MPs Figure . We confWe first characterized the integrin repertoire of Skov3, APOCC and E4+ECs Figure . Among tIntegrins are involved in MPs uptake through phosphatidyl serine (PS) interaction with lactadherin . BlockinIn its inactive form, β-catenin is located at the cytoplasmic side of the cell membrane as an element of cadherin-complexes . In endoβ-catenin can be activated through different cascades . Wnt sigβ-catenin Ser552 and 675 sites can be phosphorylated by PKA (protein kinase A), Akt or PAK1/Rac1 –40. We fWe demonstrated the endothelial cell activation of β-catenin pathway by microparticles derived from ovarian cancer cells. Thus, revealing that integrins αVβ3 play a crucial role in MPs uptake and that MMPs activity leads to VE-Cadherin disruption and the release of β-catenin. Finally within the cytoplasm, Rac1 and Akt could mediate β-catenin phosphorylation and its translocation to the nucleus.While Wnt signaling represents the major regulator of β-catenin, it is now well accepted that β-catenin is not just an element of Wnt cascade . Over thThe involvement of cancer derived MPs in endothelial β-catenin activation has been described for the first time in myelogenous leukemia cells . The autHere, we described a mechanism explaining ovarian cancer-MPs uptake in ECs. We demonstrated that β-catenin activation was dependent on MPs internalization through αVβ3 integrins. Interestingly, αVβ3 integrins are expressed at low levels on quiescent ECs, but strongly induced on angiogenic ECs present in cancer . We illuEven if αvβ3 integrins have already been illustrated to participate to the disruption of VE-cadherin localization at ECs junctions –66, our de novo expression of genes involved in the neo-angiogenesis process. Altogether, our results support the fact that MPs released from cancer cells could be an important actor of angiogenesis driven by cancer cells. MPs secretion might be of clinical importance as large amounts of MPs were observed in ascites from advance-stage ovarian carcinomas [Overall our results indicate that ovarian cancer MPs could have several effects on ECs. First, loss of VE-cadherin and β-catenin at the intercellular junctions induced by the MPs could be responsible for increased motility as well as the permeable vasculature observed in solid tumor . Additiorcinomas . A clearOvarian cancer cell lines Skov3 were purchased from ATCC and cultured following ATCC recommendations . A primary ovarian cancer cell line was derived in our laboratory from ascites of a patient with Stage III serous adenocarcinoma (APOCC). The cell lines were cultured in DMEM high glucose , 10% FBS , 1% Penicillin-Streptomycin-Amphotericyn B solution (Sigma), 1X Non-Essential Amino-Acid  and 1% L-glutamine. Cultures were incubated in humidified 5% CO2 incubators at 37°C and the media was replaced every 3 days.To avoid bias due to the use of serum, we used our model of HUVECs with autonomous Akt-activation surviving in the absence of FBS and cytokines (ECs) as a surrogate for tumor-associated endothelium , 73–75. MPs isolation was performed using ExoQuick™-TC  according to the manufacturer recommendations. Briefly, 48-h-supernatants of 80% serum-starved confluent tumour cells were collected, and centrifuged (4°C) at 3,000 g for 15 min to remove cells and cell debris. Supernatant aliquots of 5 mL were mixed with 1 mL of ExoQuick™-TC and incubated at 4° C overnight. The mixture was centrifuged at 1,500 g for 30 min and the MPs pellets were collected.The final pellet containing purified MPs was either re-suspended in media for treatment of cell cultures or lysed for protein extraction or labeled for cytometry analysis or microscopy imaging. The protein concentrations of MPs were measured by Bradford assay (Biorad). Cancer cells MPs will be referred to as CC-MPs.Migration was assessed by wound closure assay as previously described . Cells cA Matrigel-based capillary-genesis assay was performed on E4+EC to assess the ability of these cells to form an organized tubular network as previously described . E4+EC wCells were plated at 50000 cells per well in a 6 well plate in medium without FBS with or without MPs. Cells were then counted with a hemocytometer for the following six days every two days. Two wells were counted per condition. For the co-culture, only the green cells (MDA-GFP) were counted. The experiment was performed in triplicate.Fluorescence (FL) was quantified on a SORP FACSAria2 (BD Biosciences) as previously described , 79. DatTo quantify MP uptake we used stained MPs (WGA AF594) and treated cell cultures for 6 or 24 h at 37°C or 4°C. Then cells were harvested and single cell suspension was analyzed by flow cytometry or fixed for confocal microscopy imaging.ECs treated with MPs as detailed in specific experiments were fixed in 3.7% formaldehyde. Slides were mounted in a mounting media SlowFade® Gold Antifade Reagent (Invitrogen). Fluorescence Imaging was performed using a Zeiss confocal Laser Scanning Microscope 710 (Carl Zeiss). Post-acquisition image analysis was performed with Zeiss LSM Image Browser Version 4.2.0.121 (Carl Zeiss).The ImageXpress platform  is a widefield automated microscope capable of fluorescent, transmitted light, and phase-contrast imaging of fixed- or live-cells. The system optical drive includes a 300-W Xenon lamp as broad-spectrum white light source and 2/3 chip cooled CCD camera and optical train for standard field of view imaging and a transmitted light option with phase contrast. After incubating endothelial cells with CCs-MPs (from 15 minutes to 24h), cells were stained for beta catenin or Ve-Cadherin. 100 pictures were taken per wells (4 wells per condition) and images were analysed with MetaXpress® High-Content Image Analysis Software .Western blot was carried out as previously described . ImmunosThe activity of MMPs inside the OCC-MPs was determined by gelatin zymography, 10% polyacrylamide gel polymerized together with gelatin (1 mg/ml) (Novex by life technologies Lot no.:14051365).Cells are lysed using 2x tricine SDS sample buffer . After electrophoresis, gels were placed in renaturing buffer  for 30mins at RT and then in developing buffer  for 30 minutes at RT. After an overnight incubation at 37°C in developing buffer, gels are stained with blue stain (Invitrogen Simply blue Safestain TM Cat no.: LC6060) for 1h at RT. After 1 to 2h of destaining process, gels are finally developed, and the Gelatinolytic activities appearing as clear zone were quantitated.Total RNA was extracted from cell cultures using Trizol. After genomic DNA removal by DNase digestion , total RNA 1μg) was reverse transcribed with oligodT (Promega) using the Superscript III First-Strand Synthesis SuperMix (Invitrogen). PCR analysis was performed as previously described  with a Mμg was reAll quantitative data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed with SigmaPlot 11 . A Shapiro-Wilk normality test, with a p = 0.05 rejection value, was used to test normal distribution of data prior further analysis. All pairwise multiple comparisons were performed by one way ANOVA followed by Holm-Sidak posthoc tests for data with normal distribution or by Kruskal-Wallis analysis of variance on ranks followed by Tukey posthoc tests, in case of failed normality test. Paired comparisons were performed by Student's t-tests or by Mann-Whitney rank sum tests in case of unequal variance or failed normality test. Statistical significance was accepted for p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***). All experiments were performed in triplicates.


Stem cells are a powerful resource for cell-based transplantation therapies, but understanding of stem cell differentiation at the molecular level is not clear yet. We hypothesized that the Wnt pathway controls stem cell maintenance and neural differentiation. We have characterized the transcriptional expression of Wnt during the neural differentiation of hADSCs. After neural induction, the expressions of Wnt2, Wnt4, and Wnt11 were decreased, but the expression of Wnt5a was increased compared with primary hADSCs in RT-PCR analysis. In addition, the expression levels of most Fzds and LRP5/6 ligand were decreased, but not Fzd3 and Fzd5. Furthermore, Dvl1 and RYK expression levels were downregulated in NI-hADSCs. There were no changes in the expression of ß-catenin and GSK3ß. Interestingly, Wnt5a expression was highly increased in NI-hADSCs by real time RT-PCR analysis and western blot. Wnt5a level was upregulated after neural differentiation and Wnt3, Dvl2, and Naked1 levels were downregulated. Finally, we found that the JNK expression was increased after neural induction and ERK level was decreased. Thus, this study shows for the first time how a single Wnt5a ligand can activate the neural differentiation pathway through the activation of Wnt5a/JNK pathway by binding Fzd3 and Fzd5 and directing Axin/GSK-3ß in hADSCs. Undenditions , 9. Seconditions , 11, incnditions , 13, chonditions .β-catenin by the glycogen synthase kinase-3β (GSK3β). The accumulation of the stabilized β-catenin in cytoplasm is increased and enters the nucleus where it induces transcription of target gene expression. Noncanonical pathway, which is independent of β-catenin, mediates several cellular processes through monomeric GTPases of the Rho family, mitogen-activated protein kinase like Jun N-terminal kinase (JNK), and changes in intracellular calcium levels [Wnts are a family of glycoproteins that can signal through distinct intracellular pathways and regulate many developmental events in vertebrates and mammals , 15. As m levels , 16–20.β-catenin expression [The Wnt signalling pathway plays an important role in the development of the central nervous system and regulates the formation and function of neuronal circuits by controlling neuronal differentiation . Severalpression . Wnts alpression , 23. Howpression  and figu2. The hADSCs used in the present study were from passages 2 to 5. To induce neural differentiation, hADSCs were grown in DMEM containing 1% FBS and supplementary 100 ng/mL basic fibroblast growth factor  for seven days and were then incubated in 10 μM forskolin  for the next seven days. After that, the cells were subjected to RT-PCR analyses, real time RT-PCR assay, and western blot study.In this study, we used human ADSCs, which were already isolated and characterized in our previous study . In brieTo analyse the relative expression of different mRNAs, the amount of cDNA was normalized based on signals from the ubiquitously expressed GAPDH , 24. TotμL.For real time RT-PCR analyses, cDNA was synthesized using total RNA which was previously prepared from primary hADSCs and NI-hADSCs using TRIzol protocol. Real time RT-PCR was performed with SYBR Green Premix Ex Taq (Takara Bio Inc.) and Thermal Cycler Dice Real Time System (Takara Bio Inc.). Each sample was analysed in four replicate reactions of 20 β , GSK3β , β-catenin , or GAPDH . Following several washes, membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody . The signals were detected by enhanced chemiluminescence reagents (Santa Cruz Biotechnology) and the density was quantified using ImageJ software [The cells were cultured and Wnt signalling pathway was observed by western blot analysis. Briefly, the cells were washed twice in phosphate-buffered saline  and lysed in a lysis buffer including protease inhibitors and phosphatase inhibitors. Protein concentration in cell lysates was determined by Quant-iT Assay Kit . An equal amount (15 mg) of protein for each sample was resolved on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . After blocking with 1% skim milk  in PBS-T, the membranes were incubated with specific antibodies for Wnt3a , Wnt5a/b , Dvl2 , Naked1 , Axin 1 , phosphor-JNK , JNK , phosphor-ERK1/2 , ERK1/2 , phosphor-PKC , PKC , phosphor-GSK3software . post hoc comparison) was used to analyse differences between groups, with P < 0.05 being considered significant.Protein and mRNA levels were quantified by measuring the optical density of each band using computer-assisted densitometry . All values were expressed as the mean ± SEM. The one-way ANOVA test  level and an activation of c-Jun N-terminal kinase (JNK). To investigate the signalling pathways by which Wnt5a influences neural differentiation, we employed phosphor-specific antibodies to identify the signalling effectors activated by Wnt5a in NI-hADSCs. Western blotting was carried out to show expression of JNK and phosphor-JNK, key molecules of the noncanonical Wnt signalling pathway. The phosphor-JNK expression level was upregulated in relation to control GAPDH in NI-hADSCs compared with primary hADSCs (β and phosphor-GSK3β protein levels were not changed in both the primary hADSCs and NI-hADSCs. The quantification data showed that the phosphor-JNK, phosphor-ERK1/2, and phosphor-PKC levels were increased in relation to total-JNK, total-ERK1/2, and total-PKC in NI-hADSCs, respectively (Figures Noncanonical Wnt signalling has been shown in two different ways such as increases in intracellular Cay hADSCs . Further Figures . These d Figures .β or anti-tumor necrosis factor-α induced differentiation of human MSCs into osteoblasts [In the last few years there have been wide interests in the role of Wnt signalling and it is also thought to play a key role in controlling stem cell fate. Thus, understanding the mechanisms that regulate Wnt signalling is of critical importance, especially in stem cell differentiation. In particular, Wnt5a/receptor tyrosine kinase-like orphan receptor 2 pathway regulated interleukin-1eoblasts , 13. In eoblasts  and a preoblasts . Here weeoblasts .β and β-catenin and found that protein levels were not changed after neural induction of hADSCs. These results depict the fact that the activation of the Wnt/β-catenin signalling pathway, which is a canonical Wnt pathway, is not involved in neural differentiation in our study.We observed that Wnt5a upregulated the Fzd3 and Fzd5 expressions and promoted the neural differentiation of hADSCs , showing2+, which downregulate PKC or calcium/calmodulin-dependent kinase II, or Wnt/JNK pathway by stimulated Wnt5a ligand [There are two pathways of noncanonical Wnt signalling, Wnt/Caa ligand , 30. Zhaa ligand . These ea ligand . InteresIn this study, we have focused on the stem cell differentiation, especially neural differentiation, through the noncanonical Wnt pathway. We have proved that the expression of Wnt5a, which is one of the noncanonical Wnts, gene level was increased after neuronal differentiation in hADSCs and protein level of downstream phosphor-JNK was also increased in the same way. Taken together, these data demonstrate that noncanonical Wnt signalling could activate the neural differentiation in hADSCs through the regulation of Wnt5a and the activation/phosphorylation of JNK pathway. in vitro. Future study will contribute to understanding the control of neural differentiation using Wnt signalling.Wnt5a promotes noncanonical Wnt signalling followed by Fzd3 and Fzd5 expressions when primary hADSCs are differentiated into neural hADSCs


Protein arginine methyltransferases (PRMTs) plays critical roles in cancer. PRMT5 has been implicated in several types of tumors. However, the role of PRMT5 in cancer development remains to be fully elucidated. Here, we provide evidence that PRMT5 is overexpressed in colorectal cancer (CRC) cells and patient-derived primary tumors, correlated with increased cell growth and decreased overall patient survival. Arginine methyltransferase inhibitor 1 (AMI-1)strongly inhibited tumor growth, increased the ratio of Bax/Bcl-2, and induced apoptosis in mouse CRC xenograt model. AMI-1 also induced apoptosis and decreased the migratory activity in several CRC cells. In CRC xenografts AMI-1 significantly decreased symmetric dimethylation of histone 4 (H4R3me2s), a histone mark of type II PRMT5, but not the expression of H4R3me2a, a histone mark of type I PRMTs. These results suggest that the inhibition of PRMT5 contributes to the antitumor efficacy of AMI-1. Chromatin immunoprecipitation (ChIP) identified FGFR3 and eIF4E as two key genes regulated by PRMT5. PRMT5 knockdown reduced the levels of H4R3me2s and H3R8me2s methylation on FGFR3 and eIF4E promoters, leading to decreased expressions of FGFR3 and eIF4E. Collectively, our findings provide new evidence that PRMT5 plays an important role in CRC pathogenesis through epigenetically regulating arginine methylation of oncogenes such as eIF4E and FGFR3. APC gene mutation is an early event in the multistep process of CRC and occurs in more than 70% of colorectal adenoma. The adenoma-carcinoma sequence is further promoted by activating mutations of KRAS oncogene and inactivating mutations of TP53 tumor suppressor gene [BRAF oncogene [Colorectal cancer (CRC) is the third most common cancer and the fourth most common cancer cause of death globally . Most caoncogene . NeverthS-adenosylmethionine (AdoMet) as methyl donor to catalyze the transfer of methyl groups to the arginine residues of histones H3 and H4 or non-histones [Arginine methylation is crucially involved in the regulation of gene expression, RNA metabolism, cell cycle, and protein function -7. Protehistones , 8. The histones , 9. PRMThistones -12. Indehistones , 13-15. histones . PRMT5-dhistones , 18. PRMhistones , 20.In this study, we aimed to characterize the role of PRMT5 in CRC and elucidate the underlying mechanism. Our data show that PRMT5 was overexpressed in approximately 75% of cases. Inhibition of PRMT5 by AMI-1 or knockdown of PRMT5 by siRNA in CRC led to the restoration of critical regulatory pathways affecting cell growth, survival, migration and tumor suppressor activity.To evaluate the expression levels of PRMT5 in CRC cells, we performed Western blot analysis to determine protein levels of PRMT5 in several CRC cell lines. In comparison with normal colonic mucosal FHC cells, all seven human CRC cell lines expressed high levels of PRMT5 Figure . To furtP > 0.05, Table To confirm the clinical significance of PRMT5 overexpression in human CRC development, we analyzed PRMT5 level and its distribution in CRC tissue array by immunohistochemical staining. The array included cancer tissue samples and corresponding NATs from 90 CRC patients . Among these patients, 46 51.1%) died of tumor related causes and 44 (48.9%) were still alive at the last follow-up , we then measured H4R3me2s expression in CRC xenograft nude mice treated with AMI-1. We found that H4R3me2s expression was significantly decreased by AMI-1 compared with the control. To exclude the effect of AIM-1 on type I PRMTs, we measured H4R3me2a level and found no significant change Figure  and 3F. PRMT5 silencing can induce apoptosis in different types of cancer , 24. To To further corroborate the role of PRMT5 in CRC cell proliferation, three different small interfering RNAs (siRNAs) against human PRMT5  was usedWe next investigated whether si-PRMT5 inhibits the growth of colorectal tumor xenografts in nude mice. On day 7 after xenograt implantation, mice carrying palpable subcutaneous SW480 tumor xenografts received intratumorally (i.t.) or intravenously (i.v.) injections of formulated si-PRMT5. As controls, tumor-bearing animals were injected i.t. or i.v. with formulated si-NC or vehicle alone. Tumors injected with formulated si-NC developed at a pace similar to those that received vehicle. In contrast, injections of formulated si-PRMT5 specifically blocked tumor growth in nude mice Figure  and 5E.In addition, to assess the safety of systemically delivered si-PRMT5, we examined serum levels of alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, alkaline phosphatase, creatinine, and creatine kinase from mice treated with formulated si-NC, si-PRMT5 or vehicle alone. As shown in It has been reported that FGFR3 and eIF4E play an important role in cell proliferation and promote tumorigenesis -27. PRMTRecent studies have shown that the methylation of histones H3 and H4 by PRMT5 plays an important role in the regulation of gene expression , 14, 16.FGFR3 is one of the receptors that promote cell survival by stimulating PI3K/AKT/mTOR signaling, and has been shown to activate AKT and ERK in human cancers , 26. To in vivo and in vitro. Thus, it appears that PRMT5 is a key histone-modifying enzyme that controls cell growth by modulating the expression of target genes through histone arginine methylation.Knowledge about the precise molecular mechanisms underlying colorectal tumorigenesis is crucial to the development of better therapy strategy for CRC patients. The dysregulation of PRMTs has been observed in diverse types of cancer and the modulation of PRMTs levels affects cancer cell growth, thus PRMTs become promising targets for therapeutic strategies . Here, wArginine methylation is a very stable mark because it is unclear if the modification can be enzymatically reversed. Two types of putative arginine demethylases have been reported: Jumonji domain-containing protein (JMJD6) and peptidylarginine deiminases (PADIs) , 31. Howin vitro, our results revealed the in vivo substrate selectivity of AMI-1, since no significant change was observed in the expression of H4R3me2a, a histone mark of type I PRMTs. These findings are not surprising, because PRMT5 is markedly upregulated in CRC. In addition, it has been reported that PRMT3 and PRMT6 are mainly overexpressed in breast, bladder and lung cancer but not CRC [Numerous efforts are currently underway to develop PRMTs inhibitors as chemical probes and therapeutic reagents , 34. PRM not CRC . These rras and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway, which are frequently activated in a diverse range of human cancer [Because PRMT5 has been shown to mediate arginine methylation of p53 to regulate its function , 17, we n cancer , 42. TheBoth FGFR3 and eIF4E have been shown to promote tumor growth, and are overexpressed in human cancers including CRC , 41, 43.PI3K/AKT/mTOR signaling regulates several cellular functions that are critical to tumorigenesis such as cellular proliferation, growth, survival and mobility . A majorin vitro and in vivo. PRMT5 overexpression represents an unfavorable prognostic marker and an attractive therapeutic target for CRC. Expanding insight into the dysregulation of oncogenes through epigenetic mechanism in colorectal tumorigenesis will yield important clues to improve our understanding of the complicated molecular pathogenesis of CRC and facilitate the development of new therapeutic regimens for CRC patients.Taken together, our results show that PRMT5 is frequently overexpressed in human CRC tissues and cell lines. PRMT5 knockdown or PRMT5 inhibitor significantly inhibits cell proliferation Forty eight flash-frozen CRC tumor samples and corresponding normal adjacent tissues (NATs) were collected between 2011 and 2012 at State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University. Tissue samples were immediately snap-frozen in liquid nitrogen. Ninety pairs of CRC tissues and adjacent NATs arrays were purchased from the National Engineering Center for Biochips . Both tumor and adjacent NAT were histologically examined. All human materials were obtained with informed consent, and this project was approved by Ethics Committee of Sichuan University. Seven human CRC cell lines, including HCT116, SW480, CX-1, SW620, LoVo, COLO 205, LS-174T and normal colonic mucosal FHC cells, were purchased from American Type Culture Collection , and cultured under conditions recommended by ATCC.® Green Supermix (Bio-Rad), and β-actin gene was used as an internal control. PRMT5, FGFR3, and eIF4E expression were determined by using qRT-PCR primers according to the provided protocol. Raw CT values were normalized to the values of β-actin to correct for equal input of RNA, and analysis of gene expressions was performed using the 2−ΔΔCt method. The qRT-PCR primers are listed in Total RNA from CRC tumor samples was extracted using TRIzol reagent . cDNA was synthesized with the iScript™ cDNA Synthesis Kit . Quantitative real-time PCR (qRT-PCR) analyses were carried out to detect mRNA expression using iQ™ SYBRTissue arrays were constructed using 90 pairs of CRC tissues and adjacent NATs. Immunohistochemical staining was performed on 4 μm sections of paraffin-embedded human CRC tissues and matched NATs. Briefly, the slides were incubated in 200 μg/mL PRMT5 antibody  overnight at 4°C. Subsequent steps were performed using Polymer Detection System for Immuno-Histological Staining  according to the manufacturer's instruction. PRMT5 expression in the tissues was evaluated by immunohistochemical staining with a PRMT5-specific antibody. A semi-quantitative scoring method according to the overall staining of the cells was applied for IHC , 50. ThePRMTs enzymatic assays were conducted using commercial PRMTs Chemiluminescent Assay Kit  following the protocols. Briefly, histones were coated in a high binding plate and incubated with histone methyltransferase (HMT) assay buffer, S-adenoslymethionine, PRMTs enzyme, and AMI-1 . Then, primary antibody was added. Finally, the plate was treated with an HRP-labeled secondary antibody followed by the addition of HRP substrate to produce chemiluminscence that can then be measured using a BioTek Synergy™ 2 Multi-mode Microplate Reader.6 SW480 cells were inoculated in 0.1 mL of serum-free medium subcutaneously in the right flank of 5–6-week old healthy BALB/c nude mice. When the tumors reached an average volume of about 60 mm3, the mice bearing too large or too small tumors were eliminated and the left were divided into groups for treatment. Turing the experiment, tumor size was measured using with calipers every three days, and tumor volumes were calculated according to the following formula: (Length × Width2) × 0.52.Male athymic BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co. Ltd.  and maintained under specific pathogen-free conditions in the National Chengdu Center for Safety Evaluation of Drugs. Mice were manipulated and housed according to protocols approved by the Institutional Animal Care and Treatment Committee of Sichuan University. A total of 1.0× 10in vivo delivery, PRMT5 siRNAs and its negative control  were conjugated with cholesterol and were administered either intratumorally (i.t.) or intravenously .Three different siRNAs for human PRMT5 were provided by Ribobio  and their sequences are listed in 3 cells were seeded per 96–well plate. After 24 h, cells were transfected with si-NC or si-PRMT5. At times indicated, proliferation assay was performed with the Cell Counting Kit-8 . For the colony formation assay, cells were placed in 60 mm dishes at 200 cells per dish and maintained in the absence or presence of PRMT5 siRNA for 14 days. Cells were fixed with fixative  for 15 min and then stained with crystal violet (0.2 g/liter) for 25 min. For cell cycle assay, cells were plated at 1 × 105 cells/well in 6-well plates. The next day, cells were treated with AMI-1 or vehicle control only for 24 h, 48 h and 72 h. The treated cells were harvested using 0.25% trypsin without EDTA and fixed in 75% ethyl alcohol. After digestion with RNase, DNA was stained with propidium iodide and analysis with a Beckman Coulter flow cytometer and Modfit software. For apoptosis assay, treated cells were stained with Annexin V and propidium iodide and evaluated using a Beckman flow cytometer.To determine cell growth rate, 2.5 × 104 cells resuspended in 100 μL non-serum culture medium were placed triplicatedly in upper chamber of insert and medium with 10% FBS was used as chemo-attractant in lower chamber. AMI-1 or vehicle was added to inner chamber. Before plating in the outer chamber, cells were incubated for 20 min with RPMI 1640 medium containing AMI-1 or vehicle. Cells were allowed to migrate for 20 h. The cells remaining on the top surface of the membrane were removed with a cotton swab. The cells on the bottom surface of the membrane were fixed and stained with 0.2% crystal violet.Assays were conducted using 8 μm pore size Transwell plate (Corning Costar). In brief, approximate 4 × 10Protein were separated in 6%-12% SDS-PAGE gels and then transferred to PVDF membrane (Bio-Rad). After blocking with 5% non-fat milk for 1 h, the membranes were incubated with the primary antibodies at 4°C overnight. After washing with 1 × TBST, the membranes were incubated with corresponding secondary antibodies conjugated to horseradish peroxidase . Immunoreactivity was visualized using ECL Western blotting detection reagents and then analyzed through scanning densitometry. The identity and suppliers of the antibodies are listed in ChIP was performed with SW480 cells transfected with si-NC or si-PRMT5 using EZ-Magna ChIP G kit  according to the manufacturer's protocol. Protein-DNA complexes were precipitated with normal IgG, anti-PRMT5, anti-Histone H3 (Sym-dimethyl Arg8) and anti-Histone H4 (Sym-dimethyl R3) antibodies at 4°C overnight with rotation. The abundance of FGFR3, GAPDH, and eIF4E promoter regions in ChIP precipitates was quantified using qRT-PCR and specific primers. The qRT-PCR primers are listed in t test. P-value less than 0.05 was considered statistically significant. Error bars, S.D. *P < 0.05, **P < 0.01 or ***P < 0.001 level.The results were presented as mean ± S.D., except where indicated. The data were subjected to student's


For this review, we will focus on the regulation of the Wnt/β-catenin signaling pathway in skin cancer. With the important role in stemness and skin CSC proliferation, the Wnt/β-catenin signaling pathway and its elements have the potential to be targets for skin cancer therapy.Cooperating with other signaling pathways, Wnt signaling controls cell proliferation, morphology, motility, and embryonic development destination and maintains the homeostasis of tissues including skin, blood, intestine, and brain by regulating somatic stem cells and their niches throughout adult life. Abnormal regulation of Wnt pathways leads to neoplastic proliferation in these tissues. Recent research shows that Wnt signaling is also associated with the regulation of cancer stem cells (CSCs) through a similar mechanism to that observed in normal adult stem cells. Thus, the Wnt/ Drosophila (wg) ..8].The epidermis forms the outermost layer of mammalian skin. Cells within the basal layer of the epidermis are the progenitors of the epithelium, which constantly proliferate to ensure renewal of the epithelium. Except for the basal layer, the bulge is an important niche for skin stem cells; the bulge is attached to the hair follicles (HFs). Many Lgr6+ cells reside above the bulge area and have been demonstrated to represent the ability to generate all cell lineages of skin . Almost β-catenin signal controls the fate of the bulge stem cells and plays an essential role in hair but not epidermal differentiation. Using cre/loxP recombining system driven by the cytokeratin (K)14 promoter to induce the conditional mutation of β-catenin in mice, Peter Wend's Laboratory demonstrates that the epidermis of young mutant animals develops normally but without hair [β-catenin is introduced by Adenovirus-mediated gene transfer of cre/loxP system demonstrated that Wnt/β-catenin plays a minor role in epidermal but not in hair development [Evidence supports that the Wnt/out hair . Howeverout hair –13. At tβ-catenin in the dermal papilla and matrix cells in which β-catenin contributes to the maintenance of HF structures [In the epidermis, mesenchymal stem cells are self-renewing and differentiate into hair follicles (HFs) when proper stimulations are performed. During this process, signals that are initiated from the dermis can induce epithelium thickening, elongation of epithelial cells, and formation of placodes that contain Wnt-responsive cells –17. In rructures .Neural crest cells represent a transient and highly migratory embryonic cell population, and neural crest stem cells (NCSCs) are characterized by their multipotency and self-renewal abilities . It is d Wnt1-Cre R26R mice found that SKP cells were the same as the cells derived from the neural crest lineage [Although a substantial number of reports have demonstrated the existence of NCSCs in adult tissues, the physiological role of these cells still remains unknown. Recently, for the first time, a study by Johnston's group  demonstr lineage . Moreove lineage , and neu lineage . After wβ-catenin signaling plays a critical role in driving neural crest cells toward a melanocyte cell fate through regulating the homeobox gene microphthalmia transcription factor (MITF) [β-catenin is in the majority of benign nevi; however, there is no nuclear β-catenin in melanoma progression [β-catenin signaling may be essential for cellular homeostasis. Consequently, a loss of Wnt/β-catenin signaling may result in the dysregulation of specific transcriptional programs in melanocytes and nevus cells, which may lead to lower survival rates in melanoma patients [Wnt/r (MITF) –32. It igression –35. Thuspatients –35.β-catenin and other signaling pathways. There are two pathways interacting with Wnt/β-catenin signaling in the epidermis: Notch and vitamin D pathways. Notch1 plays an essential role in the development and homeostasis of postnatal hair follicles [β-catenin can bind to and activate the vitamin D receptor (VDR) by cooperating with vitamin D response elements that significantly function in the postnatal maintenance of HFs [β-catenin, TCF/Lef, and VDR. In wild-type epidermis, initiation of the growth phase in HF anagen is dependent on Wnt signaling activation. HFs are kept in the telogen phase in a mouse model with the functional loss of Wnt signaling. However, the cycling of hair growth was not maintained, and HFs were degenerated in the absence of VDR. A high level of Wnt signaling activation in the presence of endogenous vitamin D or a topically applied vitamin D analog not only was able to trigger anagen, but also induced ectopic formation of HFs. Wnt activation was unable to induce differentiation of ectopic follicles without the presence of VDR  signaling [β-catenin, pharmacological inhibition of Shh signaling can block de novo hair follicle formation. Conversely, a high level of β-catenin activation can make the inhibition of Shh signaling to improve morphogenesis [β-catenin signaling [β-catenin affects hair development via Shh, and Shh can function similarly to β-catenin. Therefore, we can target not only β-catenin but also Shh when we consider therapy for skin cancer.In addition, one of the pathways that is upregulated by ignaling . Combineogenesis . The expogenesis , which rignaling , 44. ThuSkin cancer is one of the most common forms of cancer with an especially high incidence in the white population . There aβ-catenin signaling plays a critical role in skin CSCs. Recently, a population of CSCs was identified by Malanchi et al. These cells show similar molecular characteristics to normal stem cells in the bulge [β-catenin in the nucleus. Human squamous cell carcinomas also exhibit a preferential nuclear location of β-catenin. Remarkably, conditional ablation of β-catenin will result in complete regression of the tumors and terminal differentiation of the tissues through tamoxifen-induced mutagenesis in DMBA-TPA or Ras-induced tumors. Thus, Wnt/β-catenin signals are essential in normal skin to drive bulge stem cells toward the hair cell fate, whereas they play a critical role in the maintenance of skin CSCs in epidermal tumors [Wnt/he bulge . CD34+ bl tumors .β-catenin signaling contributes to the homeostasis of normal skin stem cells, and its malfunction results in production of skin cancer stem cells. Thus, we can prevent the generation of CSCs by regulating Wnt signaling specifically. Thereby, the therapy of skin cancer targeting Wnt/β-catenin signaling is an innovative strategy to cure skin cancer. Herein, several approaches will be introduced.Because of drug resistance and the relapse and metastasis of cancer stem cells, skin cancer therapy is faced with a number of technical challenges and regulatory hurdles that must be overcome. In order to avoid all of these negative effects, it is very important to search for targeted and effective treatments for skin cancer. As described above, Wnt/β-catenin transcription response [Porcupine (Porcn), a member of the membrane-bound O-acyltransferase (MBOAT) family, catalyzes the palmitoylation family of Wnt proteins . Palmitoresponse . We can β-catenin transcription, such as CBP, p300, B-cell lymphoma 9 (BCL9), and pygopus, have been identified [β-catenin was identified using a cell-based reporter screen. While it is a small molecule secondary structure-template chemical library, ICG-001 does not interact with the homologous coactivator p300. Therefore, fundamental stem and progenitor cell switching points are regulated by the switch from β-catenin/CBP to β-catenin/p300 [β-catenin targets cellular proliferation and p300/β-catenin responds to cellular differentiation, the balance of CBP/β-catenin and p300/β-catenin may be very important for a cell to decide to either maintain its level of potency or go on to differentiate and lose a level of potency. Therefore, it may be an effective therapeutic strategy to find the inhibitors of CBP/β-catenin [Several coactivators for Wnt/entified –51. The nin/p300 , 53. Sin-catenin .β-catenin signaling pathway. As for skin cancer, the Wnt/β-catenin pathway has been related to malignancy in nonmelanoma skin cancers (NMSCs) such as squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) [NSAIDs, including aspirin, flurbiprofen, sulindac, and indomethacin, function by inhibiting the activity of cyclooxygenase (COX), which is a key enzyme in the arachidonic acid cascade. Recently, many experimental and epidemiological studies in humans have provided evidence that aspirin and other NSAIDs exhibit chemopreventive effects against multiple cancer types by inhibiting the Wnt/ma (BCC) –57. Prevma (BCC) . The datma (BCC) . Regularma (BCC) –62. To oma (BCC) –66. One ma (BCC) , 67.β-catenin promotes proliferation and blocks epidermal differentiation although it stimulates hair follicle differentiation [β-catenin to promote keratinocyte proliferation, 1,25(OH)2D3/VDR promotes the ability of β-catenin to stimulate hair follicle differentiation. The mechanism involved 4 aspects: 1,25(OH)2D3 and VDR suppress transcriptional activity of β-catenin/TCF, 1,25(OH)2D3 represses mRNA expression of the β-catenin target gene of Gli1 but induces the β-catenin target gene PADI1 in vitro, VDR ablation reduces β-catenin target genes involved in hair differentiation in vivo, and 1,25(OH)2D3/VDR suppresses the transcriptional activity of β-catenin and represses the mRNA expression of Gli1 [β-catenin inhibitory proteins; for instance, disabled-2 (Dab2) is induced by retinoic acids and Dickkopf-1 and Dickkopf-4 (Dkk-1 and Dkk-4) are induced by vitamin D [1,25-Dihydroxyvitamin D3 2D3), the active metabolite of vitamin D, suppresses the proliferation while promoting the differentiation of keratinocytes through the vitamin D receptor (VDR). However, ntiation . A recen of Gli1 . Recentlitamin D , 71.β-catenin signaling. Fisetin-treated cells showed increased cytosolic levels of Axin and β-TrCP and decreased phosphorylation of glycogen synthase kinase 3β associated with decreased β-catenin stabilization. Fisetin-mediated interference with functional cooperation between β-catenin and T-cell factor- (TCF-) 2 resulted in the downregulation of positively regulated TCF targets, such as c-myc, Brn-2, and Mitf. Those data suggest that fisetin can be developed as an effective agent against melanoma because of its potential inhibitory effect on β-catenin/Mitf signaling [With the exception of chemical synthetic products, natural products, particularly traditional Chinese medicines (TCMs), are rich sources of pharmacologically active substances. Through the investigations, it has been discovered that stem cell signaling pathways could be targeted by the natural products . Epidemiignaling .V600E cooperates with Pten loss to induce metastatic melanoma [β-catenin signaling pathway is frequently upregulated in melanoma but its functional implication is unclear. Damsky and colleagues studied the functional role of β-catenin by modulating it in the BRafV600EPten−/− model. They showed that β-catenin is the mediator of melanoma metastasis to lymph nodes and lungs. In addition to its role in metastasis, β-catenin levels control cell differentiation and regulate both the MAPK and the PI3K/AKT signaling pathways [Although mice rarely develop melanoma spontaneously, the genetically engineered mouse models of melanoma have been generated by different groups. Compared to xenograft hosts, mice used for GEM models have a fully functional immune system. Therefore, they serve as reliable and repeatable models to study the role of the immune system cells in melanoma biology and drug resistance , 74. Danmelanoma . This mopathways . This moβ-catenin signaling plays a critical role in both normal stem cells and CSCs. Over the past few years, remarkable progress has been achieved in research on Wnt/β-catenin signaling and its inhibitors. The pharmaceutical industry has placed high expectations on a number of clinical candidates that target Wnt signaling. There are many novel drugs and natural products targeting Wnt/β-catenin signaling pathway that have been developed.Stem cell research has a significant potential to enhance our understanding of cancer stem cells and thus revolutionize tumor therapy. Wnt/


Banana is the fourth most important food commodity of the world and forms the staple food of majority of people in the tropical and subtropical regions. Banana production is severely constrained by Fusarium wilt disease that causes enormous loss. The present study developed transgenic banana plants overexpressing native cell death genes to impart Fusarium wilt resistance. Since banana is predominantly a vegetatively propagated crop, genetic engineering is the most viable option for development of resistance against important diseases of this crop. Fusarium oxysporum f. sp. cubense (Foc). Since there are no established edible cultivars of banana resistant to all the pathogenic races of Foc, genetic engineering is the only option for the generation of resistant cultivars. Since Foc is a hemibiotrophic fungus, investigations into the roles played by different cell-death-related genes in the progression of Foc infection on host banana plants are important. Towards this goal, three such genes namely MusaDAD1, MusaBAG1 and MusaBI1 were identified in banana. The study of their expression pattern in banana cells in response to Foc inoculation  indicated that they were indeed differentially regulated by fungal inoculation. Among the three genes studied, MusaBAG1 showed the highest up-regulation upon Foc inoculation. Further, in order to characterize these genes in the context of Foc infection in banana, we generated transgenic banana plants constitutively overexpressing the three genes that were later subjected to Foc bioassays in a contained greenhouse. Among the three groups of transgenics tested, transformed banana plants overexpressing MusaBAG1 demonstrated the best resistance towards Foc infection. Further, these plants also showed the highest relative overexpression of the transgene (MusaBAG1) among the three groups of transformed plants generated. Our study showed for the first time that native genes like MusaBAG1 can be used to develop transgenic banana plants with efficient resistance towards pathogens like Foc.In order to feed an ever-increasing world population, there is an urgent need to improve the production of staple food and fruit crops. The productivity of important food and fruit crops is constrained by numerous biotic and abiotic factors. The cultivation of banana, which is an important fruit crop, is severely threatened by Fusarium wilt disease caused by infestation by an ascomycetes fungus  Fusarium oxysporum f. sp. cubense (Foc), the causative agent of Fusarium wilt disease of banana  is a highly prized banana cultivar preferred for its unique fruity aroma and golden yellow colour. Introduction of Fusarium wilt resistance in cv. Rasthali therefore remains an important objective.By the year 2050, the world's population is expected to be double the present number, making food security for all the most important social issue over the next 35 years. To feed this surge in the population, the annual food output will have to be at least doubled in future. To address this enormous task, agricultural productivity should be increased in a sustainable way. The most important constraints in achieving this target are the numerous biotic stress factors that cumulatively contribute to a significant lowering of the productivity of important food and fruit crops to below what their actual genetic yield potential can confer. Banana is counted among the most important food security crops in the world after rice, wheat and maize. Although the potential of banana as a cash and export commodity is generally considered to be huge, diseases and pests have hindered efforts towards increasing its production in the last few decades . Fusariuf banana , is a hef banana  beyond Sf banana  is all tf banana ]. Owing f banana . GeneticF. oxysporum have a short biotrophic phase inside the host plant which is followed by complete necrotrophy of the infected plant  is an ER resident protein which takes part in the UPR response, thereby assisting in restoration of cellular homoeostasis by facilitating efficient protein folding  is a membrane-bound protein that blocks the activity of BAX protein by an unknown mechanism leading to the suppression of cell death processes induced during fungal infections, by fungal toxins, salicylic acid, reactive oxygen species and during ER stress (BI-1 (OsBI-1) gene was shown to be down-regulated in rice suspension cells upon treatment with the cell wall extract of rice blast fungus Magnaporthe grisea (Arabidopsis BI-1 (AtBI-1) gene showed resistance to cell death caused by the above-mentioned elicitor treatment. BAX inhibitor-1 (TaBI-1) in wheat was identified as a negative cell death regulator and conferred resistance against stripe rust  goes beyond its capacity, hence several of them are left misfolded or unfolded, causing an unfolded protein response (UPR) ultimately leading to PCD . Among tR stress . BI-1  or beauvericin (BEA) or simply by inducing host cell death initiating proteins thereby leading to PCD. A recent report illustrated that overexpression of Supporting Information.Primers used in the present study are listed in Rasthali) leaves using Concert Plant RNA Reagent and RNeasy Plant Mini Kit as described above. Accuscript Reverse Transcriptase was used to make cDNA from this total RNA. Full length coding sequences of MusaDAD1, MusaBAG1 and MusaBI1 were amplified from the cDNA prepared as above using Pfu Ultra AD DNA Polymerase  and subsequently sequenced. Predicted protein sequences of three genes cloned were aligned with their closest homologues using ClustalW2 program . Evolutionary relationships of the three genes were studied using a MEGA 5 software tool.Total RNA was extracted from young banana  maintained in M2 medium  and BEA . Total RNA was extracted from the treated as well as control untreated cells using Concert Plant RNA Reagent . The extract obtained after chloroform extraction was cleaned and treated with DNase using the RNeasy Plant Mini Kit . This RNA (∼5 μg) was then used to make first-strand cDNA using Oligo (dT)12–18 primer (Invitrogen) and AccuScript Reverse Transcriptase  following the manufacturer's instructions. Three independently treated cell aliquots for each treatment were pooled prior to RNA isolation to ensure reproducibility of results. cDNAs derived from the treated and untreated controls were then diluted appropriately and used for quantitative real-time RT–PCR using SYBR Green Extract-N-Amp PCR ReadyMix  and Rotor-Gene Q platform (Qiagen). Primers specific for the three identified genes  were used in these reactions alongside primers specific for Musa acuminata elongation factor 1α (EF1α) coding sequence so as to allow gene expression normalization and subsequent quantification. Ct values obtained using the Rotor-Gene software were analysed using REST-MCS utility  and incubating it at room temperature for 4 weeks. Two-month-old greenhouse acclimatized transgenic banana plants derived from independent transformation events were replanted in the mixture of soil and fungal mass culture in a 1 : 1 ratio and kept under greenhouse conditions (Seven-day-old culture of Foc Race 1 grown on potato dextrose agar (PDA) was inoculated in PDB medium and allowed to grow at 30 ± 1 °C for 5 days. The fungal spores were separated from the mycelium by sieving the culture through four layers of cheese cloth. The spores were washed with sterile distilled water and adjusted to a spore density of 8 × 10−1 in a 0.9 % (w/v) agarose TAE gel. DNA was then transferred on to a positively charged nylon membrane by a capillary method using 20× SSC buffer. Transferred DNA was UV crosslinked and subsequently immobilized by baking at 120 °C for 30 min. Restricted and immobilized genomic DNA was then allowed to hybridize with DIG-labelled probes targeted against a hygromycin phosphotransferase coding sequence present towards the left border in T-DNA of the three binary vectors used to transform banana cells. Chemiluminescent detection of hybridization signals was subsequently carried out in a chemiluminescence detector enabled gel documentation system using DIG High Prime DNA Labeling and Detection Starter Kit II  according to the manufacturer's instructions.Genomic DNA was isolated from young leaves of the selected transgenic lines as well as untransformed controls as described previously. Genomic DNAs (∼20 µg) were digested overnight with HindIII restriction enzyme at 37 °C. Digested DNAs were separated overnight at a field strength of 1.25 V cmMusa acuminata elongation factor 1α (EF1α) gene was also amplified along with the three overexpressed genes to allow gene expression normalization and subsequent quantification. Ct values obtained using the Rotor-Gene software were analysed using REST-MCS utility  as described above. MusaDAD1, MusaBAG1 and MusaBI1. Their coding sequences were amplified using leaf derived cDNA of banana cv. Rasthali and they were subsequently sequenced to confirm the presence of the complete ORF. The 348 bp CDS of MusaDAD1 codes for a 115 amino acid long protein have three putative transmembrane domains similar to its close homologues Supporting Information][see . Like MusaDAD1, MusaBI1 also had high levels of homology with its close relatives, except at the N-terminal.Based on the nomenclature of their closest homologues the three genes identified as part of this study were named F. oxysporum has been accepted to have a hemibiotrophic mode of nutrition during its presence on the plant host, the roles of different host PCD related genes during the infection and colonization process assume importance. Thus, we studied the differential regulation of the three identified PCD related genes  in banana in response to Foc infection using two Fusarium toxins namely FA and BEA or by utilizing the active Foc culture (aFoc) or an autoclaved filtrate of Foc culture (fFoc). For the three genes studied in this way, striking similarities were noticed in the expression pattern obtained using either BEA, aFoc or fFoc  in our studies, possibly indicating a vital role for this gene in the response of banana to Foc invasion.Given that Zea mays polyubiquitin promoter and nos 3′ UTR to obtain p1301-MusaDAD1, p1301-MusaBAG1 and p1301-MusaBI1 binary vectors, respectively  in the respective transgenic lines as compared with the untransformed control plants  were identified and overexpressed in transgenic banana plants. The plants overexpressing MusaBAG1 demonstrated the best resistance towards Foc infection in greenhouse bioassays.Owing to the recent spread of highly virulent Foc Race 4 beyond South-East Asia and Australia, the threat of a major disease epidemic in major banana-growing areas of the world, mainly Africa and Asia has become very real. Genetic engineering of elite banana cultivars for resistance to Fusarium wilt is the only pragmatic goal as there are no established Foc Race 4 cultivars readily available right now. In the present study, three genes putatively involved in cell-death-related processes in banana  were identified in banana by homology searching (using genes derived from rice and Arabidopsis as query) in the EST databases of banana. Expression profiling of these genes in banana cells treated with known Foc derived toxins (FA and BEA) or the active culture/autoclaved filtrate (aFoc/fFoc) indicated differential regulation of the three genes in response to these four treatments. The fact that BEA, aFoc and fFoc treatments led to similar expression profiles for all the three genes studied indicated that they probably activated common signalling pathways upon inoculation. Further, since these effects were reported in just 6 h post-inoculation, this suggested that BEA probably acts in the initial stages of the infection process. Also, the fact that MusaBAG1 was induced to significant levels in BEA-treated cells (similar to those which were treated with aFoc and fFoc) correlated well with our later observations wherein transgenic banana plants overexpressing MusaBAG1  were found to be highly resistant to Foc Race 1 infection. Conversely, FA induced the three genes studied in a diagonally inverse pattern as compared with the other three treatments. Fusaric acid, which down-regulated MusaBAG1 in the treated cells, therefore, appears to work towards the induction of the PCD and thereby the necrotropic phase of Foc life cycle on the host banana plant. Although FA induced the expression of MusaBI1 to significant levels, the fact that MusaBI1 overexpressing plants did not show impressive Foc resistance indicated that unlike MusaBAG1, the role of MusaBI1 in the establishment of Foc infection on host banana plants is not very significant. As regards the MusaDAD1 gene regulation, although we could not find any significant change in the expression level of this gene in three of four treatments preformed, past reports have shown significant down-regulation of this gene in Foc susceptible as well as resistant plants upon inoculation with Foc spores  in MusaDAD1 overexpressing plants.Since there are multiple confirmed reports describing the manipulation of host PCD machinery by an invading fungal pathogen in plants , a detaiMusaBAG1 overexpressing plants. This was probably the result of lower steady state levels of the MusaBAG1 gene in uninfected untransformed control plants. Concurrently, the best resistance against Foc was also noticed in MusaBAG1 overexpressing plants. So, although all the three genes studied here have been associated with improved resistance against specific pathogens in model plants like Arabidopsis, we propose a far greater role for the MusaBAG1 gene in the control of PCD in banana plant as compared with the other two genes studied. Further, in the face of stiff resistance towards development of novel transgenic plants with genes derived from animal systems , use of native genes like MusaBAG1 for incorporating efficient resistance towards pathogens like Foc is expected to be far more acceptable to the scientific community as well as to the general populace. These plants can potentially resist Foc Race 4 infection also. But since Foc Race 4 has still not been reported from India , no bioassay against Foc Race 4 could be performed with the plants developed during this study.Among the three genes studied as part of this study, we could get the maximum relative overexpression in MusaBAG1 for development of Fusarium wilt resistance in cultivars like Rasthali could be the best way to improve the production of local cultivars where huge losses have been reported due to Fusarium wilt in the past.In conclusion, use of native genes like This work was funded by the Department of Atomic Energy, Government of India.S.B.G., U.K.S.S. and T.R.G. conceived and designed the experiments and wrote the paper; S.B.G. performed the experiments; U.K.S.S. and S.B.G. analysed the data; and T.R.G. and U.K.S.S. contributed reagents/materials/analysis tools.None declared.MusaDAD1, MusaBAG1 and MusaBI1 from banana have been submitted to GenBank with accession numbers KJ636052, KJ636053 and KJ636054, respectively.The nucleotide sequences of Supporting Information is available in the online version of this article –The following Table S1. Primers used in the present study.Fig. S1. Sequence and phylogenetic analysis of MusaDAD1.Fig. S2. Sequence and phylogenetic analysis of MusaBAG1.Fig. S3. Sequence and phylogenetic analysis of MusaBI1.


These effects are thought to be mediated by an iodinated arachidonic acid derivative, 6-iodolactone (6IL), and one of the proposed mechanisms is that 6IL activates Peroxisome Proliferator-Activated Receptors type gamma (PPARG). These receptors have been implicated in the inhibition of carcinogenic processes, in addition to their classical role in maintaining lipid and glucose homeostasis. The aim of this study was to determine whether PPARG participates in the 6IL antiproliferative and apoptotic effects on the mammary cancer cell line MCF-7.Molecular iodine  context, and stable PPARG-knockdown MCF-7 cells , were used to corroborate the PPARG participation. 6IL effects on proliferation (measured by Trypan Blue exclusion) and apoptosis (phosphatidylserine identification by flow cytometer) were evaluated in conditions of chemical inhibition (GW9662) and silencing (RNA interference). A wound-healing assay was conducted on wild-type and stable PPARG-knockdown MCF-7 cells to evaluate the antimigrational effect of 6IL. Caspase-8 activity was evaluated to determine if the extrinsic pathway is involved in the effects of 6IL and I2-induced apoptosis.Antiproliferative and pro-apoptotic 6IL effects require the activation of PPARG. In addition, wound-healing assays show that 6IL is able to inhibit MCF-7 cell migration and that PPARG plays a role in this phenomenon. Finally, the data exclude the participation of the extrinsic apoptotic pathway in 6IL- and I2 effects are mediated by 6IL, and they provide further support for the use of I2 as coadjuvant in breast cancer treatment.These results support the previously proposed mechanism, in which the I Moleculpoptosis ; on the 2 as 6IL generate antitumor effects through similar pathways. Both trigger a p53/p21-mediated cell growth arrest, and both induce Bax-caspases and AIF/PARP-1 apoptosis [PPARG) and inhibition of PPAR alpha (PPARA) expression [At the molecular level, both Ipoptosis . In MCF-pression . Moreovepression –13. Whilpression , 15, PPApression , 17 is ppression –22. Thespression , 24. Witpression . Thus, wTo demonstrate that PPARG activation plays a key role in the 6IL effects, we inhibited PPARG/6IL complex formation by blocking the PPARG ligand-binding domain with its highly specific antagonist GW9662. To determine the appropriate concentration of GW9662, i.e., a concentration that inhibited PPARG/6IL complex formation without causing the antiproliferative effects previously reported , we perfGFP- and PPARG-adenoviral vectors, as measured by GFP intensity and mRNA amplification, respectively. Figure PPARG overexpression was confirmed by fatty acid synthase (FASN) expression, a PPARG-regulated gene  was assessed  and RNAi-PPARG (RNAi) MCF-7 cells were treated with 10 μM 6IL over a 96-h period, and their proliferation rates were evaluated. Figure PPARG MCF-7 cells had the same proliferation rates, whereas RNAi-PPARG MCF-7 cells exhibited significantly faster proliferation. The 6IL supplement inhibited proliferation of WT and Scramble-PPARG MCF-7 cells, whereas it had no effect on the PPARG-knockdown group (RNAi-PPARG). Figure PPARG cells and no effect on RNAi-PPARG MCF-7 cells.To confirm the stable transfection of RNAi against sed Fig. . PPARG sbes Fig.  and Westbes Fig. . Wild tyPPARG MCF-7 cells treated with 6IL showed significant inhibition of migration, around 60 %; however, 6IL did not change the migration rate of RNAi-PPARG MCF-7 cells -independent mechanism [2 reacts with cell components, but I2 is known to bind covalently to proteins and lipids [2 after its conversion to 6IL is supported by the following evidence: 1) 6IL can be identified in both in vitro and in vivo models after administration of I2 [PPARG is increased in I2- or 6IL-supplemented conditions (possibly through an autoregulation mechanism) [2 is only observed in cancer cells, which contain elevated concentrations of AA [285 in the PPARG ligand-binding domain with an IC50 in the nanomolar range [50 of 6IL [PPARG. By its nature, the adenoviral system forces the cell to maintain unregulated expression; therefore, it is possible that the GW9662 concentration was not sufficient to saturate the PPARGs due either to their overexpression from the vector or to well-established, positive, autoregulatory mechanisms that maintain a high and continuous expression of PPARG [PPARG-overexpressing cells. Thus, partial PPARG blockage may unmask this dual action of 6IL, in which only a few free PPARG receptors are required for the antiproliferative effect (maybe cell cycle arrest), but additional available receptors are needed to induce apoptosis. Similar dose-dependent responses of PPARG ligands have been described in the literature previously, showing induction of arrest at low concentrations, whereas at high levels they trigger apoptosis [PPARG-overexpressing cells, 6IL administration also induces increased expression of FASN, a PPARG-regulated gene that is related to differentiation [A significant body of data from eriments , 26–29, eriments –33, and eriments –36 suppoechanism , 8, 37 techanism . It has d lipids . The iodd lipids , 39, andchanism) ; 3) the ns of AA . Previouns of AA , 29. GW9ar range , as comp0 of 6IL . This veof PPARG , 42. In poptosis , 44. Morpoptosis . Interesntiation , 47. Thintiation .PPARG MCF-7 cells was two-fold greater than that of MCF-7 control cells, corroborating similar reports in other cancer cells [PPARG knockdown are insensitive to both the antiproliferative/apoptotic as well as to the anti-migratory effects of 6IL are clear indications that these receptors have an important role in the antineoplastic actions of I2 or 6IL. The next step is to determine if 6IL affects the cell’s invasive characteristics, since the activity of some metalloproteinases and other invasive markers has been blocked by PPARG ligands in several cancers [In relation with silencing the expression of PPARG by molecular tools, our results show that the proliferation rate of RNAi-er cells . The fin cancers , 50.2 or 6IL induces apoptosis effects through at least two pathways: AIF/PARP1 and Bax-caspases [et al. [FasL gene, thereby inducing extrinsic apoptosis in MCF-7 cells; this observation was reinforced recently by similar findings in human colorectal cancer cells [2 or by 6IL treatment; therefore, it seems that the apoptotic effects of I2 and 6IL are mediated exclusively by intrinsic apoptosis pathways.Finally, it has been described that Icaspases ; Bonofig [et al.  reports er cells . In the FASN expression strengthens previous observations that 6IL also exerts differentiating effects . 6IL in2 prior to experiments.Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, puromycin and trypsin solution were supplied by GIBCO . Arachidonic acid (AA) (purity >99 %) was used to synthesize 6IL was from Calbiochem . GW9662 (purity >98 %), a specific PPARG antagonist, was obtained from Sigma-Aldrich . All other chemicals were of the highest grade of purity commercially available. The breast cancer cell line MCF-7 was obtained from ATCC®. The cells were maintained in Basal Medium  at 37 °C and 5 % CO2, stirred for 4 h at room temperature and protected from light. The solution containing crude product was concentrated under low nitrogen flow to 0.5 ml and separated on preparative TLC using the solvent system CH2Cl2/MeOH (97.5:2.5). The reaction products, AA (rf: 0.5) and 6IL (rf: 0.88) standards were visualized by iodine vapors, and the 6IL synthesized was eluted with the same solvent and concentrated under low nitrogen flow.6IL was synthesized with a modification of the method of Monteagudo . BrieflyPPARG (AdPPARG) were provided by Dr. Steals . Adenovirus expressing GFP (AdGFP), provided by Dr. Hernandez-Gutiérrez , was used to confirm infection and as control. Viruses were multiplied in HEK-293 cells . Adenoviral titer was determined by the MOI (multiplicity of infection) test . Prior to treatment with GW9662 and/or 6IL, MCF-7 cells were infected at an MOI of 50 for 24 h in basal medium.Recombinant adenoviruses expressing human PPARG, a puromycin resistance gene, a tGFP gene and a U6 promoter was obtained from Origen Technologies, Inc.  (RNAi-PPARG cells). A similar plasmid but with a scrambled sequence of the short RNAi hairpin against PPARG (Scramble-PPARG cells) was used as internal control. MCF-7 cells were transfected with 5 μg of plasmid and Xfect™ Transfection Reagent  according to the manufacturer’s instructions, and they were maintained in culture for 48 h; after this time the transfected cells were selected with 1.0 μg/mL puromycin daily over 2 weeks and expanded. Transfection of the cells was confirmed by tGFP expression using an IX50 Olympus microscope with inverted reflected light fluorescence system , or by an Agilent 2100 bioanalyzer using an On-Chip flow cytometry system . Knock-down of PPARG expression was evaluated by quantitative real time PCR (qPCR) with the specific Hydrolysis Probes  shown in Table Retroviral silencing plasmid (HuSH™) containing a short, specific hairpin with RNA interference for PPARG-MCF-7-infected cells, on Scrambled- or RNAi-PPARG-MCF-7-transfected cells and on wild-type MCF-7 cells. The cells were cultured at a density of 5 × 105 cells in 60-mm culture dishes  for qPCR assays; 4 × 104 cells/well in 24-well culture plates for proliferation assays, 2 × 105 cells in 60-mm culture dishes for apoptosis experiments, 3 × 106 cells in 100-mm culture dishes for Western blot assays, 8 × 105 cells in 60-mm culture dishes for wound-healing assays, and 5 × 106 cells in 100-mm culture dishes for caspase-8 activity assays. All cells were seeded in complete basal medium and incubated for 24 h before treatment; at the beginning of experiments the medium was changed to low serum (0.5 %)-containing medium. The cells were treated for 24, 48, 72 or 96 h, (depending on the experiment) at 37 °C with 10 μM 6IL, 0.5 μM GW9662 or vehicle (ethanol) diluted before application in low serum-containing medium. In the GW9662/6IL-treated group, GW9662 was administered 2 h prior to 6IL treatment.The experiments were carried out on AdACTB, a non-regulated housekeeping gene. All measurements were performed in triplicate.Total RNA was extracted from cells using TRIzol® reagent  according to the manufacturer’s instructions. Two micrograms of total RNA was reversed transcribed using the Superscript II system . qPCR was performed on the sequence detector system Roto-Gene 3000  using SYBR green as a marker for DNA amplification. The reaction was performed with 1 μL cDNA template and the Maxima SYBR Green/ROX qPCR Master Mix , using 40 cycles of three-step amplification  and the gene-specific primers listed in Table Pellets of whole protein were obtained from lysed cells using RIPA buffer with complete Mini, EDTA-free protease inhibitor cocktail tablets . The extracted proteins were quantified by the Bradford method  and analyzed by Western blot. SDS polyacrylamide gel electrophoresis was performed as described , using s4/well) were cultured in DMEM supplemented with 10 % FBS on 24-well culture plates. Control cells were treated with 0.1 % ethanol (solvent for 6IL). After a 72-h treatment, the cells were harvested with trypsin-EDTA 0.05 % . Trypan Blue (0.4 %) was added; those cells that excluded the dye were counted with a hemocytometer under the microscope ; proliferation is reported as fold change with respect to control.The effects on proliferation of 10 μM 6IL, 0.5 μM GW9662 and the GW9662/6IL mixture were analyzed using the Trypan Blue dye-exclusion assay. The cells , and 1 μL of Annexin V  was added and incubated at room temperature for 10 min; after centrifugation at 2500 rpm for 3 min the supernatant was removed, and 1 μL each of Calcein-AM and Fluorolink Cy5 in 200 μL of Binding Buffer were added to the pellet and incubated for 10 min. Finally, the cells were collected by centrifugation and resuspended in Cell Buffer (provided by the manufacturer). A 10-μL aliquot of cells from each treatment was placed on the cell fluorescence LabChip® together the solutions provided in the kit. The apoptotic cells are reported as a percentage of the number of events; this population is delimited in a standardized region by the program Cell-Chip Agilent 2100 Expert software .Apoptosis was assessed by detecting phosphatidylserine on the external surface of the cell membrane with annexin V conjugated to the Cy5 fluorophore. To mark cells with intact cell membranes, Calcein-AM was used. Apoptosis was assessed by an Agilent Bioanalyzer 2100 and apoptosis kit following the manufacturer’s instructions . Briefly, after treatment the cell density was adjusted to 1 × 10PPARG MCF-7 cells, Scramble-PPARG MCF-7 cells and wild-type MCF-7 cells, 8 × 105 in each case, were seeded on 60-mm culture plates and incubated at 37 °C and 5 % CO2. After 24 h, the attached cells were scratched three times in parallel with a 1000-μl pipette tip (~377 μm) and treated with 10 μM 6IL or vehicle (ethanol) for 24 h. After this time, three new parallel wounds were performed as width control; the number of migrating cells was obtained by counting the number of cells inside this width control. The count was measured by Leica Application Suite Version 2.8.1., and the images were captured with a 10× objective lens using a Leica DM 2500 microscope and DFC 420 camera . The results are presented as number of migrating cells of treated (6IL) vs. control (Ctrl) groups (mean ± SD). At least three fields were analyzed for each plate, and each group was analyzed in three independent experiments.RNAi-6 MCF-7 cells were collected after a 48-h incubation with I2 or 6IL. The cell pellet was lysed, and an aliquot containing 200 μg of protein was incubated with DTT (10 mM) and a colorimetric substrate, IETD-p-nitroanilide;  at 37 °C for 120 min in the dark. The samples were then read in the spectrophotometer at 405 nm. The absorbance of pNA from a treated sample was compared with an untreated control to determine the fold increase in caspase-8 activity.The caspase-8/FLICE activity was measured using a commercial kit  according to manufacturer’s instructions. Briefly, 5 × 10P < 0.05 were considered statistically significant. In the figures, an asterisk indicates significant differences with respect to the control (P < 0.05), and different letters indicate significant differences between groups (P < 0.05).One- or two-way ANOVA was performed to determine the significance of differences between groups, followed by Tukey’s test for the significance of differences among multiple experimental groups. Data are expressed as mean ± standard deviation (SD), and values with


However the relationship between the core pluripotent factor, Kruppel-like factor 4  and Wnt/β-catenin signaling, remains ambiguous in J1 mESCs. DNA microarray analysis revealed that CHIR-treatment promoted pluripotency-maintaining transcription factors and repressed germ layer specification markers. CHIR also promoted genes related to the development of extracellular regions and the plasma membrane to maintain pluripotency of J1 mESCs. Among the CHIR-regulated genes, Klf4 has not been reported previously. We identified a novel cis element in the Klf4 gene that was activated by β-catenin in J1 mESCs. We determined that β-catenin interacted with this cis element, identifying Klf4 as a β-catenin target gene in this context. Moreover, several microRNAs that targeted the 3′-UTR of Klf4 mRNA were identified, with miR-7a being down-regulated by CHIR in a β-catenin-independent manner in J1 mESCs. These data collectively suggest that CHIR enhances Klf4 expression by repressing miR-7a expression or canonical Wnt pathway activation.Understanding the mechanisms that regulate pluripotency of embryonic stem cells (ESCs) is important to ensure their safe clinical use. CHIR99021 (CHIR)-induced activation of Wnt/ The -catenin . The stand Nanog , 7. ReceB6 mESCs , 9. ThusKlf4, a member of the Kruppel-like factor (Klf) family of conserved zinc finger transcription factors, establishes an ‘‘authentic” and ‘‘metastable” pluripotent state in various pluripotent cell types [Klf4 also mediates the basic nuclear organization at the Oct4 locus and maintains a high-order chromatin structure, which contributes to maintaining the pluripotency of ESCs [Klf4 is indispensable to maintaining the self-renewability and pluripotency of mESCs [Klf4 and take over the functions of LIF in mESCs.ll types –12. Klf4 of ESCs . As a diof mESCs . In seruof mESCs . These rKlf4 as a downstream target of CHIR, whose expression and functions are regulated by miR-7a and the Wnt/β-catenin signaling pathway.MicroRNAs (miRNAs) are evolutionarily conserved, small noncoding RNAs consisting of 21–25 nucleotides, that have essential roles in the self-renewability of ESCs –19. For Nanog, Klf4, Tbx3, Tfcp2l1, Nr5a2, Nr0b1, and Esrrb, and repress germ layer specification markers such as Gata3, Nodal, Otx2, Pax6, Notch1, and Neurod1 [Tfcp2l1 (Oct4 mRNA expression level was not influenced by CHIR treatment (We compared the expression profiles of J1 mESCs (GEO ID Number: GSE40959) treated with dimethyl sulphoxide (DMSO) or 3 μM CHIR. CHIR can promote the expression of pluripotent factors such as [Tfcp2l1 , while t[Tfcp2l1 . Howeverreatment . Meanwhireatment . We thenreatment . Among treatment . Moreovereatment . This chKlf4 mRNA expression. The differential expression level of Klf4 was confirmed by RT-qPCR in J1 mESCs. As shown in Klf4 mRNA were elevated 2-fold compared with control DMSO-treated cells. The changes in Klf4 mRNA expression correlated with altered Klf4 protein abundance after 3 μM CHIR treatment in mESCs  cells for further detection. F9 EC cells were treated with CHIR at final concentrations of 3, 10, 15 and 20 μM respectively. After 24 h treatment of F9 EC cells, levels of endogenous lf4 mRNA  and protlf4 mRNA  expressiKlf4 expression in J1 mESCs. We considered β-catenin as the key regulator, because this protein works as a dominant downstream transcription factor of Gsk3β. Previous studies have demonstrated that β-catenin activates canonical Wnt/β-catenin signaling by binding to the promoter of the gene of interest in a T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-dependent manner [As indicated above, Klf4 expression was responsive to CHIR and BIO-activated signaling. We therefore explored the mechanism by which CHIR influences t manner , 28.Klf4 expression is induced by the activated canonical Wnt signaling, we used a construct with seven tandem copies of the consensus Tcf/Lef binding site (TopFlash)  into J1 mESCs. RT-qPCR analysis revealed that the depletion of β-catenin was significant   for the er assay . Further-catenin , which t-catenin  and form of Klf4  mediatedted ESCs . These rKlf4 5′ flanking region for sequences corresponding to the β-catenin-Tcf/Lef DNA-binding consensus sequences to determine the mechanism by which β-catenin increased Klf4 expression in J1 mESCs. We noted a potential Tcf/Lef binding site (−1102/−1095) in the Klf4 promoter according to the prediction results by PROMO v8.3 [Klf4 promoter (−1124/+240) responded to the CHIR signaling depending on the Tcf/Lef binding sites. However, the truncated promoter (−1081/+240) and the Klf4 promoter-mut (−1124/+240) were slightly elevated after CHIR treatment, this phenomenon cannot disregard the indirect regulatory role on Klf4 by other factors that respond to CHIR signaling. To further characterize the DNA-binding sequence that is recognized by β-catenin, we performed chromatin immunoprecipitation assays. As shown in β-catenin was observed. These results collectively indicate that β-catenin directly binds to the Klf4 promoter and activates the expression of Klf4.We then screened the mouse OMO v8.3 , 30. We Klf4 expression, we investigated the effects of miRNAs on the regulation of Klf4. miRNAs that potentially targeted the 3′-UTR of Klf4 were predicted using multiple databases including TargetScan, Pic Tar and miRanda  reader, binding to specific methylated and/or unmethylated elements in mESCs [ in ESCs . Moreovereatment . We specin mESCs , 39. TheKlf4 and found that miR-7a could inhibit Klf4 expression. The 3′-UTR of Klf4 that we used, contains two putative miR-7a binding sites, consistent with previous studies [β-catenin could not repress miR-7a expression, suggesting a β-catenin-independent manner between CHIR signaling and Klf4 regulation mediated by miR-7a. Recent studies showed that miRNAs are initially transcribed as long, capped and polyadenylated primary miRNA transcripts. The RNase III enzymes, Drosha and Dgcr8 process the pri-miRNAs into approximately 72 bp precursor miRNAs, which are further processed by the RNase Dicer to produce the mature miRNAs [Klf4 expression by transcriptionally activating Klf4 in a β-catenin-dependent manner and by repressing the expression level of miRNAs that target Klf4 in a β-catenin-independent manner.Post-transcriptional modifications of stemness factors mediated by sumoylation , ubiquit studies , 32. We e miRNAs . Moreovee miRNAs , similare miRNAs . In addie miRNAs –48. Thusβ-mercaptoethanol, 1× non-essential amino acids, 2 mM GlutaMax, 50 U/mL penicillin, 50 μg/mL streptomycin , and 1000 U/mL LIF . The culture of murine F9 EC cells  and HEK 293FT cells  were performed as previously described [J1 mESCs purchased from the American Type Culture Collection  were cultured without feeders on tissue culture plates coated with 0.1% gelatin. The cells were cultured in knockout Dulbecco’s modified Eagle’s medium supplemented with 15% (v/v) knockout serum replacement, 0.1 mM escribed , 50. TraCHIR99021  was dissolved in dimethyl sulfoxide (DMSO) and added to cell medium at a final concentration of 3 μM for 24 h unless otherwise specified in J1 mESCs. While F9 ECs were treated with CHIR at a final concentration of 3 μM, 5 μM, 10 μM, 15 μM and 20 μM respectively. Cell medium with an equal volume of DMSO was used as a control.Klf4 forward primer, 5′-GGCGAGTCTGACATGGCTG-3′; Klf4 reverse primer, 5′-GCTGGACGCAGTGTCTTCTC-3′; Nanog forward primer, 5′-CACCCACCCATGCTAGTCTT-3′; Nanog reverse primer, 5′-ACCCTCAAACTCCTGGTCCT-3′; Tfcp2l1 forward primer, 5′-CAGCCCGAACACTACAACCAG-3′; Tfcp2l1 reverse primer, CAGCCGGATTTCATACGACTG-3′5′-; Axin2 forward primer, GGGGGAAAACACAGCTTACA-3′5′-; Axin2 reverse primer, 5′-TTGACTGGGTCGCTTCTCTT-3′; Gapdh forward primer, 5′-GTGTTCCTACCCCCAATGTGT-3′; Gapdh reverse primer, 5′-ATTGTCATACCAGGAAATGAGCTT-3′; For miRNA RT-qPCR analysis, the total RNA (2 μg) was reverse transcribed using a miScript II RT kit  with 5 × miScript HiSpec buffer to obtain mature miRNA. Real-time PCR was performed in triplicate for each sample using miScript SYBR Green PCR kit (Qiagen). We used the endogenous reference RNA RNU6B to normalize the amount of template added. The following primers were used: miR-7a primer 5′-GGGGGGGTGGAAGACTAGTGA-3′; U6 Reverse primer 5′-CTCGCTTCGGCAGCACA-3′; Universal miRNA Forward primer 5′-TGAATCGAGCACCAGTTACGCATGCCGAGGTCGACTTCCTAGA-3′; Relative expression of genes was evaluated and expressed as 2−ΔΔCT following previously described procedures.Total RNA was extracted from treated cells using Trizol Reagent (Life Technologies) according to the manufacturer’s instructions. Total RNA (1 μg) was reverse- transcribed using a PrimeScript RT reagents kit . Messenger RNA expression was determined by real-time PCR with an ABI StepOne Plus PCR system  using SYBR Premix Ex Taq II (TaKaRa). PCR amplification cycles were programmed for 30 s at 95°C, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s. After each cycle, SYBR green fluorescence was monitored, and the melting curve was analyzed to ensure that a single PCR product was obtained. Data were collected after each annealing step. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an endogenous control to normalize for the differences in the amount of total RNA in each sample. The following primers were used: J1 mESCs were treated with DMSO or 3 μM CHIR for 24 h on gelatin-coated 12-well plates. The medium was discarded, and the cells were washed twice with phosphate-buffered saline before being fixed and permeabilized with immunostaining fix solution  for 10 min. After blocking with immunostaining blocking buffer (Beyotime) for 1 h, the cells were incubated with primary antibody in dilution buffer (Beyotime) overnight at 4°C and then with Alexa Fluor 555-secondary antibody (Beyotime) for 2 h at room temperature in the dark. After each step, the cells were washed thrice with immunolstaining wash buffer for 5 min before the next step. DAPI  staining was performed after secondary antibody incubation for 10 min at room temperature. The primary antibodies and dilutions used were as follows: rabbit anti-Klf4 , rabbit anti-Klf4 , mouse anti-Oct4 , rabbit anti-Cdx2 , and rabbit anti-Nanog . All reagents not indicated were purchased from the Beyotime Institute of Biotechnology (Beyotime). Immunofluorescence staining was visualized and imaged by a confocal microscope .Gene classification and biological functional annotations were performed using the online DAVID database v6.7 , 52. GenKlf4 (−1124/+240) containing a Tcf/Lef binding site  was amplified from the genomic DNA of ES cells by PCR. The PCR primers were as follows: forward primer: GGTACCGGGAAAAGGAGTAATAAGACTAAAA-3′5′-GG (underlined letters indicate a KpnI restriction site) and reverse primer: AAGCTTCCGCCAGGTGAGAATGGCCG-3′5′-CCC (underlined letters indicate a Hind III restriction site). It was then cloned into the luciferase reporter vector pGL4.10 , yielding pGL-Klf4 (−1124/+240) WT. To disrupt the Tcf/Lef-binding motifs, forward primer GGTACCGGGAAAAGGAGTAATAAGACTACCAACACGACTGC-3′5′-GG  and reverse primer: AAGCTTCCGCCAGGTGAGAATGGCCG-3′5′-CCC (underlined letters indicate a Hind III restriction site) were used to generate pGL-Klf4 (−1124/+240) Mut. 5′-deletion construct was generated by PCR using the forward primer GGTACCCTCCCGGTAGGCAAGGGCCTGAGGT-3′5′-GG (underlined letters indicate a KpnI restriction site) and reverse primer AAGCTTCCGCCAGGTGAGAATGGCCG-3′5′-CCC (underlined letters indicate a Hind III restriction site), yielding pGL-Klf4 (−1081/+240). The full-length coding sequence of β-catenin was amplified from mESC cDNA by PCR. The PCR primers were as follows: forward primer: CTCGAGCGATGGCTACTCAAGCTGACCTGATG-3′5′-CCG (underlined letters indicate an XhoI restriction site and the bold fonts were inserted to make sure the correctness of open reading frame) and reverse primer: GCGGCCGCTTACAGGTCAGTATCAAACCAGGC-3′5′-ATTT (underlined letters indicate a NotI restriction site). It was then cloned into the pCMV-Myc vector, yielding pCMV-Myc-β-catenin. To obtain stabilization expression of the β-catenin protein, we constructed the mutation form of β-catenin (pCDNA3.1-β-catenin S37A). To generate a miR-7a expression vector, a fragment carrying pre-miR-7a was amplified from genomic DNA of J1 ESCs and cloned into the EcoRI and NotI sites of the pCDH-CMV-MCS-EF1-coGFP vector . Expression vectors of miR-128, miR-152, and miR-363 were constructed using the same method. The PCR primers were as follows: miR-7a forward primer: GAATTCGTCAGTTGACCCTTAGCTTTATGT-3′5′-CG (underlined letters indicate a EcoRI restriction site) and reverse primer GCGGCCGCACAGCACCATCATTATCTGTCCT-3′5′-ATAAGAAT (underlined letters indicate a NotI restriction site); miR-128 forward primer: GAATTCGCAGCTTCTCCTTATGTGCTTAT-3′5′-CG (underlined letters indicate a EcoRI restriction site) and reverse primer GCGGCCGCGATGTCTAGTAATGAGTTTGGCATG-3′5′-ATAAGAAT (underlined letters indicate a NotI restriction site); miR-152 forward primer: GAATTCGAGATTCTGGCGGGACGAGGGAGGA-3′5′-CG (underlined letters indicate a EcoRI restriction site) and reverse primer GCGGCCGCTCCAGCGGGGAGGGGTTACCTTGGC-3′5′-ATAAGAAT (underlined letters indicate a NotI restriction site); miR-363 forward primer: GAATTCTTCAGCATCCATGCCCATTCAT-3′5′-CG (underlined letters indicate a EcoRI restriction site) and reverse primer GCGGCCGCCACCCTGAGTGAGTTCCAGGACA-3′5′-ATAAGAAT (underlined letters indicate a NotI restriction site); The 3’-UTRs of Klf4 (NM_010637.3) that contain two putative miRNA binding sites [CTCGAGATCCCACGTAGTGGATGTGACCCAC -3′5′-CCG (underlined letters indicate a XhoI restriction site) and reverse primer: GCGGCCGCAACTTATTTCTCACCTTGAGTATGC-3′5′-ATAAGAAT (underlined letters indicate a NotI restriction site). All plasmids were confirmed by sequencing.Promoter sequence of ng sites , were amTotal protein of J1 mESCs or F9 EC cells was extracted, and its concentration was determined using the bicinchoninic acid protein assay (Beyotime). Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then the proteins were transferred to the polyvinylidene fluoride membranes (Millipore). After blocking for 3 h at room temperature in 10% nonfat dry milk in TBST containing 0.05% Tween-20, the membranes were incubated with primary antibodies overnight at 4°C and then after washes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Peroxidase activity was detected through autography using SuperSignal west pico substrate . Primary antibodies included rabbit anti-Klf4  and anti-β-catenin . Rabbit monoclonal anti-PCNA (Abcam. 1:1000). Rabbit monoclonal anti-GAPDH (Sigma. 1:5000).For reporter assays, transfection efficiencies were normalized to Renilla plasmid pRL-TK  values, which served as an internal control. DNA concentrations were kept constant with an empty expression vector. The cells were harvested after transfection, and luciferase activity was measured using the Dual-Luciferase Reporter (DLR) assay system (Promega) according to the supplier’s recommendations. Briefly, the cells were transfected with reporter constructs, and a Renilla luciferase plasmid was co-transfected as an internal control by Lipofectamine™2000 according to the manufacturer’s instructions. The cells were lysed with 100 μL of passive lysis buffer (Promega) after transfection. The cell lysate was vortexed and then briefly centrifuged. A 20 μL aliquot of the cell lysate was assayed for luciferase activity using VICTORX5 Multilabel Plate Reader . All transfections were repeated thrice.The pSuperTOPFlash luciferase reporter was constructed by inserting seven copies of the TCF/LEF binding site (AGATCAAAGG) into the pTA-luc vector, while pFOPFlash was previously constructed using second amplification from M51 Super 8x FOPFlash vector (Addgene plasmid # 12457) and cloned into the XhoI and BglII sites of the pTA-luc vector . These constructs contain a firefly luciferase reporter under the control of seven repeats the wild-type or mutant Tcf binding site upstream of minimal TA promoter. pFopflash containing mutated Tcf binding site was used as a negative control. The pRL-TK luciferase reporter construct was used as an internal standard of transfection. Transfection of plasmid DNA and assay for luciferase activity were performed as described previously.β-catenin knockdown in J1 ES cells, short interfering RNAs (siRNAs) that target mouse β-catenin and negative control siRNA-NC were purchased from Shanghai GenePharma . siRNA sequences were: si-β-catenin: 5′-GCCUUAGUAAACAUAAUGA-3′. For RNA interference experiments, J1 mESCs were transfected with the indicated siRNAs  using Lipofectamine 2000.For β-catenin  and rabbit anti-IgG (pierce) were used for immunoprecipitation. ChIP enrichment was performed by qPCR. Fold-enrichment was determined by normalizing threshold cycle values of β-catenin ChIP against IgG ChIP. The following target primers were used for ChIP-qPCR: Klf4 forward primer, 5′-ACTGAGGGTAGTGGGGAATGG-3′Klf4 reverse primer 5′-GTGATCCTGCGCTGGGAAGAG-3′.The protocol for chromatin immunoprecipitation assays (ChIP) has been described previously . BrieflyThe data in graphs are expressed as the mean ± SD. The difference between two groups was compared by a two-tailed paired Student’s t-test, and significance was set at P-values < 0.05. The difference between three or more groups was compared by an analysis of variance (ANOVA), and significance of differences was determined by post hoc testing.S1 Fig: J1 mESCs were treated with 3 μM CHIR or equal volume of DMSO in the presence or absence of LIF for 24 h, RT-qPCR was used to detect the expression of Klf4 and Map2. Data are presented as the mean ± SD of three independent experiments. .(TIF)Click here for additional data file.S2 FigThis construct contain a firefly luciferase reporter under the control of seven repeats the wild-type Tcf binding site upstream of minimal TA promoter (pTA). Abbreviation luc represent firefly luciferase gene.(TIF)Click here for additional data file.S3 Fig: CHIR treatment or β-catenin overexpression promotes cytosolic β-catenin expression. J1 mESCs were treated with 3 μM CHIR or equal volume of DMSO (A), or transfected with pCDNA3.1-β-catenin s37a / pCDNA3.1 control plasmid (B) for 48 h, Cell cytosolic lysates were extracted and the expression of β-catenin was analyzed by western blot. Relative expression levels were compared with Gapdh.(TIF)Click here for additional data file.S4 FigMiR-7a expression vector pCDH-mir-7a and their negative control pCDH-GFP were transfected into J1 mESCs, and miR-7a expression was detected by RT-qPCR. U6 was used to normalize template levels. Data are presented as the mean ± SD of three independent experiments (**p < 0.01).(TIF)Click here for additional data file.S1 TableFold change (FC) values are provided in comparison with the control J1 mESCs which were maintained in standard ESC medium without the addition of CHIR99021. .(DOC)Click here for additional data file.S2 TableTargetscan, Pic tar, Microsom Targets and DTANA-lab microT 3.0 were used to predict the potential miRNAs that may target Klf4 in mouse.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.