Interestingly, β-catenin is concurrently activated by loss of Nrf1α in a way similar to β-catenin-driven liver tumor. However, a presumable relationship between Nrf1 and β-catenin is not yet established. Here, we demonstrate that Nrf1 enhanced ubiquitination of β-catenin for targeting proteasomal degradation. Conversely, knockdown of Nrf1 by its short hairpin RNA (shNrf1) caused accumulation of β-catenin so as to translocate the nucleus, allowing activation of a subset of Wnt/β-catenin signaling responsive genes, which leads to the epithelial-mesenchymal transition (EMT) and related cellular processes. Such silencing of Nrf1 resulted in malgrowth of human hepatocellular carcinoma, along with malignant invasion and metastasis to the lung and liver in xenograft model mice. Further transcriptomic sequencing unraveled significant differences in the expression of both Wnt/β-catenin-dependent and Wnt/β-catenin-independent responsive genes implicated in the cell process, shape, and behavior of the shNrf1-expressing tumor. Notably, we identified that β-catenin is not a target gene of Nrf1, but this CNC-bZIP factor contributes to differential or opposing expression of other critical genes, such as CDH1, Wnt5A, Wnt11A, FZD10, LEF1, TCF4, SMAD4, MMP9, PTEN, PI3K, JUN, and p53, each of which depends on the positioning of distinct cis-regulatory sequences  in the gene promoter contexts. In addition, altered expression profiles of some Wnt/β-catenin signaling proteins were context dependent, as accompanied by decreased abundances of Nrf1 in the clinic human hepatomas with distinct differentiation. Together, these results corroborate the rationale that Nrf1 acts as a bona fide dominant tumor repressor, by its intrinsic inhibition of Wnt/β-catenin signaling and relevant independent networks in cancer development and malignant progression.Our previous work revealed that Nrf1 Drosophila Cnc protein, the Caenorhabditis elegans Skn-1, the vertebrate activator nuclear factor-erythroid 2 (NF-E2) p45, NF-E2-related factor 1 , Nrf2, and Nrf3, as well as the repressors Bach1 (BTB and CNC homology 1) and Bach2 [cis-regulatory homologues  within the gene promoter regions [On the oxygenated earth, all of the cellular life forms have evolutionarily established a series of integral cytoprotective systems against various stresses, such that these living organisms are allowed for ecological adaption to the changing environments during development, growth, and other life processes . Hence, nd Bach2 , 7, togend Bach2 –8. Such  regions , 7.retrotranslocation machinery into the cytoplasmic side. Therein, Nrf1 is deglycosylated to generate a transient deglycoprotein, which is further proteolytically processed by cytosolic proteasomes and/or DDI-1/2 proteases to give rise to a mature N-terminally-truncated CNC-bZIP factor, before being unleashed from ER membranes to translocate the nucleus and mediate transcriptional expression of ARE-driven genes  [In mammals, Nrf1 and Nrf2 are two principal CNC-bZIP proteins with similar, but different, structural domains , 9. By tubunits) –15. By cubunits) . Such di−/−Nrf2 knockout mice are viable and fertile, without any obvious defects and pathological phenotypes occurring during embryonic development and postnatal growth [−/−Nrf2 mice do not develop any spontaneous cancer, but they are more susceptible than wild-type mice to chemical carcinogens [bona fide tumor-promoting effects on carcinogenesis, cancer progression, metastasis, and resistance to therapy [Nrf1 are employed to create distinct animal models with significant pathological phenotypes [−/−Nrf1 knockout in mice leads to embryonic lethality at E6.5 to E14.5, resulting from severe oxidative stress damages [−/−Nrf1 : −/−Nrf2) [−/−Nrf1 mice are manifested with certain typical pathologies, each of which resembles human nonalcoholic steatohepatitis and hepatoma [However, Nrf2 has been generally accepted as a master regulator of ARE battery gene expression , 16, thol growth , 18. So cinogens . Subsequcinogens , 21. Con therapy , 23. Suc therapy . This isenotypes –30. Glob damages –27. ThisNrf2−/−) . Furtherhepatoma , 29, typhepatoma , and neuhepatoma , 34. Theα  by its Nfe2l1 gene editing from hepatoma cells leads to aberrant accumulation of Nrf2 [−/−Nrf1α-derived tumor in the invasion and metastasis [ΔN in the presence of Nrf1 [−/−Nrf1α cells, the hyperactive Nrf2 accumulation was determined to result from substantial decreases in protein and mRNA levels of Keap1, GSK-3β, and most of the 26S proteasomal subunits, so that this CNC-bZIP protein degradation is almost abolished. Further mechanistic insights into −/−Nrf1α-derived malignance discovered that significantly decreased expression of the tumor repressor PTEN leads to the reversed activation of its downstream AKT oncogenic signaling, as also accompanied by augmented expression of COX-2 and other inflammatory cytokines in −/−Nrf1α, but not −/−Nrf2, cells [α contribute to the −/−Nrf1α phenotype is unclear.Our recent work has unraveled that knockout of the human full-length Nrf1 of Nrf2 , 35. Destastasis , 36. Thi of Nrf1 . In Nrf1−, cells . Such beα- and β-catenins (encoded by CTNNA1 and CTNNB1). The latter β-catenin is a versatile player of the Wnt signaling involved in liver development, health, and disease [β-catenin signaling cascades is a genetic predisposition to hepatocellular carcinoma, in which the CTNNB1 mutation appeared to occur earlier during child liver carcinogenesis, whereas the NFE2L2 mutation was acquired later [β-catenin-driven liver tumors, activation of Nrf2-target antioxidant genes by a mutant β-catenin or another component (Axin1/GSK-3β) of Wnt signaling also appears to create a protumorigenic environment [β-catenin-target genes, along with dysregulation of other critical genes for EMT-related cell shape, cancer invasion, and metastasis behavior, also occurs in −/−Nrf1α-derived tumor [β-catenin is not yet established to date.It is of crucial significance to note the involvement of the epithelial-mesenchymal transition (EMT) in cancer invasion and metastasis, which is modulated by cadherins and  disease –39. Cleaed later –42. In βironment , 44. Simed tumor , 36. As β-catenin ubiquitination for targeting the proteasome-mediated degradation pathway. Conversely, silencing of Nrf1 by its short hairpin RNA (shNrf1) interference caused the accumulation of β-catenin and its translocation into the nucleus. Consequently, a subset of Wnt/β-catenin signaling responsive genes were activated, leading to the putative EMT and related changes in cell shape and behavior. Such silencing of Nrf1 further promoted the malgrowth of human hepatocellular carcinoma, along with malignant invasion and metastasis to the lung and liver in xenograft model mice. Further transcriptomic sequencing identified significant differences in the expression of Wnt/β-catenin-dependent and Wnt/β-catenin-independent responsive genes in shNrf1-expressing cells. Of note, it was identified that β-catenin is not a direct target gene of Nrf1, but the CNC-bZIP factor contributed to the differential or even opposing expression profiles of other critical genes, such as CDH1, Wnt5A, Wnt11A, FZD10, LEF, TCF4, SMAD4, MMP9, PTEN, PI3K, PDK1, JUN, ILK, and p53, each of which depends primarily on the positioning of distinct cis-regulatory ARE and/or AP1-binding sites within the gene promoter regions. In addition, altered expression profiles of some Wnt/β-catenin signaling proteins were context dependent, as accompanied by the decreased expression of Nrf1 in the clinic human hepatomas with distinct differentiation. Collectively, these corroborate the rationale that Nrf1 acts as a bona fide dominant tumor repressor, by intrinsic inhibition of the Wnt/β-catenin and their independent signaling networks involved in cancer development, progression, and malignancy.In this study, we demonstrate that overexpression of Nrf1 enhanced μg/mL of polybrene overnight, before they were allowed for a recovery in a fresh media and continued to incubate for 48-72 h. The positive clones of stably expressing cell lines were selected by 2 μg/mL puromycin (Invitrogen) for being used in other experiments.Four human liver cancer cell lines HepG2, MHCC97H, MHCC97L, and HEK-293T cell lines were maintained in the State Key Laboratory of Cancer Biology, the Fourth Military Medical University. A human immortalized hepatocyte cell line HL7702 and another house liver cancer cell line Hepa1-6 were provided as gifts from the Chinese Academy of Sciences Shanghai Cell Bank . All shNC- and shNrf1-expressing cell lines were herein established by a lentivirus-transducing system, which were purified by microfiltration from 293T cells that had been cotransfected with a target vector for shNrf1 or a scrambled shRNA as a negative control  along with three packaging vectors, as instructed in a packing kit . Then, Hepa1-6, HepG2, MHCC97H, and MHCC97L cells were plated in 6-vial plates and transduced with the packaged lentivirus in 8 v/v) fetal bovine serum , supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin  in a 37°C incubator with 5% CO2 humidified air. If required, transient transfection of the cells with some indicated plasmids alone or in combination was also performed in the TurboFect Transfection Reagent (Thermo Scientific) or another Lipofectamine®3000 Transfection Kit , according to the manufacturer's instructions. The cells were then allowed a 24 h recovery from transfection in the fresh medium before being experimented elsewhere. Notably, most of the key reagents and resources used for the following “wet” experiments were listed in All experimental cell lines were grown in Dulbecco's Modified Eagle Medium  containing 10% (n = 3 × 3), after being normalized by the mRNA level of β-actin, as an internal standard control.Experimental cells were subjected to the extraction of total RNAs by using an RNAsimple kit . Then, 500 ng of total RNA served as a template for the cDNA synthesis by using a RevertAid RT Reverse Transcription Kit . The new synthesized cDNA products were further used as the templates of real-time quantitative PCR (qPCR) in the GoTaq®qPCR Master Mix  containing each pair of the indicated primers . The supernatants of cell lysates were collected before their protein concentrations were determined using a BCA protein assay kit . Equal amounts (20 μg) of protein extracts were then subjected to separation by SDS-PAGE gels containing 8% to 10% polyacrylamide. The resolved proteins were transferred onto PVDF membranes (Millipore). The transferred membranes were blocked by incubation in 5% bovine serum albumin at room temperature for 1 h and then incubated with primary antibody overnight at 4°C. After washing three times, the blots were recognized by the corresponding secondary antibodies for 1 h and also detected by enhanced chemiluminescence with the Odyssey Imaging System (LI-COR Biosciences). The intensity of blots was quantified by the ImageJ software or the Quantity One 4.5.2 software.Experimental cells were treated with CHX and/or MG132 for distinct lengths of time (as described in details for the relevant figures) before being harvested. The cells were homogenized in RIPA lysis buffer  containing 2 Subcellular fractionation of shNrf1- and shNC-expressing cells was conducted according to the previous procedure as described by our group , 45. The4) were seeded in each well of a 48-vial plate and allowed for growth to ~70% confluence. The cells were cotransfected with 100 ng of the firefly luciferase reporter called TOPflash (driven by the consensus β-catenin/Tcf4-binding site) or its mutant control plasmid called FOPflash, together with 5 ng of Renilla luciferase reporter (pRL-CMV), plus 10 pmol of indicated small interference RNA targeting for Nrf1  or a scrambled negative control RNA . At 48 hours after transfection, the cells were subjected to the dual luciferase reporter assay (E1910 from Promega) on a TD-20/20 Luminometer , according to the manufacturer's instruction. After the luciferase activity was normalized to the Renilla luciferase values, fold activation of the putative Wnt/β-catenin signaling was quantified by the luminescence ratio of TOPflash to FOPflash reporters. Subsequently, significant differences in the reporter activity between siNrf1 and siNC were statistically analyzed.Human 293T cells (2 × 105) grown in 60 mm dishes were cotransfected for 48 h with pcDNA-3×HA-Ub (5 μg) and pcDNA3.1-V5His-Nrf1 (5 μg), along with additional 5 μg of either pcDNA-Flag-β-catenin or pcDNA-EGFP. The cells were treated with 10 μmol/L of MG132 for 4 h before being lysed on ice in the RIPA buffer supplemented with a proteinase inhibitor cocktail (Roche). The cell lysates were precleared with 20 μL of protein A/G Agarose (Santa Cruz), before being subjected to immunoprecipitation with 2 μg of anti-V5 antibody or mouse IgG (as a blank control), that were incubated overnight at 4°C. The immunoprecipitated beads were further incubated with 30 μL of protein A/G Agarose at 4°C for 6 h, before being washed for 5 times in buffer . Lastly, 30 μL of the loading buffer was added in the washed immunoprecipitates, followed by Western blotting with distinct antibodies against Ub, Flag, and V5 epitopes.Human 293T cells (5 × 105 cells in 200 μL of serum-free DMEM) were injected through the tail veins of nude mice  that had been divided into two groups. At 6 weeks after cell inoculation, these animals were sacrificed and then subjected to the pathological and histochemical examinations. The volume of metastatic tumors in the murine lung and liver were calculated by the formula π/6 × length × width2.Either shNrf1- or shNC-expressing HepG2 cells  were sufficiently suspended in 200 μL of serum-free DMEM and then inoculated subcutaneously into the right upper back region of male nude mice  at a single site. The procedure of injection into all the experimental mice was completed within 30 min, and the subsequent formation of the subcutaneous tumor xenografts was observed. Once the tumor xenografts emerged, the sizes of these ongoing tumors were successively measured once every two days, until the 42nd day when these mice were sacrificed and their transplanted tumors were excised. Thereafter, distinct sizes of those growing tumors were also calculated by a standard formula  and are shown graphically (n = 7 per group). The tumor tissues were also subjected to the pathohistological examination and Western blotting.Mouse xenograft models were also made by subcutaneous heterotransplantation of the human hepatoma HepG2 cell lines expressing shNrf1 or shNC into nude mice (as described above). Equal amounts of the indicated cells  in accordance with the United Kingdom Animal (Scientific Procedures) Act (1986) and the guidelines of the Animal Care and Use Committees of Chongqing University and the Third Military Medical University, both of which had been subjected to the local ethical review (in China). All the related experimental protocols had been approved by the University Laboratory Animal Welfare and Ethics Committee .μm slices were prepared. Firstly, the sections were dewaxed in the pure xylene twice (each for 5 min) and then washed in 100% ethanol twice (each for 5 min) to eliminate xylene, followed by rehydration in a series of gradient concentrations of ethanol with distilled water. Subsequently, they were stained with the routine hematoxylin and eosin (H&E) and visualized by microscopy. As for immunohistochemical staining, after the indicated tissue samples were dewaxed and rehydrated, they were treated with 3% hydrogen peroxide before being boiled in the microwave for 15 min in a citrate buffer (pH 6.0) to retrieve the putative antigen. The slides were blocked with 1% bovine serum albumin for 60 min and then incubated at 4°C overnight with the primary antibodies against Nrf1 (saved in our group) and TCF4 (both at a dilution of 1 : 50). Thereafter, the primary antibody-stained slides were reincubated with a biotin-conjugated secondary antibody for 60 min at room temperature, before being visualized by the peroxidase-conjugated biotin-streptavidin complex . In similar experimental settings, the negative staining controls were also set up by replacing the primary antibody with the normal nonimmune serum diluted in PBS. The resultant images were acquired under a light microscope  equipped with a DC350F digital camera.Murine subcutaneous xenograft tumors derived from shNrf1- or shNC-expressing human hepatoma cells, along with several human liver cancer and adjacent tissues , were fixed with paraformaldehyde (4%) and embedded in paraffin before the sections of 5 β-catenin, and Cyclin D1, each of which was diluted at 1 : 100, and with the secondary antibody against rabbit IgG, which had been conjugated with horseradish peroxidase. The stained sections were developed with a 3,3′-diaminobenzidine kit , according to the manufacturer's instruction.Further examination of the tumor metastatic liver and lung tissues was performed as described above. In brief, the tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. The sections were subjected to routine pathohistological examination by H&E staining. The immunohistochemical staining was also conducted by incubation of indicated sections with distinct primary antibodies against human Nrf1 , 4) of experimental cells were grown in 24-well plates. After reaching 80% confluence, the cells were cotransfected with each of those indicated firefly luciferase plasmids  or an empty plasmid with no enhancer to be encompassed, along with a Renilla luciferase plasmid and an expression construct for Nrf1, β-catenin, or Ub alone or in combination, in the Lipofectamine-3000 mixture. After transfection for 24 h, the cells were harvested by adding 200 μL of lysis buffer in each well. The cell lysates were subjected to the reporter assay by the dual-luciferase reporter system. Of note, the Renilla luciferase expressed by the pRL-TK plasmid served as an internal control for transfection efficiency. The luciferase activity was measured by the dual-luciferase reporter assay system . The resulting data were normalized and calculated as a fold change (mean ± SEM) relative to the activity of the control group . All the data presented in this study represent at least three independent experiments undertaken on separate occasions which were each performed in triplicate. Significant differences in the transcriptional activity were determined by statistical analysis. For a detailed description of the β-catenin/Tcf-driven TOPflash reporter and its mutant FOPflash in the luciferase assays, please see the relevant supplementary information. In addition, all the core sequences of ARE and AP1-binding sites were shown in Equal numbers (5 × 102 (fold change) ≥ 1 were herein taken as the threshold, in order to be identified as each of DEGs. The pathway enrichment analysis was also performed by using the online KEGG database (https://www.kegg.jp/). In addition, the putative interaction networks of Nrf1-related genes involved in carcinogenesis, migration, metastasis, and metabolism were annotated with the sequencing results by the Cytoscape software.After total RNAs were extracted by using an RNAsimple kit , the integrity of purified RNAs was also validated by an Agilent Bioanalyzer 2100 system . For each sample, equal amounts of total RNAs were collected from three independent experiments and pooled together for RNA-sequencing (RNA-Seq). Subsequently, RNA-Seq was carried out by the Beijing Genomics Institute  on an Illumina HiSeq 2000 sequencing system  after the sample library products are ready for sequencing. After the RNA-Seq quality was examined by removing the “dirty” raw reads, which contain low-quality reads and/or adaptor sequences, the clean reads were generated and stored as the FASTQ format. Thereafter, the clean reads were mapped to the reference of the human genome (GRCh37/hg19 from UCSC database) by using SOAP2, before distinct gene expression levels were calculated by using the RPKM (reads per kilobase of feature per million mapped reads) method. Notably, those differential expressed genes (DEGs) were further identified by the Poisson distribution model method (PossionDis), which was developed referring to “the significance of digital gene expression profiles” by BGI. Both FDR ≤ 0.001 and the absolute value of logt-test, Fisher exact test, and multiple analysis of variations (MANOVA), as appropriate. The resulting value of p < 0.05 was considered a significant difference. Furthermore, another statistical determination of the “dry” sequencing analysis was also carried out as described by Wang et al. [The “wet” experimental data provided in this study were represented as a fold change (mean ± SD), each of which represents at least 3 independent experiments that were each performed in triplicate. Significant differences were statistically determined using the Student g et al. .α/TCF11 and derivative isoforms between 140 kDa and 100 kDa in a noncancerous human liver HL7702 and other four human hepatoma-derived cell lines  were mostly silenced by shNrf1  in the Nrf1-silencing HepG2 cells  [β-catenin is allowed for release from physical interaction with E-cadherin to translocate the nucleus and acts as a coactivator to bind one of its partner transcription factors LEF or TCF . The resulting β-catenin/TCF complex can enable target genes to be transcriptionally activated upon induction of Wnt signaling. The TCF-binding motif (5′-AGATCAAAGG-3′) is widely used for the Wnt/TCF reporter, such as pTOPflash [β-catenin/TCF transactivity was significantly augmented by knockdown of Nrf1  , 51, 52.TOPflash . Herein, of Nrf1 . The proll lines . This wain vivo, here, we injected the human shNrf1- or shNC-expressing HepG2 cells (2 × 106 in a solution of 200 μL) into nude mice through their tail veins. Then, six weeks later, these mice were sacrificed and dissected. The anatomical observations showed that all the mice became the human hepatoma xenograft bearers in their lungs and livers  were inoculated subcutaneously into nude mice. The incubation period of tumorigenesis before the injected in situ emergences of visible tumor xenografts derived from shNrf1-silenced cells were strikingly shortened by 40% of the control values obtained from shNC cells  chase analysis of both protein degradation, particularly in the presence of MG132. Therefore, shNrf1- or shNC-expressing HepG2 cells had been cotreated with CHX (100 μg/mL) and MG132 (5 μmol/L) for the indicated lengths of time before Western blotting was conducted to determine whether β-catenin stability was influenced in Nrf1-silenced cells. As anticipated, the results revealed that silencing of Nrf1 caused a highly increased expression level of β-catenin  were observed in the cytoplasm of Nrf1-silenced cells -3×HA-Ub and then treated with MG132. The in vivo ubiquitination of β-catenin immunoprecipitates (IP) with anti-Flag antibody was visualized by Western blotting with HA (UB) antibody  of the 20S core proteasomal particle were downregulated in the shNrf1-expressing cells, which was roughly similar to those obtained from −/−Nrf1α (HEA157) cells , GSK3B, FBXW11, RBX1, SENP2, NLK, PPP2R1B, PRKACA, PRKX, LRRF1P1, CTBP1, SMAD3, SMAD4, MYC, and TP53. These collective data revealed that both Wnt/β-catenin-dependent and Wnt/β-catenin-independent signaling responsive genes were dysregulated in Nrf1-silenced cells  and also renders all the Wnt proteins hydrophobically tethering to the cell membranes, thus, determining Wnt production, secretion, and range of action [Transcriptomic analysis demonstrated that 34 of differential expression genes (DEGs) were obviously upregulated, while other 31 DEGs were downregulated or silenced by shNrf1 (ed cells  and . Off action .CTNNB1 (encoding β-catenin) was almost unaffected by shNrf1  was observed  , and further decrease was caused by its mutant , C10orf106 , PTPRD, APOC1, M1AP, INA, CTSS, FBLN2, BARX1, ZNF525, CPPED1, RYR1, SPINK5, MAP1LC3A, CT45A4, CT45A5, HBE1, HBG2, OR51B5, ICAM5, RNF5P1, and KCNQ2  were downregulated or completely blocked by −/−Nrf1α  were almost unaffected by −/−Nrf1α, when compared with wild-type controls. Further comparative analysis unraveled that silencing of Nrf1 led to obvious down-expression of 27 genes critical for cancer cell adhesion and ECM remodeling, of which 8 genes  were almost unaltered by −/−Nrf1α, but with an exception of 3 genes  that were strikingly upregulated by this knockout of −/−Nrf1α  . Clo−/−Nrf1α-derived HEA157 cells  were downregulated by −/−Nrf1α  were upregulated by −/−Nrf1α to varying extents. Rather, expression of additional 15 genes  was unaffected by −/−Nrf1α in HEA157 cells, albeit they were significantly altered by shNrf1, when compared to those control values obtained from wild-type +/+Nrf1 cells   were augmented by shNrf1 or −/−Nrf1α  was elevated by shNrf1 or pe level , E5. By /− cells , E6. AccNrf1α−/− , D6. How Figures , D5. Con Figures  D7 and .p53 was substantially blocked or abolished by shNrf1 or −/−Nrf1α  was significantly augmented by shNrf1, but not by −/−Nrf1α, albeit most of its basal and increased abundances were markedly repressed by Nrf1α or TCF11 , whilst concurrent activation of other tumor repressors, such as PTEN and p53, is also triggered by this CNC-bZIP factor.In the present study, we have corroborated the axiomatic rationale that Nrf1 is endowed with a dominant tumor-preventing function against human liver cancer development and malignant progression. Such tumor-repressing effect of Nrf1 is aroused by its intrinsic inhibition of Wnt/tm1uabLcrf1) in the mouse leads to a failure to form the primitive streak and mesoderm, dying at E6.5–E7.5 [−/−Nrf1 was also construed in a non-cell-autonomous way because the deficient embryonic stem cells (ESCs) were rescued in the chimeric mice during in vitro differentiation. Another non-cell-autonomous defect in global −/−Nrf1 embryos (created by knocking-in to yield rPGK-neoNrf1 such that its DNA- binding domain-encoding sequence was disrupted in most of the distinct Nrf1 isoforms) leads to the lethality at mid-late gestation  from severe anemia, which results from abnormal maturation of precursor cells in fetal liver microenvironment [−/−Nrf1 ESCs to adult hepatocytes was traced during organ development of chimeric mice [−/−Nrf1:−/−Nrf2 [−/−Nrf1, but not −/−Nrf2 [Nrf1α [de novo synthesis of compensatory proteasomes is determined by Nrf1, but conversely, its deficiency leads to an impaired expression profile of proteasomal subunits and thereby caused an aberrant accumulation of Nrf2 [β-catenin , another subset of ARE-driven genes were transactivated predominantly by Nrf2 in adaptive response to endogenous oxidative stress arising from knockout of −/−Nrf1, because their transactivation was terminated by double knockout of −/−Nrf1 : −/−Nrf2 [Nrf1α [As a matter of fact, Nrf1 is essential for the mature of fetal hepatocytes contributing to the adult liver because of widespread apoptosis of  embryos . Furtherhepatoma . Signifi Nrf1−/− . Besidesteatosis . Further Nrf2−/− . Overall Nrf2−/− . Togethe− [Nrf1α , 36.Nrf1α [shNrf1-driven tumor appears to be a little more severe than the case of −/−Nrf1α (with aberrant accumulation of Nrf2), by comparison of subcutaneous tumorigenesis in speeds and sizes, as accompanied by cancer metastasis and cachexia syndromes  [+/+Nrf1/2-bearing tumors are also strikingly ameliorated by Nrf2−/−ΔTA (with genomic deletion of transactivation domains of Nrf2), but roughly unaffected by caNrf2−/−ΔN  [shNrf1-derivated cells  . By contof Nrf2) . Collectof Nrf2) ; the latβ-catenin is seriously impaired by silencing of Nrf1 so that it is accumulated and translocated into the nucleus, leading to Wnt/β-catenin-mediated transcriptional activation of target genes. Notably, β-catenin is a key core component of the Wnt signaling cascades; this canonical pathway has been also accepted as a highly conserved and tightly controlled molecular mechanism that regulates important physiological and pathological processes in development, health, and disease  of multicellular organisms from early metazoan to human [−/−β-cat in mice leads to embryonic death at ~E7.0 from no formation of a primitive streak for mesoderm [tm1uabLcrf1 [−/−Wnt3, −/−Lrp5, −/−Lrp6, or −/−β-cat mice were obtained [β-catenin signaling pathway, along with Nrf1, are essential for gastrula development. Furtherly, hepatocyte-specific conditional knockout of −/−β-cat or other genes indicates that the Wnt/β-catenin pathway is critical for the formation of adult liver from the early embryonic stages and its homeostatic maintenance by dictating relevant cell fates and polarity during development and growth [β-catenin because it is subjected to proteasomal degradation , along with ILK, by knockdown of Nrf1 also causes β-catenin to be rapidly released from the plasma membrane-tethered adhesion complex and translocated into the nucleus. Thereby, the total nonphosphorylated protein of β-catenin is accumulated in the nucleus, resulting in aberrant expression profiles of β-catenin-associated transcription factor  target genes. Accordingly, an increase in the β-catenin/TCF-mediated TOP/FOP reporter gene activity is caused by silencing of Nrf1 .Insights into the pathobiological mechanisms of Nrf1-deficient hepatoma have discovered that ubiquitin-mediated proteasomal degradation of to human , 52. Glomesoderm ; this isf1tm1uab . Similarobtained –64. Thesd growth , 39. Ratβ-catenin signaling is constitutively activated by knockdown of Nrf1. However, such deficiency of Nrf1 led to downregulation of TCF4  in human Nrf1-silenced hepatoma xenografts and metastatic tumor tissues. Such seemingly confusing result appears to be coincident with the previously “surprising” finding by Shulewitz et al. [bona fide down-expressed in Nrf1-silenced cells, the LEF/TCF family factors are also functionally redundant in the human Wnt/β-catenin signaling activation stimulated by deficiency of Nrf1, as identified by Hrckulak et al. [β-catenin signaling and relevant responsive effects on target gene transcription, cell division cycle, cell proliferation, and differential fates, as well as cell polarity, cell adhesion, and cytoskeleton  within distinct gene promoters, enabling recruitment of distinct transcription factor complexes with cognate partners existing in different differentiated cancer cells. In addition, it cannot be ruled out that potential positive and negative feedback loops are encompassed within this pivotal signaling-to-gene regulatory network.As discovered by our data, the Wnt/z et al. . Therebyk et al.  that TCFβ-catenin signaling can polarize cells at their contact sites, orienting the axis of cell division while simultaneously programming daughter cells to adopt diverging differential fates in a tempo-spatially stereotyped way [β-catenin signaling network  is implicated in Nrf1-deficient hepatoma development and malignant progression.Significantly, the Wnt/yped way . The couyped way ). Therebβ-catenin stability and its cooperative transcription factors to control downstream gene expression. Thus, Wnt/β-catenin signaling could represent a critical target for cancer, particularly while certain mutagenesis had been acquired in a host of cancer development. Of note, the solid evidence that has been provided in the present study and our previous work [β-catenin signaling, as accompanied by transcriptional induction of EMT, and relevant morphological changes in cell shape. Such EMT also promotes cancer cell migration, invasion, and distant metastasis, e.g., to the liver and lung, particularly when cell adhesion junction with the ECM-receptor interaction networks had been remodeled by activated MMP and inactivated cadherins . Our evβ-catenin pathways and other signaling networks involved in human hepatoma development and progression. Herein, aberrant activation of this Wnt/β-catenin signaling by impairment of the core proteasomal subunits is much likely to play a pivotal role in orchestrating Nrf1-deficient liver carcinogenesis, progression and malignancy. As such, the pathological event occurs in particular dependence on severe endogenous oxidative stress and potential damages resulting from Nrf1-deficient cells, albeit hyperactivation of antioxidant factor Nrf2  is implicated in Nrf1-deficient liver cancer development and malignant behavior, albeit the detailed mechanisms are required to be further determined in the future. Overall, unraveling the unique function of Nrf1, which is distinctive from Nrf2, in liver cancer malignancies is likely to lead to novel preventive and therapeutic strategies to be paved against human cancer.In summary, our evidence corroborates that Nrf1 functions as a dominant tumor repressor by intrinsic inhibition of the Wnt/


The canonical Wnt pathway transcriptional co-activator β-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated β-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of β-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/β-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/β-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter–enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, β-catenin’s direct transcriptional role is restricted to the induction of NPCs, where rising β-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program. The now classical model of canonical Wnt signaling invokes two transcriptional states . In the Wnt9b from the ureteric epithelium and NPC-specific production of β-catenin also results in the failure of NPC differentiation (The adult (metanephric) mammalian kidney arises from distinct cell populations within the intermediate mesoderm . All nepntiation , whereasntiation , or genentiation . Geneticntiation . Genomicntiation , and subntiation . Thus, cIn this study, we employed an in vitro model to investigate the genomic regulatory mechanisms underlying the diverse action of canonical Wnt signaling in NPC programs. In this system, maintenance and expansion of NPCs, or their commitment to a nephrogenic program, are controlled by varying levels of CHIR99021 (CHIR)  supplemeA low level of CHIR (1.25 µM) is an essential component in NPEM medium supporting the expansion of NPCs while maintaining the nephron-forming competence . Within As expected, low CHIR conditions maintained Six2, a key determinant of the NPC state, but did not induce expression of Jag1 , a NotchPax2, Wt1, Hoxa/d11, and Sall1  . We alsond Sall1 . In contl1  enrichment analysis of differentially expressed genes  with DAVID . CompariSix2 expression in uncommitted NPCs  and Wnt4 activation on NPC differentiation . In low CHIR conditions, the Six2DE shows an open and active configuration: an ATAC-seq peak, flanked by H3K27ac peaks with Pol II engagement at the enhancer and within the gene body. In high CHIR, ATAC-seq, H3K27ac, and Pol II ChIP-Seq signals were reduced, correlating with the downregulation of gene expression  enriched in uncultured NPCs relative to NFC as they reflect a general NPC-specific signature. NPC-specific DARs were significantly enriched in transcription factor binding sites for Six, Pax, and Hox factors consistent with the critical roles of Six1/2, Pax2, and Hox11 paralogues in NPC programs . FunctioJun, Junb, and Jund in high CHIR . Of the four genes, Tcf7l1, Tcf7l2, and Tcf7 transcripts were expressed at low  or moderate  levels in low CHIR NPC maintenance conditions. CHIR-mediated induction of NPCs resulted in a significant downregulation of Tcf7l1 expression, while expression of both Tcf7 and Lef1 was markedly upregulated , though over half were unique to a data set . We assiExamining chromatin features and RNA Pol II engagement, we observed stronger enrichment of active enhancer markers (H3K27ac and RNA Pol II) in low CHIR on set 1 sites predicting indirect Tcf7l1 engagement than set 2 sites, where Tcf7l1 was predicted to bind DNA directly through TCF/LEF motifs . Thus, dTo examine genome organization and chromatin interactions at a higher level in self-renewing and induced NPC, Hi-C analysis  was perfAnalysis of two Hi-C data sets, using HiCCUPS within Juicer Tools , replicaWnt4, Lhx1, Emx2, Bmp7, and Cxcr4, which associate with NPC differentiation in vivo. Of these, only Wnt4 enhancer elements have been rigorously defined through transgenic studies  while in high CHIR, Tcf7 and Lef1 replace Tcf7 and Tcf7l2, with a concomitant decrease in Six2 and increase in β-catenin association  and dramatic increase in expression of mRNAs for activating forms (Tcf7 and Lef1), promoting an inductive program. (3) Direct binding of inhibitory Tcf7l1 and Tcf7l2 engagement at target motifs within putative cis-regulatory elements prefigures engagement of Tcf7 and Lef1 in a chromatin landscape primed for the transcriptional activation of the nephron-forming program. (4) High CHIR invokes a switch from inhibitory to activating TCF/LEF engagement at enhancers promoting nephron differentiation consistent with β-catenin controlling TCF/LEF target engagement. (5) In addition to enhancer–promoter loops pre-established in uncommitted NPCs associated with switching of inhibitory to activating TCF/LEF binding signatures on high CHIR induction, TCF/LEF/ β-catenin interactions at de novo sites may play additional roles in the inductive process, including positive feedback in the Lef1 program promoting nephrogenesis.(1) In low CHIR, NPC maintenance and expansion conditions, the mitogenic activity mediated through CHIR stimulation of Wnt signaling appears to be independent of TCF/LEF binding to TCF/LEF motifs in target genes. (2) CHIR-mediated elevation of β-catenin is accompanied by reduced expression of mRNAs for transcriptional inhibitory TCF factors  culture, canonical Wnt signaling, induced by CHIR, supports long-term self-renewal of ESCs . Tcf7l1 tor Tcf7 .In hair follicle stem cells (HFSC), Wnt induces a transition of the stem cells from the quiescent to the proliferative state. Tcf7l1 and Tcf7l2 are preferentially expressed in HFSC, while Lef1 and Tcf7 are preferentially expressed in the differentiated HFSC, that is, hair germ cells . TCF7L1/Wnt4 and Lef1 expression , 9 μLAnti-CD140-PE , 9 μLAnti-Ter119-PE , 8 μLAnti-CD326-PE , 8 μLThe cells and antibodies were incubated at 4℃ for at least 30 min without agitation, then washed three times with 1 mL AutoMACS running buffer resuspending cells at each step in 80 μL the same buffer. To remove unwanted cells, 20 μL of anti-PE beads were added to the cell suspension for 30 min at 4℃, cells were washed three times in 1 mL of running buffer, and finally cells resuspended in 0.5 mL of AutoMACS running buffer and sent through the AutoMACS program as described in the published protocol to remove non-NPC cell types enriching for NPCs  1:25 in APEL medium and incubated at room temperature in a laminar flow cabinet for cell culture for at least 1 hr. NPC Around 50,000 cells were added to a well for each imaging, RNA-seq or ATAC-seq study. For ChIP-Seq and Hi-C experiments, we cultured 1,500,000 cells in 6-well plates. For all culture experiments data was collected 24 hr after cell seeding.Total RNA was reverse-transcribed with SuperScript IV VILO Master Mix with ezDNase Enzyme (cat #: 11766050). qPCR was performed with Luna Universal qPCR Master Mix Protocol (New England Biolab #M3003) on a Roche LightCycler 96 System. p-Values were obtained by performing t-test between replicates of samples indicated. Primers used in RT-qPCR are listed as follows:Six2:CACCTCCACAAGAATGAAAGCGF: CTCCGCCTCGATGTAGTGCR: Cited1:AACCTTGGAGTGAAGGATCGCF: GTAGGAGAGCCTATTGGAGATGTR: Wnt4:AGACGTGCGAGAAACTCAAAGF: GGAACTGGTATTGGCACTCCTR: Jag1:CCTCGGGTCAGTTTGAGCTGF: CCTTGAGGCACACTTTGAAGTAR: Fgf8:CCGAGGAGGGATCTAAGGAACF: CTTCCAAAAGTATCGGTCTCCACR: Lhx1:CCCATCCTGGACCGTTTCCF: CGCTTGGAGAGATGCCCTGR: Pax8:ATGCCTCACAACTCGATCAGAF: ATGCGTTGACGTACAACTTCTR: Tcf7l1:CCCGCTGACACCTCTCATCF: ACAGTGGGTAATACGGTGACAGR: Tcf7l2:AACGAACACAGCGAATGTTTCCF: CACCTTGTATGTAGCGAACGCR: Tcf7:AACTGGCCCGCAAGGAAAGF: CTCCGGGTAAGTACCGAATGCR: Lef1:TGTTTATCCCATCACGGGTGGF: CATGGAAGTGTCGCCTGACAGR: To perform immunofluorescence staining, cell cultures were fixed with 4% PFA in PBS for 10 min, then washed with PBS twice before blocking in 1.5% SEA block  in TBST (0.1% Tween-20 in TBS). After minimally 30 min at room temperature, switched to primary antibody (diluted in blocking reagent) incubation in 4° overnight. After washing three times with TBST, switched to secondary antibody (diluted in blocking reagent) incubation for minimally 45 min in room temperature, blocking light. This was followed by three washes with TBST, then the cells were kept in PBS for confocal imaging. For freshly isolated cells , cells wTo separate, protein samples containing at least 1 million cells were boiled with β-mercaptol and ran in SDS-PAGE gels casted from 30% acrylamide/Bis solution 29:1  using the Mini-PROTEAN system (Bio-rad). Afterwards, the gel was transferred in Mini Trans-Blot Cell (Bio-rad) system to PVDF membranes . The membrane with protein was blocked with I-block  in TBST (0.1% TritonX-100 in TBS) at room temperature for 45 min before switching to primary antibody (diluted in blocking reagent) incubation in 4° overnight. Subsequently, the membrane was washed three times and switched to secondary antibody incubation (diluted in blocking reagent) for 45 min at room temperature. This was followed by three washes with TBST before drying the membrane and adding HRP substrate . Finally, the membrane was used on Autoradiography Film  to visualize location of protein.In total, 50,000–100,000 cells were collected for each RNA experiment. RNA was isolated with RNeasy micro kit . mRNA-Seq libraries were prepared with KAPA Stranded mRNA-Seq Kit . The libraries were subsequently sequenced with Illumina NextSeq500 model with pair-end 75 bp setting.mRNA-Seq reads were aligned with STAR  to mm10 To identify differentially expressed genes, count tables of the two groups of data being compared were processed through DESeq2  to obtaiGO enrichment analysis was performed with DAVID .Fixation. Freshly isolated NPCs were fixed in 1 mL AutoMACS running buffers (for each 3–5 million cells). Cultured NPCs were fixed in NPEM medium before scraping. In both cases, cells were fixed with final 1% formaldehyde  for 20 min at room temperature.Chromatin preparation. 3–5 million cells were processed for each chromatin preparation. Chromatin preparation includes cell lysis and nuclei lysis, which were done with SimpleChIP Sonication Cell and Nuclear Lysis Buffers  following manufacturer’s instruction.Chromatin fragmentation. For chromatin fragmentation, lysed nuclei were sonicated with Branson Ultrasonics Sonifier S-450 using a double-step microtip. Each sample was resuspended in 1 mL nuclear lysis buffer in a 15 mL conical tube, embedded in water-filled ice. Sonication was performed at 20% amplitude for 4 min, with 3 s of interval after each 1 s of duty time.Immunoprecipitation. 1 million-equivalent fragmented chromatin was used for each immunoprecipitation experiment. Immunoprecipitation was done with SimpleChIP Chromatin IP Buffers  following the manufacturer’s instruction with the following details. The amounts of antibody used were case-dependent. In general, 2 μg or 1:50 to 1:100 antibody was used for each precipitation. Chromatin with antibody were rotated overnight at 4°C before 1:40 protein A/G agarose beads  were added and incubated for another 6 hr to overnight. After washing and elution, antibody-precipitated input DNA were purified with minElute reaction cleanup kit , reconstituting to 35 µL EB buffer.ChIP-qPCR. qPCR was performed with Luna Universal qPCR Master Mix Protocol (New England Biolab #M3003) on a Roche LightCycler 96 System. For each reaction, 0.5 out of 35 µL ChIP or input DNA was used. The qPCR primers used are listed below:Six2-DE:ggcccgggatgatacattaF: cgggtttccaatcaccatagR: Wnt4-DE:GACCCATAAGGCAGCATCCAF: CTTGCTGGGCAGAGATGAAR: Non-ChIP:tctgtgtcccatgacgaaaaF: ggaagtcatgtttggctggtR: Sequencing. ChIP-Seq libraries were prepared with Thruplex DNA library prep kit . The libraries were sequenced with Illumina NextSeq500 model using single-end 75 bp setting.http://broadinstitute.github.io/picard/index.html). Peak calling was performed with MACS2 . Subsequently, ATAC-Seq reads from all samples concerned were counted in ±250 bp bins centering on the merged peaks, generating a count table. Hierarchical clustering was generated based on fold enrichment calculated from the count table.De novo Motif discovery was performed with Homer . GO analWe generated about 700 million raw reads for each sample. The reads were aligned by bwa , then duTo identify loops that are consistently present in both replicates, we extracted loops whose coordinates of anchors are within 10 kb between replicates.To find TSS of genes and peaks connected by loops, we look for peaks that are within 5 kb from center of one of the loop anchors and TSS that are within 15 kb from center of the other loop anchor.Cortical cells were dissociated from E16.5 kidneys as described in To enable calculation of standard error and finding of statistically differentially expressed genes and DARs, we generated three samples per condition for RNA-Seq and ATAC-Seq; due to restriction on resource, we generated two samples per condition for ChIP-Seq and Hi-C experiments.Each replicate experiment was performed with a 1–2-month interval. To us, biological replicates are the same assays performed over different litters of mice, and technical replicates are the same biological material  that were assayed multiple times. For sequencing data (stated above), we generated biological replicates only and no technical replicates. For qPCR, we did generate three technical replicates per biological replicate, with three biological replicates per condition in total. We did not encounter any outliers and exclude any data.All of our sequencing data, except for the scRNA-Seq data, has been submitted to GEO (GSE131119). In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:We are excited to publish your findings and believe your study will significantly enhance our understanding of the dosage dependent role for Wnt signaling in stem cell biology. Moreover, we believe that your study will also reinforce and expand our understanding of Wnt signaling in kidney development.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Edward Morrisey as the Senior and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Melissa H Little (Reviewer #1).Thank you for submitting your article "A β-catenin-driven switch in TCF/LEF transcription factors promotes commitment of mammalian nephron progenitor cells" for consideration by The reviewers have discussed the reviews with one another and the Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments may be required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Most of the requested revisions can be addressed without the need for additional experiments. Of note, the editors do not feel that the request for additional Western blotting noted by reviewer 2 is required for the revision.Reviewer #1:This is a detailed and careful analysis of the mechanism by which cultured mouse nephron progenitors respond to low versus high levels of CHIR, a GSK3β inhibitor used to mimic canonical Wnt activation. It includes transcriptional profiling, ATAC-Sed ChIP seq and Hi-C analyses. The work is a collaboration between two groups who have established the duality of the action of Wnt signalling on nephron progenitors at the cellular and molecular level and developed protocols for their isolation and maintenance in vitro using a support medium called NPEM. Having titrated two concentrations of CHIR as representing the low (NPC maintenance ) and high (commitment) inducing states, they investigate how this translates into transcriptional output in vitro, revealing a shift in the interactions between b-catenin and specific TF complexes at a range of target genes  and novel genes. They relate this back in some instances to previously proposed targets. The work is logical, careful, comprehensive and extensive. The claims made are well supported by the data. In essence, this study both supports the authors prior findings but clarifies the interaction of b-catenin with specific chromatin complexes and target genes. There is no particular need to ask for any further studies.Reviewer #2:I am not a bioinformation and I will therefore restrict my comments to the biology behind the data. In general the manuscript is very well written and the majority of conclusions drawn supported by the data. However, there are a number of points that need to be clarified.1) The study is based on MACS isolated cells that are treated with varying concentrations of CHIR, but I was unable to find a detailed description of the experimental set up. How long were the cells cultured before they were harvested for the analysis? What was the plating density? These are important parameters that can dramatically influence the cellular response. Please provide a dedicated paragraph in the Materials and methods section.2) Figure 1A evaluates expression of different proteins by inferring levels from immunofluorescence intensity. Western blots are much more accurate for a quantitative analysis and should be shown instead. .3) Axin2 is considered a direct target of β-catenin and is often used as a read out for canonical signaling. Axin2 appears to be expressed in renal progenitors, albeit at relatively low levels . How does expression, occupation of enhancer sites and looping at this gene change under the different conditions?4) The Hi-C studies allows drawing interesting conclusions about chromatin looping under two different CHIR conditions and clearly shows that certain genes are "primed" on the chromatin level to engage in transcription, whereas others only make contact with their enhancers upon High CHIR treatment. In this context, Figure 5C examines the distal enhancer of Wnt4 and the authors demonstrate a clear switch of enhancer occupation from TCF7l1/2 and SIX2 towards TCF7 and LEF1. β-catenin seems already bound to this enhancer under low CHIR conditions, but increases under High CHIR . Figure 5D, however, suggests that under low CHIR conditions b-catenin does not bind to the WNT-DE. This is confusing and needs to be clarified5) Schuijers et al., 2014, identified two classes of β-catenin binding sites in murine intestine and two different cell lines and concluded that the vast majority of β-catenin transcriptional activity is mediated through TCF/LEF transcription factors. This paper needs to be cited and discussed.6) The study uses CHIR to mimic different levels of β-catenin activity. CHIR is an inhibitor of GSK3β, a central component of a protein destruction complex that not only influences β-catenin stability, but also other signaling and cellular pathways. The authors acknowledge this shortcoming in their Discussion and suggest themselves that "Wnt-ligand mimetics offer a promising future approach to confirm CHIR-β-catenin-centered findings". The paper would gain impact, if some of the most important findings could be verified by testing target site occupation on some key sites using WNT surrogates (ChIP-qPCR experiments).7) Under low CHIR conditions only 234 β-catenin ChIP-seq peaks were detected and the authors hypothesize that WNT signaling may regulate proliferation (at least partly) independently of DNA binding. In this context the Wnt/STOP model should be discussed (DOI: 10.1016/j.molcel.2014.04.014), a mechanism that protects proteins from GSK3-dependent degradation, but does not seem to involve β-catenin activity.Reviewer #3:Aspects of the manuscript that require further clarification/revision:1) Details on in vitro NPC culture conditions and incubation hours with low or high CHIR are not provided and could be included in the Materials and methods. This information is important when different studies are compared.2) Data presented in supplementary filesSupplementary files should contain more details, including titles with a description of the data shown, abbreviations, as the authors have shown under file "Notes" in Supplementary file 3 or 6– Supplementary file 2 is not cited in the text (likely in the first Chapter of Results?).– Concerning Supplementary file 3, cited several times in the manuscript, it is hard to find the results indicated.To simplify inspection of the lists of DE genes shown, it would be helpful to add an additional file or supplementary file with selected differentially expressed genes for each condition indicating the fold-change cut off value applied ( >1 or other) and the adjusted p-value (FDR).3) Related to the previous comment, Figure 1D and Figure 1—figure supplement 1 show the Top 5 enriched GO terms on the basis of lists of selected transcripts that are not shown .Similarly Figure 1—figure supplement 1 shows GO terms of Low CHIR>NPC and NPC >Low CHIR of additional lists of transcripts. In addition is indicated "* genes were selected with a different DE threshold" but it is not shown which is such threshold (Panel E).4) It is unclear how the selection of the Top5 GO-terms was performed because in many cases they are redundant Figure 1—figure supplement 1D Low CHIR > NPC (494 transcripts): top 5 GO terms are redundant as they are in Figure 4—figure supplement 2E.5) The authors indicate that "high CHIR led to a down regulation of regulators and markers of self-renewing NPCs, including Six2 , Cited1 , Osr1  and Eya1 , and a concomitant increase in expression of genes associated with induction of nephrogenesis, such as Wnt4, Jag1, Lhx1, Pax8 and Fgf8 . Trends in gene expression from mRNA-seq were confirmed by RT-qPCR analysis ". However this is not fully supported by the data presented in Figure 1C, Supplementary file 3 and Figure 1—figure supplement 1B, which do not show a significant increase in Pax8 and Ffg8 transcript levels.Please comment whether these differences in the expression of these differentiated genes in High CHIR are due either to the different ex vivo inductive culture conditions or another alternative explanation.6) “Figure 2—figure supplement 1. ATAC-Seq data analysis –(A) Hierarchical cluster of R-square values between normalized ATAC-Seq reads within merged peaks from all samples.”To obtain a more global picture of the process, it would further informative if the genome-wide ATAC-sequencing heatmaps are associated to the corresponding GO analysis on an additional column highlighting the signal pathways of each condition.Indeed, NPC self-renewing is known to depend on additional growth factor signaling pathways, such as FGF and BMP. The interactions of the Wnt-pathway with other spatially and temporally restricted signaling pathways may in addition affect the effects of Wnt-β-catenin on the expansion of nephron progenitors, which the authors found here to be independent of direct β-catenin/chromatin engagement and probably involving other mechanisms.7) Supplementary Figure 3C. Immunoblots of TCF/LEF family factors in NPC cultures should be improved , and the MW size markers indicated8) A recent publication of Hilliard et al., 2019 Development reported the differential chromatin accessibility in mouse NPCs at embryonic day E13/ E16 and postnatal P2 and as well as the chromatin landscape in young and old NPCs. This study is not commented. The NPC culture conditions used by Hilliard et al. for ChIP-seq analyses were different than those used here (and in addition not well detailed), limiting the comparisons. However, ATAC-seq was performed on freshly isolated E16 NPCs. It would be important to discuss and compare these previous results of Hilliard et al. with those obtained here. Reviewer #2:I am not a bioinformation and I will therefore restrict my comments to the biology behind the data. In general the manuscript is very well written and the majority of conclusions drawn supported by the data. However, there are a number of points that need to be clarified.1) The study is based on MACS isolated cells that are treated with varying concentrations of CHIR, but I was unable to find a detailed description of the experimental set up. How long were the cells cultured before they were harvested for the analysis? What was the plating density? These are important parameters that can dramatically influence the cellular response. Please provide a dedicated paragraph in the Materials and methods section.Indeed, these are critical points, we apologize for not having this information in the original manuscript and thank the reviewer for spotting this omission. The relevant information is now included in the Materials and methods section of the revised manuscript.2) Figure 1A evaluates expression of different proteins by inferring levels from immunofluorescence intensity. Western blots are much more accurate for a quantitative analysis and should be shown instead. .While Western blots give a population-based view, each cell is its own data point, and quantifying single is a reasonable approach as evidenced by the wealth of quantitative approaches at the single cell level. The Western blot in Supplementary Figure 3C has now been quantified and placed in the new Figure 3—figure supplement 2. The result is consistent with the original conclusion from immunostaining data in the original manuscript .3) Axin2 is considered a direct target of β-catenin and is often used as a read out for canonical signaling. Axin2 appears to be expressed in renal progenitors, albeit at relatively low levels . How does expression, occupation of enhancer sites and looping at this gene change under the different conditions?Referring to new Supplementary file 2 (replacement of old Supplementary file 3), the mean TPM for Axin2 transcript is 4.6 in low CHIR and 41.7 in high CHIR: an approximately 10-fold change with an adjusted p-value of 1e-17. In line with this trend, TPM of Axin2 is 1.0 in no CHIR condition, which is significantly lower than that in the low CHIR condition (p = 1e-24). The data is consistent with an activation of canonical Wnt signaling activation. We did observe strong binding of β-catenin, Tcf7 and Lef1 near Axin2 promoter and a distal site 30 kb upstream in high CHIR condition only. This agrees with RNA PolII loading and H3K27 acetylation of the promoter region in high but not low CHIR. Tcf7l2 binding is present in both conditions but weaker in the high CHIR condition. However, we did not capture a loop to the Axin2 promoter in either condition in the Hi-C date. A caveat of Hi-C studies is an under-representation of promoter-proximal interactions which are difficult to distinguish from the high expectation of interactions for near-adjacent genomic sequences. For readers’ convenience, we provided a table of all loops connecting to at least one gene’s promoter (new Supplementary file 5).4) The Hi-C studies allows drawing interesting conclusions about chromatin looping under two different CHIR conditions and clearly shows that certain genes are "primed" on the chromatin level to engage in transcription, whereas others only make contact with their enhancers upon High CHIR treatment. In this context, Figure 5C examines the distal enhancer of Wnt4 and the authors demonstrate a clear switch of enhancer occupation from TCF7l1/2 and SIX2 towards TCF7 and LEF1. β-catenin seems already bound to this enhancer under low CHIR conditions, but increases under High CHIR . Figure 5D, however, suggests that under low CHIR conditions b-catenin does not bind to the WNT-DE. This is confusing and needs to be clarifiedWe thank the reviewer for pointing out this discrepancy. We have adjusted Figure 5D to make it reflect the data where weak β-catenin binding is present in some sites in low CHIR condition.5) Schuijers et al., 2014, identified two classes of β-catenin binding sites in murine intestine and two different cell lines and concluded that the vast majority of β-catenin transcriptional activity is mediated through TCF/LEF transcription factors. This paper needs to be cited and discussed.We thank the reviewer for bringing this point to our attention. We have modified the text in describing the NPC ChIP-seq data in the Results as follows: “In both low and high CHIR conditions, β-catenin association overlapped extensively with the binding of cognate TCF/LEF factors specifically enriched in each condition . […] This is consistent with previous findings in murine intestinal studies that localization of β-catenin is primarily dependent on TCF/LEF factors ”.6) The study uses CHIR to mimic different levels of β-catenin activity. CHIR is an inhibitor of GSK3β, a central component of a protein destruction complex that not only influences β-catenin stability, but also other signaling and cellular pathways. The authors acknowledge this shortcoming in their Discussion and suggest themselves that "Wnt-ligand mimetics offer a promising future approach to confirm CHIR-β-catenin-centered findings". The paper would gain impact, if some of the most important findings could be verified by testing target site occupation on some key sites using WNT surrogates (ChIP-qPCR experiments).A major strength of the current work is an extensive analysis of large, unbiased datasets. It is not clear what value is really added by a limited and selected analysis of Wnt-ligand mimetics to the current work? A thorough evaluation of small-molecule versus protein mimetic is better suited to an independent future study.7) Under low CHIR conditions only 234 β-catenin ChIP-seq peaks were detected and the authors hypothesize that WNT signaling may regulate proliferation (at least partly) independently of DNA binding. In this context the Wnt/STOP model should be discussed (DOI: 10.1016/j.molcel.2014.04.014), a mechanism that protects proteins from GSK3-dependent degradation, but does not seem to involve β-catenin activity.We appreciate the reviewer bringing this interesting reference to our attention. Indeed, the work provides evidence of CHIR action that is independent of β-catenin and might contribute to the pro-proliferation function of Wnt signaling. We have referenced this paper in the Discussion: “Additionally, CHIR-mediated inhibition of GSK3 outside of β-catenin regulation might play a role …”.Reviewer #3:Aspects of the manuscript that require further clarification/revision:1) Details on in vitro NPC culture conditions and incubation hours with low or high CHIR are not provided and could be included in Materials and methods. This information is important when different studies are compared.See response to reviewer 2 – we added this information to the Materials and methods and thank the reviewer for pointing out the omission.2) Data presented in supplementary filesSupplementary files should contain more details, including titles with a description of the data shown, abbreviations, as the authors have shown under file "Notes" in Supplementary file 3 or 6– Supplementary file 2 is not cited in the text (likely in the first Chapter of Results?).– Concerning Supplementary file 3, cited several times in the manuscript, it is hard to find the results indicated.To simplify inspection of the lists of DE genes shown, it would be helpful to add an additional file or supplementary file with selected differentially expressed genes for each condition indicating the fold-change cut off value applied ( >1 or other) and the adjusted p-value (FDR).Supplementary file 2 is a TPM table that lists TPM values of all genes observed in each sample. The data from this table was used in 1C, 3A and S3B, and can be used independently of any specifically mentioned data in this paper for interested readers. To make it clearer and more reader friendly, in this revision we merged Supplementary file 2 and 3 to generate a new Supplementary file 2, and we have added more description of the table in the “Notes” sheet which are intended to make the data clearer to the reader. Any reader can search for name of genes they are interested in and see their expression levels, fold changes and p-values between two specific conditions, referring to the “Notes” sheet.3) Related to the previous comment, Figure 1D and Figure 1—figure supplement 1 show the Top 5 enriched GO terms on the basis of lists of selected transcripts that are not shown .Similarly Figure 1—figure supplement 1 shows GO terms of Low CHIR>NPC and NPC >Low CHIR of additional lists of transcripts. In addition is indicated "* genes were selected with a different DE threshold" but it is not shown which is such threshold (Panel E).We thank the reviewer for pointing out these omissions. The threshold used in Figure 1—figure supplement 1E has been added to the new Supplementary file 2 “Notes” sheet. We identified some discrepancies between in the GO term figures  and Supplementary file 2 and have now corrected these and added adjusted p-values instead of p-values in the figures mentioned. There are no changes in the conclusions from correcting these errors.4) It is unclear how the selection of the Top5 GO-terms was performed because in many cases they are redundant Figure 1—figure supplement 1D Low CHIR > NPC (494 transcripts): top 5 GO terms are redundant as they are in Figure 4—figure supplement 2E.We are not sure what the reviewer refers to as redundant, as we did not see any redundancy among the GO terms. Also, Figure 4—figure supplement 2E does not contain any GO terms; maybe there is a typo here. The selection to Top5 GO terms is simply by the rank of their p-value or adjusted p-values from low to high.5) The authors indicate that " high CHIR led to a down regulation of regulators and markers of self-renewing NPCs, including Six2 , Cited1 , Osr1  and Eya1 , and a concomitant increase in expression of genes associated with induction of nephrogenesis, such as Wnt4, Jag1, Lhx1, Pax8 and Fgf8 . Trends in gene expression from mRNA-seq were confirmed by RT-qPCR analysis ". However this is not fully supported by the data presented in Figure 1C, Supplementary file 3 and Figure 1—figure supplement 1B, which do not show a significant increase in Pax8 and Ffg8 transcript levels.Please comment whether these differences in the expression of these differentiated genes in High CHIR are due either to the different ex vivo inductive culture conditions or another alternative explanation.Fgf8 is significantly upregulated in the high CHIR condition as compared to in the low CHIR condition . However, it was not identified as a “differentially expressed gene” in this comparison because the TPM of the highest transcript did not pass 5 – our criteria for inclusion  though the change in expression is significant. For Pax8, we agree, this is an error, there is no significant change between low and high CHIR conditions and we have accordingly corrected the statement. In contrast, there is a significant change in Pax8 expression between the low CHIR and no CHIR conditions , which suggests Pax8 expression maybe hyper-sensitive to CHIR concentration. Interestingly, mouse genetic studies suggest up-regulation of Pax8 is the first response in NPC commitment.6) “Figure 2—figure supplement 1. ATAC-Seq data analysis – (A) Hierarchical cluster of R-square values between normalized ATAC-Seq reads within merged peaks from all samples.”To obtain a more global picture of the process, it would further informative if the genome-wide ATAC-sequencing heatmaps are associated to the corresponding GO analysis on an additional column highlighting the signal pathways of each condition.Indeed, NPC self-renewing is known to depend on additional growth factor signaling pathways, such as FGF and BMP. The interactions of the Wnt-pathway with other spatially and temporally restricted signaling pathways may in addition affect the effects of Wnt-β-catenin on the expansion of nephron progenitors, which the authors found here to be independent of direct β-catenin/chromatin engagement and probably involving other mechanisms.We understand the reviewer’s intention to bring together the GO terms and a genomic manifestation of the ATAC-Seq data, which might enhance the visual perception. However, the heatmap  shows the R-squared values between each sample and does not reflect information on particular regions. On the other hand, the GO term results shown in Figure 2C and Figure 2—figure supplement 1C were based on the differentially accessible regions between the specified conditions. Therefore, there is not a direct connection between the heatmap and the GO terms. Instead, we came up with heatmaps showing signal of the DA regions across sample and attached corresponding GO terms and motifs found and added this to a revised Figure 2C with the intention of generating a visual representation similar to the reviewer’s suggestion. We have also improved our workflow and Supplementary file 3 for better statistical rigor and making the data more accessible to the reader with accompanying minor changes in Figure 2 and figure 2—figure supplement 1.7) Supplementary Figure 3C. Immunoblots of TCF/LEF family factors in NPC cultures should be improved , and the MW size markers indicatedWe have included the MW size marker in the immunoblots . Although the Tcf7 result looks reasonable to us, we are aware there are background bands for Tcf7l1. We have tried several different antibodies but this one gives the best result. Unfortunately, there isn’t anything we can think of to improve the result.8) A recent publication of Hilliard et al., 2019 Development reported the differential chromatin accessibility in mouse NPCs at embryonic day E13/ E16 and postnatal P2 and as well as the chromatin landscape in young and old NPCs. This study is not commented. The NPC culture conditions used by Hilliard et al. for ChIP-seq analyses were different than those used here (and in addition not well detailed), limiting the comparisons. However, ATAC-seq was performed on freshly isolated E16 NPCs. It would be important to discuss and compare these previous results of Hilliard et al. with those obtained here.We now refer to the Hilliard et al., 2019 study in the revised manuscript in “CHIR-mediated induction modifies the epigenomic profile of NPCs” part of the Results section: “Statistical assessment (Supplementary file 2) determined…”. In the Hilliard et al., 2019 study, a major focus was the identification of epigenetic differences that may relate to maturation of the Six2+ NPC state between E16 and P2, shortly before the NPC population is lost. Consequently, ATAC-seq data in this work aimed to identify epigenetic differences reflecting elevated Wnt/ β-catenin signaling activity as NPCs underwent developmental aging. While there are some expected similarities, i.e., an enhanced differentiation signature in P2 vs. E16 also observed in high CHIR vs. low CHIR condition. Importantly, the in vivo data are interpreted as reflecting, at least in part, elevated Wnt-signaling. However, this is an interpretation in contrast to our study which directly modulates the canonical Wnt-signaling pathway to determine an effect on NPCs. Given these differences, we have not incorporated additional analyses of the Hilliard et al., 2019 data.


Cure Index revealed that incorporation of 0.1 wt.% of Co-PEG-MNPs into epoxy led to Excellent cure at all heating rates, which uncovered the assistance of bulk modification of nanoparticles to the crosslinking of model epoxy nanocomposites. Isoconversional methods revealed higher activation energy for the completely crosslinked epoxy/Co-PEG-MNPs nanocomposite compared to the neat epoxy. The kinetic model based on isoconversional methods was verified by the experimental rate of cure reaction.Surface modification of nanoparticles with functional molecules has become a routine method to compensate for diffusion-controlled crosslinking of thermoset polymer composites at late stages of crosslinking, while bulk modification has not carefully been discussed. In this work, a highly-crosslinked model polymer nanocomposite based on epoxy and surface-bulk functionalized magnetic nanoparticles (MNPs) was developed. MNPs were synthesized electrochemically, and then polyethylene glycol (PEG) surface-functionalized (PEG-MNPs) and PEG-functionalized cobalt-doped (Co-PEG-MNPs) particles were developed and used in nanocomposite preparation. Various analyses including field-emission scanning electron microscopy, Fourier-transform infrared spectrophotometry (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD) and vibrating sample magnetometry (VSM) were employed in characterization of surface and bulk of PEG-MNPs and Co-PEG-MNPs. Epoxy nanocomposites including the aforementioned MNPs were prepared and analyzed by nonisothermal differential scanning calorimetry (DSC) to study their curing potential in epoxy/amine system. Analyses based on  Enhancement of properties of thermoset resins via surface modification of nanoparticles has been practiced over the years ,2,3. Ove2+  vs. ln(1-α). Crosslinking reaction of the neat epoxy and two nanocomposites progressed autocatalytically, as the maximum points derived from the results in α < 0.4.In the next step, to determine the reaction model, we used the Malek method was also used for determination of kinetic model by considering the maximum points of Malek parameters of m), z(α) = (αp∞)y(α) =  and (10), respectively, and the conversion at the maximum point of DSC curves (pα).A more accurate y(α) and z(α) are normalized with respect to their maximum values to take values between 0 and 1  Malek master plots and according to the data in mα is lower than pα and p∞α < 0.632), it was concluded that a two-parameter autocatalytic kinetic model can be considered for EP, EP/PEG-MNPs and EP/Co-PEG-MNPs.By comparing n and m are non-catalytic and autocatalytic reaction orders, respectively. The values of the orders of reactions (n and m) as well as the pre-exponential factor (lnA) are determined from Equations (A2) and (A3) found in αE (αĒ) from Friedman and KAS methods is calculated, as given in Two-parameter autocatalytic kinetic model was defined by Sestak–Berggren as follows:m+n) took a value higher than one, demonstrating the complexity of the curing reaction in the studied systems [From the data in  systems .ln(A), and consequently higher αE values were obtained (Collison in Arrhenius principle is defined as the number of contacts between molecules per unit volume). Since viscosity effect is dominantly acting against curing in such systems, more collisions would be liable for an improved curing at later stages of cure reaction.The presence of both PEG-MNPs and Co-PEG-MNPs nanoparticles in the epoxy matrix increased the reaction rate compared to the neat epoxy. Moreover, once PEG-MNPs and Co-PEG-MNPs were incorporated into the epoxy, the interaction between reactive groups with the curing moieties in the epoxy system increased the possibility of collision in the system, as reflected in higher values of dα/dt), it is calculated as:To obtain the kinetics parameters for the studied rate of cure reaction (dα/dt obtained from Equation (12) with the experimental values. Fortunately, the predicted dα/dt appropriately fitted the experimental data.gT of the EP, EP/PEG-MNPs and EP/Co-PEG-MNPs were obtained in the reheating cycle from 20 to 250 °C at β of 10 °C·min−1 for the samples, and the results are reported in The values of gT of epoxy was slightly increased by addition of 0.1 wt.% of PEG-MNPs, suggesting enhanced interaction between the PEG functional groups on the surface of MNPs and the curing moieties. In comparison with neat epoxy, addition of 0.1 wt.% Co-PEG-MNPs resulted in increase of gT from 101 to 105 °C. This rise in gT of EP/Co-PEG-MNPs nanocomposite is due to the increase in the interfacial area between the Co-PEG-MNPs and epoxy leading to higher crosslinking density, as apparent from the higher ΔH value of this system. It can be realized that the presence of Co-PEG-MNPs with higher amount of PEG on its surface compared to that of MNPs caused hindrance of segmental mobility of epoxy chains due to the formation of denser epoxy crosslinked network, in which more active sites are present on the surface of Co-PEG-MNPs for reacting with epoxide group [The de group .ΔH* values. The nanocomposites cured at all heating rates took Excellent cure label according to the Cure Index. Both Friedman and KAS isoconversional methods proved higher αE for the epoxy system in the presence of Co-PEG-MNPs nanoparticles. Well-achieved confidence between the experimental rate of cure reaction and the predicted values from the Friedman and KAS methods denoted the appropriateness of these models. The gT data indicate that presence of Co-PEG-MNPs in epoxy matrix with higher amount of PEG on its surface compared to MNPs caused hindrance of segmental mobility of epoxy chains, which featured an increase in the gT value from 102 to 105 °C due to the formation of denser epoxy crosslinked network. This approach can be applied to develop multifunctional polymer composites in view of the fact that surface and bulk characteristics can affect network formation as well as the properties of polymer composites in different ways.Surface-bulk modification of nanoparticles was applied in making highly-crosslinked epoxy nanocomposites. The galvanostatic cathodic deposition method was applied in synthesis of the PEG capped-MNPs and Co-doped MNPs as model nanoparticles. FTIR, XRD, FESEM, VSM and TGA analyses were performed to confirm positioning of elements and molecules at the surface and in the bulk of particles. FESEM micrographs proved spherical particles formed having an average diameter of 15–20 nm. The FTIR bands proved the presence of PEG on the surface of both MNPs and Co-doped MNPs. Epoxy nanocomposites containing 0.1 wt.% PEG-coated MNPs and Co-doped MNPs were then prepared and studied by nonisothermal DSC for evaluating their curing potential. Co dopant mainly positioned in the bulk layers of MNPs, which led to change in the reactivity of the surface such that more PEG was anchored on the surface of Co-doped MNPs. It was proved by TGA data that Co-doped MNPs have 6.2% more PEG on their surface, which resulted in increased


Exosomes from human umbilical cord mesenchymal stem cells (hucMSC-Ex) have been suggested as novel nanomaterials for regenerative medicine. Here we explored the roles of hucMSC-Ex through regulating Yes-associated protein (YAP) in renal injury repair by using rat unilateral ureteral obstruction (UUO) models. Our study identified mechanical stress induced YAP nucleus expression and stimulated collagen deposition and interstitial fibrosis in the kidney. Then, infusion with hucMSC-Ex promoted YAP nuclear cytoplasmic shuttling and ameliorated renal fibrosis in UUO model. Interestingly, hucMSC-Ex delivered casein kinase 1δ (CK1δ) and E3 ubiquitin ligase β-TRCP to boost YAP ubiquitination and degradation. Knockdown of CK1δ and β-TRCP in hucMSC decreased the repairing effects of hucMSC-Ex on renal fibrosis. Our results suggest that hucMSC-Ex attenuates renal fibrosis through CK1δ/β-TRCP inhibited YAP activity, unveiling a new mechanism for the therapeutic effects of hucMSC-Ex on tissue injury and offering a potential approach for renal fibrosis treatment. And tubulointerstitial fibrosis is a very important and common pathological change in the progress of CKD, which seriously affects the prognosis of kidney disease2. Initiated by diabetes, obstruction, and hypertension, it is characterized by increased production of growth factors and inflammatory factors4. Its main pathological features are inflammatory cell infiltration, tubular atrophy, capillary loss and accelerated proliferation of myofibroblasts, and excessive deposition of extracellular matrix (ECM)6. In recent years, more and more evidences show that EMT changes of renal tubules are the main pathway of myofibroblasts production in renal diseases7. Long-term tubulointerstitial fibrosis reduced the regenerative potential of the kidneys and led to a substantial decline in renal function8. At present, there is lack of effective treatment that can prevent the renal fibrosis progression10. Thus, it is imperative to enhance the understanding towards the pathogenesis of tubulointerstitial fibrosis and consequently find new therapeutic approaches12.The development of renal interstitial fibrosis (RIF) is linked to progressive renal injury and chronic kidney disease (CKD)14. MSCs have been reported to improve renal function, reduce renal damage, and inhibit chronic renal fibrosis17, where tissue damage was mainly repaired through paracrine mechanism18. Exosomes are cell-secreted membranous nano vesicles that mediate cell communication by delivering a variety of molecules, including nucleic acids, proteins and lipids, from donor cells to target cells20. MSC-derived exosomes have been shown to exert therapeutic effects on spinal cord injury21, multiple sclerosis22, infarcted hearts23. Our research focused on human umbilical cord MSC-derived exosomes (hucMSC-Ex), it was beneficial to liver fibrosis24, diabetic melitus25, and skin burn26. HucMSC-Ex could repair cisplatin-induced AKI by ameliorating oxidative stress and cell apoptosis, promoting cell proliferation in vivo and in vitro. hucMSC-Ex pretreatment promoted autophagy of renal tubular epithelial cells and reduced cisplatin-induced renal toxicity by transporting 14-3-3ζ protein28. However, whether hucMSC-Ex could attenuate renal fibrosis remains to be studied.Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have been widely used in tissue regeneration30. Along the Hippo pathway yes-associated protein (YAP) is an important effector protein, playing an indispensable role in fibrosis31. As a co-factor YAP regulates TGF-β1 signaling by retaining activated Smad2/3 in the nucleus33. YAP expression could be upregulated by a variety of factors such as mechanical forces, leading to activation of myofibroblasts and excessive deposition of ECM34. YAP was a tissue mechanosensor that the interaction of YAP and ECM formed a feed-forward loop resulting in kidney fibrosis35, indicating that YAP may be a new target for anti-fibrosis therapy. CK1δ and β-TRCP were the kinase ubiquitin system of YAP protein degradation. Firstly, the protein substrate YAP was phosphorylated by CK1δ kinase at the serine site, and then recognized by E3 ubiquitin ligase transprted ubiquitin molecules which binded to the substrate, then hydrolyzed by proteasomes and promoted YAP degradation. These led us to research the intrinsic relationship between hucMSC-Ex, kinase ubiquitin system and YAP.Being a key pathway involved in CKD progression, Hippo signaling controls organ size and regulates tissue regenerationIn this study we explored whether hucMSC-Ex could alleviate renal fibrosis induced by mechanical stress  model). HucMSC-Ex infusion in rats showed that it could migrate to damaged kidney tissue and alleviated the damage of kidney tissue. Our results showed that hucMSC-Ex could transport CK1δ and β-TRCP system to promote YAP ubiquitination and degradation, therefore inhibiting YAP activation, lessening collagen deposition, and alleviating renal fibrosis. These findings provide a new strategy for therapeutic treatment of renal fibrosis.The SD rats  age were 8 weeks and the weight were 250 g. The temperature of the housing condition was kept at 25 °C, the relative humidity was 50%, which provided 12 h of light and 12 h of darkness. After 14 days, SD rats were anesthetized with 10% chloral hydrate. Briefly, under general anesthesia, the left ureter was ligated at the ureter-pelvic junction with 4-0 silk through a left flank incision. The right kidney was sham operated and the ureter was not ligated as a control. After intervention, all animals were euthanized and their kidneys were harvested on the 14th day after operation. Normal rats were fed with regular diets. Animal Ethics Committee of the University of Jiangsu approved all animal protocols (2014280).23. The protein concentration, as the quantification of exosomes, was determined by using a BCA protein assay kit . The final amount of exosomes used for in vitro cell study was 160 μg/ml. For in vivo animal study, 200 μg exosomes were used for each animal. The morphology of the extracted exosomes was observed by using transmission electron microscopy . The size of exosomes was analyzed by measuring the rate of Brownian motion using the NanoSight LM10 system .Exosomes were extracted and purified as previously describedA cryo-TEM observation of exosomes solutions was carried out in a controlled-environment vitrification system. The climate chamber temperature was 25–28 °C, and the relative humidity was kept close to saturation to prevent evaporation from the sample during preparation. Two milliliters exosomes sample solution at room temperature was placed on a carbon-coated holey film supported by a copper grid and gently blotted with filter paper about 3 s to obtain a thin liquid film (20–200 nm) on the grid. The grid was quenched rapidly in liquid ethane at −180 °C and then transferred to liquid nitrogen (−196 °C) for storage. The acceleration voltage was 200 kV, and the working temperature was kept below −170 °C. The images were recorded digitally with a charge-coupled device camera (Gatan) under low-dose conditions with an under focus of approximately 3 μm.24, and human lung fibroblasts Cells (HFL1) maintained in a low-glucose Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum . Rat renal proximal tubular (NRK-52E) cells were purchased from Stem Cell Bank of Chinese Academy of Sciences and maintained in H-DMEM  containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37 °C with 5% CO2. The cells at passage 3 were used for the following studies.Umbilical cords were obtained from the affiliated hospital of Jiangsu University with the permission of mothers and were freshly processed within 2 h. HucMSCs were isolated as previously described36. Briefly, firstly glass coverslips were etched using 0.1 NaOH, rinsed with ddH2O, incubated in 0.5% gluteraldehyde in phosphate-buffered saline (PBS), and then acrylamide/bis-acrylamide mixtures polymerized between the functionalized coverslip and a glass slide coated with dichlorodimethylsiloxane (Sigma-Aldrich).Hydrogels were prepared as previously described on 25 mm coverslips were utilized25.For histologic analysis, the kidneys were prepared by perfusion of the rat through the left ventricle and slides of the kidney were prepared, fixed in 4% paraformaldehyde, embedded in paraffin and then cut into sections. The sections were stained with hematoxylin and eosin (HE) and Sirius Red and Masson staining, and the histological changes of renal tissues were observed under a microscope . The expression and localization of YAP , α-SMA , CK1δ , and β-TRCP  in paraffin-embedded sections were measured as previously describedRenal tissue section placed in 4% paraformaldehyde at 4 °C for 12 h. Then permeabilized with PBS solution containing 0.15% Triton X-100 for 30 min and incubated with 5% bovine serum albumin (BSA) for 1 h to block non-specific antibody binding. Kidney slices were then incubated with the following primary antibodies: YAP , α-SMA , Collagen I  at 4 °C for 24 h. The slices were washed with PBS and then incubated with two secondary antibody, Alexa Fluor 555-conjugated donkey anti-mouse IgG and FITC-conjugated goat anti-Rabbit IgG  overnight at 4 °C. The nucleus was stained with Hoechst33342 . The slides were visualized with a confocal microscope .1, Collagen I, FAP , YAP, CK1δ , β-TRCP , β-actin . The secondary antibodies were HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies .Total protein was extracted from tissues and cells by using RIPA lysis buffer with proteinase inhibitors. The protein concentration was determined by using a BCA protein assay kit. Equal amounts of protein were separated on 12% SDS-PAGE gel and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blockade with 5% skim milk for 1 h, the membranes were incubated with primary and the HRP-conjugated secondary antibodies and detected by using ECL detection system . The primary antibodies were as follows: CD9, CD63, Alix , α-SMA , TGF-βCells were pretreatment 5 h with 20 µM MG132 before immunoprecipitation. Cells were washed using PBS and subsequently lysed in Western/IP lysis buffer , followed by addition of carrier beads. Immunocomplexes were washed five times with NETN buffer before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.The adenovira CK1δ shRNA and β-TRCP shRNA vector were generated by the vector ADV1(U6/CMV-GFP) with CK1δ and β-TRCP shRNA oligonucleotides.CK1δ shRNA oligonucleotide sequences are: 5′-GGGCAAGCTCTATAAGATTCTTCCTCGAGGAAGAATCTTATAGAGCTTGCTTTTT-3′. β-TRCP shRNA oligonucleotide sequences are: 5′-CCGGGCGTTGTATTCGATTTGATAACTCGAGTTATCAAATCGAATACAACGCTTT-3′. The sequences of control shRNA are: 5′-CCGGGCAAGCTGACCCTGAAGTTCATCTCGAGATGAACTTCAGGGTCACGTTGCTTTTTG-3′. Recombinant adenovirus was produced by co-transfecting HEK293T cells with ADV1-CK1δ-shRNA or ADV1-β-TRCP-shRNA and PU1563 plasmid using Lipofectamine2000 . The efficiency of CK1δ an-d β-TRCP knockdown was evaluated by using real-time quantitative RT-PCR and western blot. HucMSCs were transduced with the prepared adenovirus . The stable cell lines were cultured in serum-free medium for 48 h, the supernatants were collected and exosomes were isolated for further study.t-test for the comparison of two groups and one-way ANOVA Tukey test for multiple-group comparisons. *P < 0.05 was considered significant.The data were analyzed using GraphPad Prism 5 . All data are reported as the means ± standard deviation. Significant differences were evaluated using an unpaired Student’s 23. As nanoscale membrane vesicles, hucMSC-Ex size was determined to be 106.3 ± 37.3 nm (mean ± SD) by nanoparticle tracking analysis (NTA)  Fig. . ExosomeTA) Fig. , cryo-TETA) Fig. . Westernxin Fig. . To invexin Fig. . Interesxin Fig. . These i1) were significantly reduced after hucMSC-Ex intervention  after being stimulated with stiff gel (45 kPa) for 48 h , and then stimulated with stiff gel in the presence or absence of hucMSC-Ex, the down-regulation of YAP by hucMSC-Ex was restored by addition of MG132  mediated YAP degradation132 Fig. . This ex132 Fig. . TherefoTo evaluate the importance of CK1δ and β-TRCP in the repairing effect of hucMSC-Ex on renal fibrosis, we knocked down CK1δ and β-TRCP expression in hucMSC by using adenovirus-mediated shRNA transfection. Exosomes were isolated from control and specific shRNA-transfected hucMSC (shCK1δ-Ex and shβ-TRCP-Ex) Fig. , and the41. However, there are few effective treatments to prevent renal fibrosis. MSCs have been suggested as important seed cells for anti-fibrosis therapy in regenerative medicine43. Systemic injection of MSC has been shown to be effective in experimental tissue injury model studies44. MSCs transplantation have been used in clinical trials as a potential treatment for nephropathy16. The mechanisms for the therapeutic effect of MSCs are complicated and the secretion of active molecules has been suggested as a major reason18. Both MSC-derived microvesicles and exosomes have been suggested to protect against renal injury46. MSC-derived exosomes have been shown to repair tissue damage by transferring active molecules into target cells47, including mRNAs, microRNAs, and proteins. In this study, we demonstrated that hucMSC-Ex could increase CK1δ and β-TRCP expression and promote YAP ubiquitination and degradation in the kidneys of UUO rat models, which inhibited ECM deposition and alleviated renal fibrosis, indicating that hucMSC-Ex may represent a new strategy for the treatment of renal fibrosis.Renal fibrosis is caused by excessive accumulation of ECM, which is present in almost all types of CKD48. In this study we first constructed rodent disease models (UUO) to examine the alteration of YAP expression during renal fibrosis and consequently the therapeutic effect of hucMSC-Ex. In vivo and in vitro both studies showed that mechanical stress, high glucose, and TGF-β1 stimulation induced the expression of YAP in the kidneys. It is in agreement with previous research found that YAP expression and phosphorylation increased in proximal tubular epithelial cells in animal models of type 1 and type 2 diabetes49. As YAP activation increased the stiffness of ECM, which further stimulated the activation of YAP in turn, forming a feed-forward loop to promote scar tissue formation and renal fibrosis35. Here we showed that the expression of YAP increased with the aggravation of renal fibrosis in UUO models, indicating that YAP may be a potential target for anti-fibrosis therapy.Activation of YAP in the mouse can promote regeneration in poor capacity organs, remarkably, prolonged activation of YAP/TAZ triggers cancer development50. In mammalian cells, YAP phosphorylated by Lats1/2 kinase at Ser127 site could bind to 14-3-3 and anchor to the cytoplasm51. Our previously studies showed that 14-3-3ζ protein in hucMSC-Ex bound Ser127-YAP to retard cell proliferation and inhibit skin scar formation52. Ubiquitin proteasome was the most important protein degradation pathway. Ubiquitin ligase E3 which determined the specific recognition of the target protein, promoted the ubiquitin degradation of proteins, and played an important role in many physiological processes in cells. Once phosphorylated by CK1δ kinase at Ser381 site, YAP could interact with the E3 ubiquitin ligase β-TRCP, leading to its poly-ubiquitination and degradation39. In UUO rat models, hucMSC-Ex treatment reduced the expression of YAP, suggesting the regulation of YAP stability by hucMSC-Ex. Due to the fact that YAP protein stability is regulated by the ubiquitin–proteasome system, we analyzed the protein component of hucMSC-Ex by LC-MS/MS. It was found that CK1δ and β-TRCP, two major ubiquitination related enzyme for YAP degradation, were enriched in hucMSC-Ex. Systemically administrated hucMSC-Ex could target the damaged kidneys in UUO rats and increase the expression of CK1δ and β-TRCP. When CK1δ and β-TRCP were knocked down, the therapeutic effect of hucMSC-Ex on renal fibrosis was greatly reduced, which suggested that hucMSC-Ex mediated CK1δ and β-TRCP in the repair of renal injury was very important.YAP expression and activity are governed by two mechanisms: cytoplasmic retention, ubiquitination, and degradationIn summary we demonstrated that mechanical stress stimulated YAP expression and accelerated the progression of renal fibrosis. HucMSC-Ex could inhibit renal fibrosis by delivering CK1δ and β-TRCP to promote the ubiquitination and degradation of YAP. As a biogenic nanotherapeutics, hucMSC-Ex could be employed to the target tissues to inhibit YAP activity and ameliorate renal fibrosis, providing a novel and effective approach for anti-fibrosis therapy.Details of experimental methods and procedures are available in the Supporting Information.Supplementary Figure legendsFigure S1Figure S2Figure S3