Prime-boost vaccination employing heterologous viral vectors encoding an antigen is an effective strategy to maximize the antigen-specific immune response. Replication-deficient adenovirus serotype 5 (Ad5) is currently being evaluated clinically in North America as a prime in conjunction with oncolytic rhabdovirus Maraba virus (MG1) as a boost. The use of an oncolytic rhabdovirus encoding a tumor antigen elicits a robust anti-cancer immune response and extends survival in murine models of cancer. Given the prevalence of pre-existing immunity to Ad5 globally, we explored the potential use of DEC205-targeted antibodies as an alternative agent to prime antigen-specific responses ahead of boosting with an oncolytic rhabdovirus expressing the same antigen. We found that a prime-boost vaccination strategy, consisting of an anti-DEC205 antibody fused to the model antigen ovalbumin (OVA) as a prime and oncolytic rhabdovirus-OVA as a boost, led to the formation of a robust antigen-specific immune response and improved survival in a B16-OVA tumor model. Overall, our study shows that anti-DEC205 antibodies fused to cancer antigens are effective to prime oncolytic rhabdovirus-boosted cancer antigen responses and may provide an alternative for patients with pre-existing immunity to Ad5 in humans.  Replication-deficient Ad5 vectors are used for gene therapy and vaccination. Tzelepis and colleagues show that seropositivity to Ad5, highly prevalent in the human population, abrogates an Ad5 vaccination response. Alternatively, they show the efficacy of αDEC205 antibody-antigen fusion in the context of an oncolytic rhabdovirus cancer vaccine targeting mouse melanoma. As knowledge of the important role played by the immune system in preventing tumor growth in healthy individuals has expanded over the last decades, immunotherapy has emerged as a viable treatment option for cancer.in situ cancer vaccine effects and relieve local immunosuppression through the induction of immunostimulatory cytokines. In this environment, dendritic cells (DCs) can phagocytose dead/dying infected tumor cells and prime an anti-tumor as well as antiviral immune response in the draining lymph node.,It is known that OVs can elicit ,NCT02285816, NCT02879760, NCT03618953, and NCT03773744).Typical vaccination regimens are generally not limited to a single dose and can be made more effective by multiple immunizations. This can involve the administration of additional homologous (matched vaccine) or heterologous (unmatched vaccine) doses.As a boosting component, oncolytic rhabdoviruses are thought to be uniquely effective because in addition to infecting tumor and breaking local immunosuppression, they efficiently, but nonproductively, infect splenic B cells, which provides an additional source for antigen presentation to DCs, resulting in secondary expansion of T cells.NCT02285816). Questions regarding the importance of vector seropositivity were raised recently following Merck’s failed phase II clinical trial of a trivalent human immunodeficiency virus (HIV) vaccine delivered in an Ad5 vector.21To prime the oncolytic rhabdovirus boost, current clinical trials employ a nonreplicating adenovirus serotype 5 (Ad5) vector expressing a shared cancer antigen  of the WTAd5 virus. After 35 days, mice were administered 108 PFUs rAd5-SIINFEKL intramuscularly (i.m.)  mice  for interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Again, there was a reduction from an average of 6% to 2% of IFN-γ- and TNF-α-producing CD8+ T cells specific to SIINFEKL detected in the splenocytes from preimmunized mice compared to control PBS  A. Generay (i.m.) B. SIINFEBS) mice C and 1D.trol PBS E and 1F.,Impaired SIINFEKL-specific immune responses following rAd5-SIINFEKL immunization of C57BL/6 mice modeling pre-existing immunity led us to consider employing an alternative priming agent that would be better able to overcome pre-existing immunity to WTAd5 and other potential alternative viral vectors. Several studies have shown the ability of chimeric aDEC205 antibodies fused to antigens, such as OVA (aDEC205-OVA) or tumor antigens, to elicit strong antigen-specific immune responses in mice when administered with an adjuvant.To generate the aDEC205 antibodies used in this study, human embryonic kidney (HEK)293T cells were cotransfected with plasmids containing the mouse aDEC205-kappa light chain and the aDEC205 heavy chain fused to the full OVA protein sequence at the carboxyl terminus (or no antigen as a control [aDEC205-empty]). The recombinant antibodies produced following transient transfection in HEK293T cell were purified on protein G Sepharose columns.+CD8a+ was confirmed with a binding assay.+CD8a+ DCs (gating strategy shown in The resulting antibodies were characterized by western blot using anti-immunoglobulin G (IgG) antibodies on SDS-PAGE under reducing conditions A. Figurein vitro restimulation of lymphocytes with the SIINFEKL peptide, showed that i.v. and i.p. routes of administration elicited statistically similar percentages of IFN-γ- and TNF-α-producing CD8+ T cells in the lung and spleen of mice immunized with aDEC205-OVA at days 10 and 21 postimmunization  and 50 μg anti-CD40 or PBS. 7 and 14 days after boost, lymphocytes were harvested from the spleen and lung and then stained with the H2Kb-SIINFEKL pentamer. At days 7 and 14 postboost, the greatest expansion of SIINFEKL-specific T cells was observed in the spleen  did not significantly impact priming responses ; no mice previously cured by any prime-boost regimen developed tumors, thus confirming that anti-SIINFEKL responses were long lasting and conferred protection against recurrent tumors.We next evaluated the therapeutic efficacy of the aDEC205-OVA/MG1-OVA prime-boost vaccine in an experimental model of lung metastasis. Briefly, 3 × 10antation A. Generaesponses , there wred i.p. B and 6D.,Cancer immunotherapy has emerged as a promising alternative to conventional cancer treatments. Therapeutic strategies that actively stimulate the immune system to reject tumors have grown to include diverse platforms, including immune-modulating antibodies,,In this study, we have further explored one such combination strategy consisting of a heterologous prime-boost, where the priming and boosting vectors share a similar tumor antigen and where the boosting vector is an oncolytic rhabdovirus.,+ T cells , SIINFEKL-specific CD8+ T cells, even though the numbers of SIINFEKL-specific T cells were similar with both primes (NCT01834248 and NCT01127464).,+ T cells.As an alternative to Ad5, we demonstrate here, in line with other studies, that i.p. or i.v. administration of aDEC205-OVA generates antigen-specific and functionally robust anti-SIINFEKL T cells, as well as humoral immunity toward OVA.h primes B. This d,Consistent with other studies, we found that heterologous boosting with an oncolytic rhabdovirus, such as MG1-OVA, amplifies antigen-specific immunity in the spleen at days 7 and 14 to a higher extent than homologous boosting, for example, with aDEC205-OVA .15,36 Alex vivo with tumor lysate or major histocompatibility complex class I (MHC-I) peptides for re-administration to patients have been studied for decades and have been shown to generate robust memory CD8+ T cell responses.,Vaccines employing DCs loaded + T cells, as well as cross present antigen to CD8+ T cells. However, this approach is also not without limitations. For example, there can be antibody/protein engineering challenges restricting the choice of antigen and how many antigens can be fused to a given aDEC205 antibody. This can be somewhat addressed by using more restricted epitopes in tandem or using multiple different chimeric antibodies.One key feature of chimeric aDEC205 antibodies is that they deliver a specific antigen directly to DCs, which in turn, present antigen and activate CD4,,Another consideration for use of chimeric aDEC205 antibodies is the requirement for an adjuvant.Altogether, our study indicates that a vaccine consisting of an aDEC205-OVA prime, followed by a rhabdovirus boost, is a promising alternative to the current heterologous prime-boost that employs Ad5-OVA as a priming agent. To our knowledge, this study is the first of its kind to showcase a combination of the well-studied aDEC205 antibody in combination with an OV. Additional studies in other tumor models and antigenic targets will be necessary to assess the applicability of this novel approach to a broad range of disease models.2 humidified incubator. All cells were tested by PCR and Hoechst staining to ensure that they are free of mycoplasma contamination.HEK 293T cells, kindly donated by the Oncolytic Virus Manufacturing Facility  for antibody production and purification, were cultured in HyQ high-glucose Dulbecco’s modified Eagle’s medium (HyClone), supplemented with 10% ultra-low IgG fetal bovine serum , 5% penicillin/streptomycin , and 5% L-glutamine (Gibco). B16-F10-OVA cells, kindly gifted by Dr. Yonghong Wan (McMaster University), were cultured in Roswell Park Memorial Institute , supplemented with 10% FBS, pen-strep, 1 M HEPES buffer, and 50 μg/mL geneticin sulfate (G148 sulfate) (Gibco). All cell lines were incubated at 37°C in a 5% CO6- to 8-week-old female C57BL/6J mice were obtained from Charles River Laboratories. All animals were handled in strict accordance with good animal practice and approved by the appropriate committee in collaboration with the Office of Animal Ethics and Compliance.The pcDNA plasmids expressing the heavy-chain aDEC205, aDEC205-OVA, and aDEC205-empty and the light-chain DEC205-kappa sequences were generated by Dr. Silvia Boscardin (University of São Paulo). The plasmid DNA was individually transformed in competent DH5-α, and DNA was purified using the QIAGEN Plasmid Maxi Kit . Transfection of 90% confluent HEK293T cells in 150 mm tissue-culture dishes, collection of antibody from culture supernatant, and antibody purification were performed as previously described.b were synthesized by New England Peptide (lot number 3001-1/48-21) and have >95% purity.Peptides corresponding to the immunodominant epitope of OVA (SIINFEKL) that binds to H-2K6 cells per condition were stained for flow cytometry.SIINFEKL-specific T cell responses were measured in blood, spleen, and lung. Briefly, saphenous bleeds of mice from hindlimb were performed, and blood (70–100 μL) was collected in sterile heparin tubes. Red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer. Spleens were excised from sacrificed mice and filtered through a 100-μm plastic cell strainer  for cell collection. The cell viability of the resulting white blood cells was determined using Trypan blue staining. Lungs were also excised from sacrificed mice after lung perfusion and dissociated using the Lung Dissociation Kit-Mouse , according to the manufacturer’s instructions. Upon resuspension in R10 buffer , the cells from blood, spleen, and lung were counted, and 1 × 10After aDEC205-OVA antibody quantification by the NanoDrop ND-1000 spectrometer, 1 μg of antibody was run on NuPAGE Novex 4%–12% Bis-Tris precast gels (Thermo Fisher Scientific) under reducing conditions using the XCell SureLock Mini-Cell System (Thermo Fisher Scientific) and transferred to nitrocellulose membranes . Blots were blocked with 2% milk and probed with a goat anti-mouse peroxidase-conjugated antibody  (Jackson ImmunoResearch Laboratories). Bands were visualized using the SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific).g, and the resulting serum in the supernatant was collected and frozen at −20°C for downstream use. Murine serum samples were evaluated for presence of OVA-specific antibodies by ELISA for all groups. 96-well enzyme immunoassay (EIA)/radioimmunoassay (RIA) microplates  were coated with albumin  at a concentration of 2 ng/μL in PBS and incubated overnight at 4°C. Plates were washed twice with PBS-Tween 20 0.02% and blocked with blocking buffer  for 1 h at room temperature (RT). Blocking buffer was removed, and serum dilutions  were added to wells and incubated for 2 h at RT. Plates were washed three times with PBS-Tween 20 0.02%, and horseradish peroxidase (HRP)-AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch), diluted 1:4,000, was added to wells and incubated for 1 h at RT. Plates were washed six times with PBS-Tween 20 0.02%, developed with substrate solution , and incubated for 20 min in the dark (RT); development was stopped by addition of 2 N sulfuric acid, and absorbance was read at 510 nm on a Multiskan Ascent plate reader (Thermo LabSystems).Murine serum was collected from blood for detection of OVA-specific antibodies. Briefly, blood (500 μL) from immunized mice was collected in sterile, 1.5 mL Eppendorf tubes. Collected blood was centrifuged for 10 min at 2,000 × 5 A549 cells per well, washed 3× with PBS, and incubated for 48 h at 37°C. To read plate, luciferin was added at a final concentration of 2 mg/mL luciferin per well and imaged/read by the Biotek Synergy Mx Microplate Reader. The antibody neutralizing unit (NU) was defined as the minimum serum dilution required to achieve at least an 80% reduction in luciferase activity, which was assumed to correlate directly to an inhibition of vector infection.A neutralization assay was performed to quantify the amount neutralizing antibodies against WTAd5, present in serum samples of preimmunized murine, and is based on the ability of serum antibodies to block adenovirus infection of A549 cells. Adenovirus used carries the firefly luciferase (Fluc) reporter gene, E1 deletion, and cytomegalovirus (CMV) promoter. 2-fold serum dilutions  were tested. In 96-well flat-bottom plates, the Ad-Fluc virus (MOI 100) was combined with different serum dilutions and incubated for 1 h at 37°C. Contents of this plate were transferred to a 96-well flat-bottom plate, previously seeded with 2 × 105 cells in 100 μL PBS. For adenovirus injections, mice were anesthetized with 5% isoflurane. WTAd5 (1010 PFUs) and rAd5-SIINFEKL (108 PFUs) were administered i.m. in 50 μL PBS. For aDEC205 injections, a solution containing 10 μg of aDEC205, 50 μg poly(I:C), and 50 μg anti-CD40 ligand (CD40L) in 150 μL of PBS was administered either i.v. or i.p. Oncolytic rhabdoviruses (MG1-OVA and VSVΔ51-OVA) were administered i.v. in 100 μL of PBS.B16-OVA lung tumors were established in 8-week-old female C57BL/6 mice by i.v. injection of 3 × 10b-SIINFEKL pentamer. For SIINFEKL-specific CD8+ T cell in vitro restimulation, 1 × 106 splenocytes and lung-resident lymphocytes were incubated in RPMI medium, supplemented with 10% FBS and 5% pen-strep containing 5 μM of SIINFEKL peptide and brefeldin A (Golgi plug) for 4 h. ICS was performed as described below.OVA-specific T cell responses were measured 7 days and 14 days postboost in blood, spleen, and lung. Splenocytes and lung-resident lymphocytes were isolated and stained for the presence of SIINFEKL-specific T cells using a H-2KThe adenoviruses were made using standard techniques.6 bulk splenocytes were incubated with graded concentrations of antibody  in a 96-well plate for 45 min (4°C). After incubation, cells were stained for flow cytometry.A flow cytometry-based binding assay was performed for evaluation of aDEC205-OVA and aDEC205-empty binding specificity to the target DEC205 receptor on DCs. Bulk splenocytes were isolated from spleens of naive C57BL/6J mice. Red blood cells were lysed, and 5 × 10After processing the tissues as described above, cells were then stained with the FVS780 viability dye  PBS for 15 min at RT. Following washes, cells were incubated with anti-CD16/32 in 0.5% BSA/PBS at 4°C to block nonspecific antibody interaction with Fc receptors. Subsequently, the following protocols were used for staining.Anti-CD11c-phycoerythrin (PE)-Cy7, anti-MHC-I-PE, anti-CD8-PE-CF594, anti-IgG-allophycocyanin (APC), anti-CD3-fluorescein isothiocyanate (FITC), and anti-CD19-FITC antibodies were added to cells and incubated for 30 min (4°C).6 cells were incubated with antibodies targeting T cell surface markers CD3-AF700 and CD8-PE-CF594 for 30 min (4°C). Cells were washed twice with fluorescence-activated cell sorting (FACS) buffer. Next, the mouse Cytofix/Cytoperm Plus (BD Bioscience) was used for permeabilization and ICS. Cells were incubated with Cytofix for 20 min to permeabilize cells for ICS (4°C). Cells were washed twice with PermWash and incubated with anti-IFN-γ-BV650 and anti-TNF-α-AF647 diluted in PermWash for 30 min (4°C).First, 1 × 10b-SIINFEKL pentamer-APC (Proimmune) in 50 μL of FACS buffer was added per well and incubated for 10 min (RT) in the dark. Cells were washed twice with FACS buffer and stained with fixable viability stain for 30 min (4°C). Subsequently, the cells were washed with FACS buffer and incubated with anti-CD16/32 in FACS buffer for 5 min (4°C). Next, cells were stained with anti-CD8-PE-CF594 and anti-CD3-AF700 for 30 min (4°C)Cells were washed with FACS buffer. In a 96-well plate, 3 μL of H-2KAfter staining, cells were washed with FACS buffer and fixed in 1% paraformaldehyde. Cells were acquired on Becton Dickinson (BD) flow cytometry (Fortessa), and analyses were performed using FlowJo software version (v.)9.Phagemid cloning vector, also known as BlueScribe SK (pBSSK)-VSVΔ51, plasmid-containing viral genome, was used to construct VSVΔ51-OVA. In brief, the OVA gene was PCR amplified from pcDNA expressing aDEC205-OVA using the following primers: forward: 5′-AATTCTCGAGATGGGCTCCATCG-3′ and reverse: 5′-CATCGCTAGCTCACTACAGATCCTC-3′. PCR amplicon was digested by XhoI and NheI and cloned into the multiple cloning site (MCS) of pBSSK-VSVd51 between G and L open reading frames (ORFs). Positive clones were screened by restriction digestion mapping and verified by sequencing.Statistical significance was calculated using Student’s t test or one-way or two-way ANOVA test, using Tukey’s multiple comparison test, as indicated in the figure legends. The log rank (Mantel-Cox) test was used to determine significant differences in plots for survival studies. Error bars represent standard error of the mean. Significance is based on a p value <0.05. Statistical analyses were performed using GraphPad Prism 6.0 and Excel.F.T. conceived the project. F.T. and J.-S.D. designed the study. All authors participated in the acquisition, analysis, and/or interpretation of data and have read and approved the final manuscript. A.J., H.K.B., and J.-S.D. drafted the manuscript with editorial contributions from F.T. S.B. provided plasmids and protocols. C.T.d.S. and A.C. performed all animal work. M.H. and K.S. constructed and rescued viral vectors. R.J. P. provided adenovirus. J.-S.D. supervised the study.The authors declare no competing interests.


The canonical Wnt (Wnt/β-catenin) signalling pathway is highly conserved and plays a critical role in regulating cellular processes both during development and in adult tissue homeostasis. The Wnt/β-catenin signalling pathway is vital for correct body patterning and is involved in fate specification of the gut tube, the primitive precursor of liver. In adults, the Wnt/β-catenin pathway is increasingly recognised as an important regulator of metabolic zonation, homeostatic renewal and regeneration in response to injury throughout the liver. Herein, we review recent developments relating to the key role of the pathway in the patterning and fate specification of the liver, in the directed differentiation of pluripotent stem cells into hepatocytes and in governing proliferation and zonation in the adult liver. We pay particular attention to recent contributions to the controversy surrounding homeostatic renewal and proliferation in response to injury. Furthermore, we discuss how crosstalk between the Wnt/β-catenin and Hedgehog (Hh) and hypoxia inducible factor (HIF) pathways works to maintain liver homeostasis. Advancing our understanding of this pathway will benefit our ability to model disease, screen drugs and generate tissue and organ replacements for regenerative medicine.  The canonical Wnt (Wnt/β-catenin) signalling pathway is an ancient and highly conserved pathway common to all metazoans . It is aWnt/β-catenin signalling plays a key role in embryogenesis, as evidenced by loss of function gene knockout studies targeting components of the Wnt/β-catenin pathway including LRP5/6  . The adult liver is the largest internal organ and performs numerous functions including energy and nutrient homeostasis, xenobiotic and pathogen elimination, bile acid production and protein secretion ,22. The The liver has evolved the capacity to regenerate large quantities of tissue mass following injury. Following a 2/3 partial hepatectomy (PHx), rat livers are able regenerate to full size in just a few days ,34,35. TThe liver is derived from the multipotent precursor cells of the definitive endoderm (DE). Fate mapping studies in the mouse embryo have indicated the presence of mesendoderm precursors that give rise to the endoderm at approximately embryonic day (E) 7.5. In vertebrates, the Wnt/β-catenin pathway participates by enhancing levels of Nodal growth differentiation factor  expression, whose activity is necessary to induce formation of the DE . HoweverThe Wnt/β-catenin signalling pathway is involved in the formation of the primitive streak and anterior–posterior axis patterning through regulation of downstream Nodal expression. Nodal ligands belong to the transforming growth factors β (TGFβ) family of secreted growth factors and signal via the ALK/SMAD cascade. During gastrulation, Nodal signalling plays a key role in the induction of mesendoderm specification and enhances the expression of GATA-binding factor 1 (GATA1), Mix-like, Sox and transcription factor forkhead box (FOX) transcription factors ,40. DepeXenopus, a maternal Wnt/β-catenin signal appears to induce high Nodal expression levels in the dorsal-anterior mesendoderm via interactions with VegT and signals from the maternal yolk syncytial layer (YSL) [In mice, Nodal protein expression in the primitive streak is controlled by Wnt3 signalling through TCF/LEF DNA-binding sites in the NODAL gene promoter ,43. Likeer (YSL) ,43. The er (YSL) . During er (YSL) . Moreoveer (YSL) ,47.Xenopus and zebrafish, activation of Wnt/β-catenin signalling on the future dorsal side of the blastula regulates Nodal signalling levels to induce expression of the haematopoietically expressed homeobox (HHEX) gene which is required for foregut development in the anterior endoderm [Following gastrulation, the DE forms into the primitive gut tube along the anterior–posterior axis from which the foregut, midgut and hindgut domains emerge . Regulatendoderm ,49. Exprendoderm ,46. Signendoderm ,50. Therendoderm ,50. Hereendoderm . Thus, Sendoderm . ConsistXenopus, Wnt ligands secreted from mesoderm cells act by promoting the expression of HHEX and prospero homeobox 1 (PROX1), which constitute two of the earliest liver-specific genes [In contrast to the requirement of Wnt/β-catenin signalling suppression for foregut development, activation of the pathway is required later in development for hepatic induction and liver bud growth . Graded ic genes ,57. Notaic genes , indicatic genes ,57. Following hepatic specification, liver bud development begins accompanied by the proliferation and expansion of the hepatic progenitor population. Wnts and other signalling molecules, including FGF and hepatocyte growth factor (HGF), are predominantly responsible for this process . Tan et Induced by signals derived from the mesoderm, bipotent hepatoblasts in the liver bud can differentiate into either hepatocytes or cholangiocytes . Wnt/β-cDue to the dual properties of being able to divide without limit and ultimately differentiate into any cell type of the body, pluripotent stem cells (PSCs) represent a powerful tool in the generation of differentiated cell types . GeneratThe Wnt/β-catenin signalling pathway is an active player during embryonic liver development and is characterized by a strictly temporally regulated profile . We can In 2008, Hay et al. reported that the combination of Activin A and Wnt3a efficiently induced differentiation towards a hepatic endoderm phenotype and generated viable HLCs . As an a2 rich blood from the hepatic artery and nutrient rich blood from the portal vein. This dual blood supply speaks to the organ’s function as a general-purpose filter and nutrient sensor. Blood entering the liver flows through sinusoidal capillaries carrying nutrients and toxins from the GI tract. Within the sinusoids, blood is processed hierarchically by cords of polarised hepatocytes which line the space of Disse, a gap which separates the hepatocytes from the sinusoidal epithelium. Microvilli projecting from hepatocyte basal membranes occupy this space facilitating exchange of nutrients, metabolites and O2 with the blood through fenestrae in the sinusoidal endothelium. This lobular structure produces concentration gradients of metabolites, hormones, proteins, O2 and by-products of metabolism ) along the porto-venous axis. Hepatocytes towards the afferent  end are, therefore, exposed to a microenvironment distinct from that at the efferent (perivenous) end. This translates to gradients of function across the porto-venous axis. For example, hepatocytes in the periportal zone exhibit activity associated with higher oxidative requirements including glycogen synthesis and storage, amino acid utilization, ammonia detoxification and beta-oxidation, while hepatocytes in the perivenous zone perform non-oxidative functions including biotransformation reactions, glutamine synthesis and glycolysis [2 is higher (65 mmHg) than in perivenous hepatocytes (30 mmHg) [Metabolic zonation is typically associated with functions served by parenchymal cells, which in the case of the liver consist of hepatocytes and comprise 80% of the liver mass and 60% of the liver’s cells . At ~20 ycolysis ,73 , in which Wnt/β-catenin signalling is constitutively activated. In contrast, periportally-associated genes were downregulated (196 of 3496 zonated genes). Under hypoxic conditions, 95 genes were upregulated in the perivenous area, while 45 were downregulated suggesting a role for pO2 in zonation [In 2017, Halpern and colleagues developed a map of gene expression in the liver using single cell RNA sequencing (scRNASeq) and single molecule fluorescence in situ hybridisation. The authors confirmed previous reports that hepatocytes were highly heterogenous and exhibited clear gradients of function. Additionally, they found that a remarkable 50% of liver-associated genes display significant hepatic zonation . Of these, some genes were expressed most highly in the intermediate zone such as hepcidin antimicrobial peptide (HAMP), HAMP2, IGF binding protein 2 (IGFBP2), major urinary protein 3 (MUP3) and CYP8b1. They also found that many perivenous genes were upregulated in APCzonation .A second study by Halpern and colleagues in 2018 focused on the possibility of zonation of non-parenchymal cells (NPCs) in the liver. They identified several distinct cell types based on enriched markers for endothelial cells, T-cells, dendritic cells, Kupffer cells, liver capsule macrophages and neutrophils. By using a paired sequencing approach based on the previously established hepatocyte gene expression map, they were able to distinguish distinct populations of NPCs at different locations within the liver lobule ,93. In tLECs comprise the central and portal veins and sinusoids and are known to regulate immune responses and secrete morphogens . EndotheWhile much is now known of the fundamental role of Wnt/β-catenin signalling in liver zonation, questions remain around how zonation arises in the developing liver, how it is re-established following injury and to what extent Wnt/β-catenin signalling maintains zonation of other metabolic functions  and non-metabolic proteins such as those involved cell-cell adherence. Ma et al. recently addressed some of these questions and extended the influence of Wnt/β-catenin signalling to include zonation of the adhesion protein E-cadherin and tight junction component Claudin2. GS and CPS1 are first expressed in the developing embryo at E15.5 and E13.5, respectively, with GS expression coinciding with the upregulation of nuclear β-catenin ,96. Clau4-mediated ablation of perivenous hepatocytes. In accordance with a recent study which found evidence for the role of Wnt/β-catenin in regeneration following CCL4-mediated injury [4 treatment and present in the intermediate zone at 2–5 days post-treatment, before resuming its periportal expression during the repair phase [Using lineage tracing of sinusoidal LECs ) the authors were able to determine that sinusoidal LECs adjacent to the central vein expressed Wnt2, Wnt9b and RSPO3 becoming limited to expression of Wnt2 alone more distally from the central vein. Furthermore, they demonstrated that ablation of sinusoidal LECs had little impact on initiation of Wnt/β-catenin dependent zonation but was involved in the maintenance of zonation in adulthood. The authors examined Claudin2 expression following CCLd injury , they deir phase . Conditiir phase . Togethe4-induced injury [Recent studies have agreed on the conclusion that it is resident hepatocytes themselves which are the major locus of hepatocyte neogenesis and reservoir of potential plasticity in homeostatic renewal and in response to injury ,14,15,98d injury . Hepatocd injury . Recentl+/Axin2+ hepatocytes adjacent to the central vein were the primary source of hepatocyte neogenesis during homeostatic renewal. New cells appeared to emanate from the perivenous region eventually populating the entire lobule [Wnt/β-catenin signalling confers a proliferative capability in the liver throughout development and adulthood by promoting transcription of genes associated with re-entry into the cell cycle including CyclinD1 and c-Myc ,107,108.e lobule . This hye lobule , implyine lobule .CreERT2/+ mice with a Rosa26LSL-Tomato/+ reporter strain to permanently label hepatocytes expressing telomerase and their progeny. In this study, they found that hepatocyte proliferation was predominantly restricted to a sparse yet uniformly distributed telomerase expressing a hepatocyte subpopulation and that this represented a stem cell-like proliferative niche. Hepatocytes expressing telomerase were observed to disproportionately repopulate the liver producing clonal clusters comprising an average 6–10 cells at 1 year [CreERT2/+ Rosa26LSL-Tomato/+ transgenic mice may bias telomerase expressing cells towards proliferation must be ruled out [A string of recent studies proffers a robust rebuttal to Nusse’s conclusion that homeostatic renewal occurs predominantly in the perivenous region with hepatocytes migrating along the acinus over time, eventually repopulating the entire lobule. Nusse’s study utilised a CreERT2 cassette to disrupt one Axin2 allele. Axin2 is both a downstream target of Wnt/β-catenin signalling as well as negative regulator, thus producing a negative feedback loop . It has t 1 year . Chen ett 1 year . In lighuled out ,116. Wheuled out ,13,14. Iuled out . Furtheruled out . There iuled out ,13,14. IAll intracellular pathways are inextricably linked with the activity of other pathways. This interdependence allows for the formation of the dynamic physical infrastructure which enables cells to communicate with each other and with their environment. Within the context of Wnt/β-catenin signalling in the liver, two pathways are of particular interest: (i) the Hh pathway and (ii) the HIF pathway . In thisLike the Wnt/β-catenin pathway, the Hh pathway is important both in embryological development controlling growth, tissue patterning, mitogenesis as well as adult homeostasis and tissue repair in vertebrates ,114,115.2 in the perivenous region is approximately half that of the periportal zone dropping from 65 to 30mmHg due to the oxidative intensive functions of periportal hepatocytes [2) have been shown to upregulate genes under β-catenin control including CyclinD1 and c-Myc [2 in the liver acinus participates in the regulation of the Wnt/β-catenin/HIF axis. HIF signalling has long been hypothesised to play an instructive role in liver zonation . Evidencatocytes . All thratocytes ,120. Hypnd c-Myc , while knd c-Myc . In 2007nd c-Myc ,123. Onend c-Myc . CollectIn this review, we have highlighted important developments in the study of Wnt/β-catenin signalling in liver development and homeostasis. The Wnt/β-catenin pathway has a conserved role in liver development where its activity is tightly regulated both spatially and temporally. During gastrulation, high levels of Nodal signalling induced by Wnt/β-catenin signals promote endoderm specification. By the end of gastrulation, suppression of Wnt/β-catenin signalling in the anterior endoderm permits induction of the foregut endoderm. Protocols facilitating the directed differentiation of iPSCs to HLCs reflect this fact with the administration of Wnt/β-catenin activators Wnt3a and Activin A or GSK3β inhibitors CHIR9021 and 1m being restricted to the first three days of culture ,71. The role of Wnt/β-catenin signalling in the maintenance of the zonation of hepatic parenchyma is now well established. Recent research has also indicated a role in the zonation of NPCs as well as an expansion of the Wnt/β-catenin pathway to incorporate the RSPO-LGR4/5-ZNRF3/RNF43 module ,78,93,95With respect to Wnt/β-catenin signalling in the context of homeostatic renewal in the liver, the controversy around exactly which cells are responsible for proliferation may be waning with consensus building around a model of proliferation which sees hepatocytes as the source of neogenesis and facultative contributor to regeneration following injury. Wang et al. suggested that perivenous hepatocytes were at least disproportionately responsible for homeostatic renewal of liver parenchyma. However, new research now makes a pan-zonal model of hepatocyte-driven homeostatic renewal seem more likely ,13,14,15By continuing to improve our understanding of this ancient and fundamental signalling pathway, we draw closer to a more complete understanding of one of the mechanisms that enabled life to transition from single to multicellular organisms. With the advent of high throughput technologies, advances in data analysis tools and ever greater resolution of gene and protein expression profiles, the integration of all pathways into a unified model must remain a goal for future research.


We use simulations and theory to determine how these molecular properties lead to chromosome compaction. High compaction can be achieved if even a small fraction of condensins have two essential properties: a long residence time and the ability to perform two-sided (not necessarily symmetric) extrusion. In mixtures of condensins I and II, coupling two-sided extrusion and stable chromatin binding by condensin II promotes compaction. These results provide missing connections between single-molecule observations and chromosome-scale organization.Chromosome compaction is essential for reliable transmission of genetic information. Experiments suggest that ∼1000-fold compaction is driven by condensin complexes that extrude chromatin loops, by progressively collecting chromatin fiber from one or both sides of the complex to form a growing loop. Theory indicates that  During mitosis in metazoan cells, each chromosome is linearly compacted ~1000-fold from a ~1-mm-long chromatin polymer globule into a ~1 µm array of chromatin loops . This reIn the first single-molecule experiments that directly imaged loop extrusion, yeast condensins were observed to extrude DNA loops in an asymmetric, ‘one-sided’ manner . In thisXenopus condensins proceeds asymmetrically rather than symmetrically  with different dynamics can generate the high degree of linear compaction observed for mitotic chromosomes in vivo. We find that asymmetric two-sided extrusion can eliminate unlooped gaps between neighboring LEFs and compact chromosomes In the model, LEFs representing SMC complexes perform loop extrusion on a polymer fiber representing the chromosome . Each LEZ-loops’  are prohZ-loops’  (moreoveZ-loops’ . Furthereriments  and lack=(1-f)-1 . This queen LEFs . FurtherPrevious computational models of loop extrusion generally assumed that all LEFs have the same average unbinding rate and that all active LEF subunits translocate at the same average speed . Since eTo determine the ability of asymmetric two-sided loop-extruding condensins  to compaEliminating or avoiding unlooped gaps between LEFs is critical to achieving a high degree of linear compaction . One-sidunlooped . In contped gaps . Based oλslow/d≫1.In simulations, we controlled asymmetry by varying the relative extrusion speeds of the active subunits, quantified by the dimensionless ratio ly large . Specifislow/d>1 , as predslow/d>1 . As showXenopus condensins by We next determined whether 1000-fold linear compaction can be achieved with the asymmetries  can achieve only ~60-fold linear compaction . The theoretical analysis predicts that the ratio of the extrusion speeds, In the scenario with long-lived two-sided LEFs (~10-fold ). The thIn the scenario with short-lived two-sided LEFs  , establiWe next used simulations to test the prediction that increasing the residence time of the two-sided LEFs relative to that of the one-sided LEFs could increase compaction in mixtures of LEFs. We hypothesized that long-lived two-sided LEFs might be able to further compact chromatin by two mechanisms. First, increasing the residence time increases the effective processivity, and thus potentially, the loop size. Second, increasing the residence time additionally allows two-sided LEFs to remain on the chromatin fiber longer than the one-sided LEFs; consequently, each transient gap between one-sided LEFs may eventually be extruded into a loop by a two-sided LEF.In both qualitative and quantitative agreement with the theory, the maximum fold linear compaction, ce times . As preds  , red boxo below) .To further increase the degree of compaction, we assumed that stably bound condensin II performs two-sided extrusion, while dynamic condensin II performed one-sided extrusion  associated with the complex. Consistent with this hypothesis, it has been observed that the yeast kleisin Brn1 and Hawk Ycg1 can act as a ‘safety belt’ that anchors condensin to DNA . HoweverXenopus condensins in their native context   and the  context . Yet anoWhat could give rise to the predicted stability of two-sided SMC complexes? One hypothesis is that condensin I performs one-sided extrusion, while condensin II performs two-sided extrusion . The meaHowever, single-molecule experiments with condensins I and II suggest otherwise, at least for human cells . It has Taking the in vitro experiments  togetherMetazoan chromosomes are composed of large (~400 kb) chromatin loops with condensin II at their bases, with smaller (~80 kb) loops mediated by condensin I nested within . ConsequThis hierarchy of loop nesting naturally emerges from the loop extrusion model for mixtures of LEFs with two different residence times. LEFs with longer residence times, ral axis . This hiral axis , mixtureral axis , and mixral axis . While nral axis , differeral axis .Furthermore, our results suggest specific properties of condensins that could regulate mitotic chromosome morphology. Previously, it was shown that mitotic chromosome morphology can be controlled by the relative ratio of condensin I to condensin II . GeneralIn the loop extrusion model, LEFs can linearly compact chromosomes by extruding chromatin into loops within approximately one LEF residence time. Chromosome morphology, as quantified by loop sizes, equilibrates over longer timescales of approximately 5–10 residence times . In mixtWith the experimentally measured residence times of condensins I and II , loop exThe principles for mitotic chromosome compaction developed here are generalizable to other chromosome organization scenarios. In particular, we previously showed that the physical principles underlying mitotic chromosome formation by loop extrusion are relevant for topologically associated domain (TAD) formation in interphase , where eWe previously showed that much like mitotic chromosome compaction, the formation of major features of interphase chromosomes, such as TADs, ‘dots’, and ‘stripes’ requires avoiding unlooped gaps, either between LEFs or between LEFs and TAD boundaries. One-sided extrusion can form TADs and stripes by enhancing local chromatin contacts , as obseulations , measureulations , and Hi-ulations . These vymmetric .Mixtures of one- and two-sided cohesins with different residence times should also be able to form TADs. We previously showed that mixtures with relatively high fractions of one-sided LEFs  .Bacillus subtilis and Caulobacter crescentus chromosomes  two-sided extrusion. Strikingly, the presence of even a small (~20%) fraction of such condensins in a mixture with other, purely one-sided condensins could be sufficient to achieve compaction under physiological conditions. It remains to be determined what mechanisms are responsible for different types of extrusion dynamics and what factors might facilitate a transition from one-sided to two-sided loop extrusion in some molecules in vivo. However, our analysis suggests that two-sided extrusion should be tightly coupled to stable chromatin binding. In particular, two-sided extrusion by stably bound condensin II is sufficient to linearly compact mitotic chromosomes in simulations. We thus hypothesize that condensins, particularly condensin II, may bind chromosomes as a dimer of condensin complexes in vivo. Such a dimer might have a longer residence time due to a larger protein-DNA interface, while also performing two-sided loop extrusion via its two protein motors.Further single-molecule, biochemical, and structural studies could also help to understand how kleisins, Hawks, and post-translational modifications might generate diverse SMC complex symmetries and dynamics, and thus, functions. Such experiments, together with the principles established by our models, could clarify how the molecular properties of loop-extruding SMC complexes compact and organize chromosomes throughout the cell cycle.https://github.com/mirnylab/one_sided_extrusion/tree/master/mitotic/;  copy archived at swh:1:rev:b27012e95d354e8deaac5bcfdcb3c36b375626ce).Stochastic simulations of LEFs on a chromatin fiber are performed as previously described  with adaThe physiological range of the ratio of the processivity to the mean separation is estimated as eviously . Processin vitro  and measin vitro . Mean se in vivo .For the general estimate of the physiological range of asymmetries, the expected range of asymmetries is calculated from the symmetry scores measured by i.e., We also considered three scenarios of asymmetric extrusion to model experiments by In the third scenario, we calculated loop-growing and loop-shrinking velocities for condensins from single-molecule experiments by  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife.Congratulations, we are pleased to inform you that your article, "The interplay between asymmetric and symmetric DNA loop extrusion", has been accepted for publication in Reviewer #2:eLife  to incorporate new experimental findings that in vivo condensins likely utilize asymmetric two-sided loop extrusion in addition to the one-sided and symmetrical two-sided extrusion examined in the previous work. By adding variable residence time and asymmetric extrusion, the authors are able to model physiologically relevant compaction consistent with experimentally determined levels and properties of Condensins I and II. These findings represent a significant advancement over the previous work, the conclusions are well-justified, and a well-written and conceived Discussion gives a valuable perspective of the current state of the field. As a result, I support the publication of this manuscript in eLife without additional data.Banigan and Mirny revisit their modeling approach previously published in Reviewer #3:Xenopus laevis egg extracts, the authors extend previous models of loop extrusion by allowing populations of loop extruders with different residence times, and active loop extruding subunits that translocate at the different speed. They show that the distribution of symmetries observed in these experiments (even though most of loop extrusion is one-sided) can account for over 1000 fold condensation (as in cells). They also considered the effects of two condensins (I and II) corresponding to purely one and two sided extrusion. Interestingly they show that a longer residence time of a two-sided condensin would also account for over 1000 fold condensation. As a consequence, their model suggests a testable prediction for the activities of condensin I and II. Overall I found the paper very clear and systematic, and their findings are an important contribution to understanding how loop extruding factors may contribute to the overall organization of chromatin, even though this work is an extension of their previous models. As such I think it fits as a research advances and support publication as is.In this paper, Banigan and Mirny study the effects of different loop-extrusion symmetries to the overall compaction of chromatin. This study is motivated by recent work showing that condensin loop extrusion is not one-sided as shown purely in vitro, but rather displays a mixed population of symmetries. From a theory point of view, one-sided loop extrusion cannot condense DNA sufficiently, so since the first in vitro demonstration of one-sided loop extrusion it has not been clear how to marry theory and experiments with the requirements of DNA condensation in cells. Based on the results from


The photosensitive precursor played a key role in these improvements permitting fewer organic species which act as defect sites after metal oxide formation. Consequently, our approach compares favorably with that of conventional fabrication process using photoresist in terms of its simplicity, cost efficiency, and electrical performance.We report a novel self-patterning method for solution-processed indium zinc oxide (IZO) thin films based on photosensitive precursors. This approach is an alternative and evolutionary approach to the traditional photoresist patterning techniques. Chelate bonds between metal ions and β-diketone compounds in ultraviolet light-exposed IZO solutions provided intrinsic photosensitivity, which resulted in a solubility difference between exposed and non-exposed regions. This difference enabled self-patterning of the IZO for thin-film transistor (TFT) fabrication. Compared with previously reported self-patterning methods based on photosensitive activators, our self-patterned IZO TFTs based on photosensitive precursors displayed excellent electrical characteristics and stability. The field-effect mobility increased from 0.27 to 0.99 cm Since the first report of InGaZnO-based thin-film transistors (TFTs), solution-processed amorphous metal oxides have been regarded as some of the most promising materials for next-generation displays because of their high mobility and transparency6.Solution-processed electronic devices are of increasing interest due to their potential lower cost and vacuum-free fabrication processes. Unlike traditional semiconductor processing, low-temperature solution processes enable the deposition of metal oxides on a broader range of substrate materials  and provide opportunities to produce flexible devices7. Most applications require metal oxides to be finely patterned to form specific active areas in devices such as TFTs, solar cells, and analog circuits11.Meeting the needs of the times, numbers of solution-based processes have been developed, however, there remain barriers to commercialization, such as the patterning method12. Therefore, various alternative patterning methods have been developed such as micro-stamping and ink-jet printing15. Among them, a self-patternable solution process is of considerable interest because of its simplicity and low cost which means no specialized equipment is necessary. For instance, self-patterning of metal oxide using selective surface wetting has been reported. However additional layer was needed for surface modification; thus, plasma treatment was required to subsequently remove this layer16.Conventional photolithography process comprises many complex steps, including photoresist coating, vacuum drying, development, etching, and strip. This complexity limits not only the design of circuits, such as the pattern density, pattern profile, and width, but also the characteristics of processes, such as the thickness and profile of the photoresist, and chemical or physical damage to the etching17. Photosensitive activators based on β-diketone compounds, such as benzoylacetone or acetylacetone (AcAc), play an important role in the presented self-patterning method. The addition of photosensitive activators to metal salts results in the formation of chelate bonds that lead to the crosslinking under ultraviolet (UV) light exposure allowing self-patterning of the metal-oxide film free of the photolithography process.In previous study, we have suggested photoresist-free solution-processed amorphous oxide semiconductor TFTs using photosensitive sol-gels and determined the mechanism of the self-patterning process3–), chloride (Cl–), or acetate (CH3COO–)) remain after film formation; these species can cause degradation of TFT electrical characteristics and reliability18.These results indicate the potential of the self-patterning method. However, some issues remain to be resolved, such as the reducing organic residues. Unwanted organic species from photosensitive activators and metal precursor ligands  IZO TFTs, while self-patterned IZO TFTs based on an activator were named extrinsic photosensitive (EP) IZO TFTs. We confirmed enhancement of the electrical characteristics and stability of the IP IZO TFTs. The ease of processing, as well as the improved electrical characteristics and reliability of the TFTs, are expected to facilitate commercial realization of the self-patterning method.Herein, we report a new self-patterning method for solution-processed In–Zn–O (IZO) TFTs that focuses on the reduction of organic residues in the film. This was achieved using photosensitive precursors, rather than photosensitive activators3CHOCCH3)3) and zinc acetylacetonate hydrate (Zn(C5H7O2)2·xH2O) were selected as precursors to prepare the IP IZO solutions (0.3 M). The precursors were dissolved in 2-methoxyethanol (2ME) at various In:Zn molar ratio  to compare the patternability and electrical characteristics of the IP IZO thin films. An EP IZO solution (0.3 M) was prepared as follows. Indium nitrate hydrate (In(NO3)3·xH2O) and zinc nitrate hydrate (Zn(NO3)2·xH2O) were dissolved in 2ME at the In:Zn molar ratio of 1:2, which was the optimal ratio in terms of TFT electrical characteristics. This is the preparation method for conventional IZO solutions. Then, AcAc was added as photosensitizer. The mixture was stirred for 1 h at 70 °C and aged another 24 h at room temperature.For solution-processed oxide thin-film, synthesis of solution should be undertaken first. Indium acetylacetonate . Images of the self-patterned IZO films were acquired using an optical microscope . The chemical properties of the IZO thin films were analyzed by X-ray photoelectron spectroscopy (XPS)  and Fourier transform-infrared spectroscopy (FT-IR) .DS) of 10.1 V as the gate voltage (VGS) was swept from –30 to 30 V. A positive bias stress (PBS) test was carried out at a VGS of 20 V and VDS of 10.1 V for 1,000 s.A Hall effect measurement system  was used to measure the semiconductor characteristics. The thickness of a patterned film was measured by atomic force microscopy (AFM) . The electrical characteristics of an IZO TFT were measured using a semiconductor parameter analyzer  in a dark box under ambient conditions. The transfer curve was measured at a fixed drain voltage  dissolved in β-diketone compounds formed metal chelate bonds by exchanging the X ligands, such as chloride (Cl–), acetate (CH10COO–), or nitrate (NO3–). UV irradiation induced π → π* electronic transitions and decomposition of these chelate ring structures, which consisted of a central metal ion surrounded by β-diketonato ligands. The partial distribution of excited electrons on the oxygen atoms reduced the coordination strength between the metal ion and the β-diketonato ligands. As a result, this gradual decomposition allowed photosensitive behavior altering its solubility in some organic agent leading to hydrolysis of the IZO thin films21. This series of reactions is depicted in Fig. In our previous research involving EP IZOs, metal salts (NO3)2 and Zn(AcAc)NO3)17. A similar absorption peak at 330 nm observed for the IP IZO solution was attributed to the AcAc-based precursor.Figure 22. Herein, a similar trend was observed: elevated field-effect mobility (μFET) from 0.01 to 1.55 cm2/Vs, reduced in Ion/off ratio from approximately 106 to 102, and increased subthreshold swing (S.S) from 0.93 to 12.49 V/dec with increasing In ratio. Table On the basis of above photoreactivity, we fabricated the IP IZO TFFs on the Si substrate as shown in Fig. FET of 0.99 cm2/Vs, Ion/off ratio of 1.00 × 109, and S.S of 0.46 V/dec were observed. Meanwhile, the EP IZO exhibited relatively inferior characteristics with μFET of 0.27 cm2/Vs, Ion/off ratio of 1.07 × 107, and S.S of 0.54 V/dec; the distributions of electrical parameters for the IP and EP IZO TFTs are summarized in Fig. Figure 16 to 8.43 × 1016/cm3 and reduced resistivity from 1.24 × 104 to 2.73 × 103 Ωcm, compared with the EP IZO thin film; these characteristics suggested fewer defects in the IP IZO phase. We previously reported that incomplete decomposition and volatilization of β-diketone photosensitive activators during metal oxide formation resulted in organics residues that degraded TFT performance. We presumed that these performance differences were derived from relatively low concentrations of residues in IP IZOs compared with the corresponding EP IZOs after film formation.A similar trend was observed in the Hall measurement data Fig. a. The IP−1 in the EP IZO spectrum were assigned to nitrogen-related vibrations of molecules  or as NO3– deformations, most of which originated from nitrate precursors of EP IZOs26. Additionally, broad peaks in the range from 2,000 to 2,700 cm−1 indicated the presence of C=O, O–H, and C–H groups, which derived from incomplete formation of metal chelate bonds between metal ligands and AcAc28. These results confirmed the presence of considerable amounts of organic residues from the metal precursors and photosensitive activator that formed during film formation29. On the other hand, most peaks in the range from 1,000 to 1,500 cm−1 were absent in the spectrum of IP IZO. In the range from 2,000 to 2,700 cm−1, some residues from acetylacetonate metal precursors still remained, and relatively small absorbance peaks were shown as already predicted. These findings indicate that uniformly formed metal chelate bonds in acetylacetonate metal precursors are important for the quality of thin films.FT-IR spectroscopy of the IZO thin films was carried out to support this hypothesis Fig. b. Peaks 5/2 and Zn 2p3/2 peaks for the In–O and Zn–O bonds, respectively. For the IP IZO thin film, a shift toward higher binding energy from 444.88 to 444.98 eV and broadening of In 3d5/2 were observed which slightly broadened the peak compared with those of the EP IZO thin film. It has been reported that oxidized states of In-O compounds have higher binding energy and broader peak than In31. In this respect, the In 3d5/2 peak represent that more oxidation and better activation was occurred during IP IZO film formation. Same tendency was also shown in Zn 2p3/2 peak, slight shift to a higher binding energy, compared with those of the EP IZO thin film; this observation was consistent with the stronger tendency of the IP IZO thin film to undergo oxide formation33. Figure 34.Additionally, the chemical and structural compositions of the EP and IP IZO films were analyzed by XPS measurements. Figure I peak at 530 eV, derived from the oxide lattice in the metal oxide phase, represents advanced conducting pathways for electrons in IP IZO . Meanwhile, the OII peak at 531 eV is associated with oxygen vacancies (VOs), and the OIII peak at 532 eV is attributed to metal–hydroxide (M–OH) or metal–oxycarbon (M–OC) groups of organic residues35. The reduction in the intensity of these two peaks  indicated an overall reduction of oxygen impurities that generate trap sites and deteriorate performance. It has been reported that carbon-related species in IZO tend to strongly attract oxygen compared with In or Zn, and interfere with metal oxide (M–O) bond formation36. These reactions result in the formation of VOs (as evidenced by the OII peak) and defect states. In this term, the smaller OII peak for IP IZO indicated fewer defect states and better M–O bonding.The OGS = 20 V and VDS = 10.1 V for 1,000 s in the dark. As a natural consequence, the IP IZO TFT showed enhanced stability with a threshold voltage shift (ΔVth) of 1.68 V, compared with 9.32 V for the EP IZO TFT.In the IP IZO solution, the only sources of carbon and oxygen impurities were the 2ME solvent and AcAc-based metal precursors. However, in the EP IZO solution, the AcAc activator was an additional impurity source in addition to the 2ME solvent and metal nitrate precursors. Consequently, these results figure out that the IP IZO thin film had enriched M–O bonding and contained fewer organic residues such as carbon or hydroxyl groups compared with the EP IZO thin film. The effect of these impurities was more discernable under bias stress test. Figure Os, a significant increase in the electrical characteristics in the transfer curve was achieved: μFET increased from 0.27 to 0.99 cm2/Vs, and S.S decreased from 0.54 to 0.46 V/dec. Furthermore, the ΔVth under PBS tests improved from 9.32 to 1.68 V. Consequently, our new self-patterning method of IZO thin films is a commercially viable technique that reduces manufacturing cost and process steps due to non-necessity of photoresist but also contribute to superior electrical characteristics without degradation caused by organic residues.In summary, we have demonstrated a novel self-patterning method for low-temperature solution-processed IZO TFTs. The AcAc based precursors provided intrinsic photosensitivity to the IZO solution, which self-patterned under UV irradiation. Compared with the conventional self-patterning method using photosensitive activators, our method provided higher-quality IZO thin films with fewer organic residues by limiting the possible sources of organic species to the 2ME solvent and the AcAc based precursors. By arranging M–O bonding of IZO uniformly with decreased VSupplementary Information


Objectives: Improper activation of Wnt/β-catenin signaling has been implicated in human diseases. Beyond the well-studied glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1), other kinases affecting Wnt/β-catenin signaling remain to be defined.Methods:To identify the kinases that modulate Wnt/β-catenin signaling, we applied a kinase small interfering RNA (siRNA) library screen approach. Luciferase assays, immunoblotting, and real-time polymerase chain reaction (PCR) were performed to confirm the regulation of the Wnt/β-catenin signaling pathway by cyclin-dependent kinase 11 (CDK11) and to investigate the underlying mechanism. Confocal immunofluorescence, coimmunoprecipitation (co-IP), and scratch wound assays were used to demonstrate colocalization, detect protein interactions, and explore the function of CDK11.Results: CDK11 was found to be a significant candidate kinase participating in the negative control of Wnt/β-catenin signaling. Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes, in a manner dependent on intact adenomatosis polyposis coli (APC) protein. Further analysis showed that CDK11 modulation of Wnt/β-catenin signaling engaged the endolysosomal machinery, and CDK11 knockdown enhanced the colocalization of Wnt/β-catenin signaling receptor complexes with early endosomes and decreased colocalization with lysosomes. Mechanistically, CDK11 was found to function in Wnt/β-catenin signaling by regulating microtubule stability. Depletion of CDK11 down-regulated acetyl-α-tubulin. Moreover, co-IP assays demonstrated that CDK11 interacts with the α-tubulin deacetylase SIRT2, whereas SIRT2 down-regulation in CDK11-depleted cells reversed the accumulation of Wnt/β-catenin signaling receptor complexes. CDK11 was found to suppress cell migration through altered Wnt/β-catenin signaling.Conclusions: CDK11 is a negative modulator of Wnt/β-catenin signaling that stabilizes microtubules, thus resulting in the dysregulation of receptor complex trafficking from early endosomes to lysosomes. Abnormal Wnt/β-catenin signaling activity has been reported in many human diseases, such as cancer3. In the absence of Wnt ligands, β-catenin is phosphorylated, ubiquitinated, and targeted for degradation in a destruction complex consisting of adenomatosis polyposis coli (APC), Axin1, and glycogen synthase kinase 3β (GSK3β) in the cytoplasm6. The binding of Wnt ligands to Frizzled (Fzd) and low-density lipoprotein receptor-related protein 6 (LRP6) results in recruitment of Dvl2, Axin1, and GSK3β, thus leading to the release of β-catenin. β-catenin then enters the nucleus and activates Wnt/β-catenin signaling9. LRP6 is phosphorylated after the binding of Wnt ligands to receptors, and Wnt/β-catenin signaling receptor complexes, consisting of Fzd, LRP6, pLRP6, Dvl2, Axin1, and GSK3β, are formed12. The receptor complexes are internalized into cells through endocytosis13 and subjected to endocytic trafficking to lysosomes, where they are degraded15, thus decreasing signaling activity. An extensive study of epidermal growth factor receptor (EGFR) has indicated that internalized EGFR can signal in early endosomes, whereas signaling terminates in lysosomes, where EGFR is degraded16. However, although the endosomal compartment has been implicated as a signaling center17, the subcellular location of Wnt/β-catenin signaling receptor complexes remains controversial13.Canonical Wnt/β-catenin signaling  is important for embryonic development and organ homeostasis in adults25. However, given the complexity of Wnt/β-catenin signaling, the precise mechanisms regulated by different kinases at different steps remain poorly understood. Thus, an extensive analysis of other kinase functions in Wnt/β-catenin signaling is warranted.Phosphorylation of signaling components by kinases has been shown to regulate various steps of Wnt/β-catenin signaling. Dozens of kinases have been demonstrated to be involved in the phosphorylation of molecules in Wnt/β-catenin signaling cascadesCDC2L1 and CDC2L226. CDK11 is versatile among CDK family members, besides cell cycle and mitosis regulation32, previous studies have shown that CDK11 is involved in the regulation of transcription and RNA splicing38, modulation of microtubule stabilization, and autophagy40. In the present study, we performed a large-scale kinase RNA interference (RNAi) screen to identify kinases that might be involved in Wnt/β-catenin signaling. Among the significant hits, CDK11 was found to be a negative regulator of Wnt/β-catenin signaling. Biochemical and functional analyses demonstrated that CDK11 interacts with SIRT2 in modulating tubulin stability, thereby affecting the cellular trafficking of Wnt/β-catenin signaling receptor complexes from early endosomes to lysosomes. Our findings suggest a new mechanism whereby Wnt/β-catenin signaling is negatively regulated.Cyclin-dependent kinase 11 (CDK11) is a serine/threonine kinase encoded by two nearly identical genes, Renilla luciferase reporter plasmid was purchased from Promega , and the CDK11-Flag plasmid was purchased from Genscript . Wnt3a was purchased from R&D Systems . Antibodies to the following were used: β-catenin, CDK11, and MEC-17 ; LRP6, pLRP6, Axin1, phospho-β-catenin (Ser33/Ser37/Thr41), GSK3β, acetyl-α-tubulin, histone deacetylase 6 (HDAC6), tubulin, and N-cadherin ; Dvl2, EEA1, and LAMP1 ; Flag, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and SIRT2 ; and TSG101 . In addition, normal rabbit IgG, horseradish peroxidase (HRP)-conjugated secondary mouse antibody, and HRP-conjugated secondary rabbit antibody (CST) were used.HeLa, HCT116, and SW480 cells were maintained in RPMI 1640 , and HEK293T cells were maintained in DMEM (Hyclone) supplemented with 10% fetal bovine serum (FBS) . The TOPFlash luciferase reporter plasmid was purchased from Addgene , the pRL-TK The siGENOME SMARTpool Library-Human Protein Kinase was purchased from Dharmacon . Cotransfection of the kinase siRNA library and luciferase reporter plasmids (at a 200:1 ratio of TOPFlash plasmid to pRL-TK plasmid in micrograms) followed the protocol of DharmaFECT® Duo Transfection Reagent .The siRNAs targeting various genes were purchased from GenePharma , and their sequences were as follows (sense strand 5′-3′): β-catenin: AGGUGCUAUCUGUCUGCUC; CDK11-1: AUGGAGUGGUCUACAGAGCAA; CDK11-2: AGAUCU ACAUCGUGAUGAA; HRS: CGUCUUUCCAGAAUUCAAA; TSG101: CAGUUUAUCAUUCAAGUGUAA; EAP20: CGAUCCAGAUUGUAUUAGA; CHMP6: AGAUCGAAA UGAAAGUGAU; LRP6: CCAUGGAUAUACAUGCUUU; and SIRT2: CAGCGCGUUUCUUCUCCUGUA. siRNA transfection was performed according to the protocol of DharmaFECT transfection reagent (Dharmacon).Plasmid transfection was carried out with ViaFect™ transfection reagent (Promega) according to the manufacturer’s instructions.Renilla luciferase luminescence was measured. The ratio of firefly to Renilla luciferase luminescence for each well was calculated as the relative luciferase activity.Luciferase activity was measured with the Dual-Glo® Luciferase Assay System (Promega) according to the manufacturer’s protocol. In brief, Duo-Glo® luciferase reagent was added to cells (75 μL/well) that had been grown in 96-well plates with complete medium. Firefly luminescence was measured after incubation at room temperature for 20 min, and equal amounts of Duo-Glo® Stop & Glo® reagent (75 μL/well) were added to the plates, which were further incubated for 20 min at room temperature. Subsequently, Cells were lysed in TRIzol reagent , and total RNA was extracted according to the manufacturer’s protocol. RNA was reverse transcribed into cDNA with a kit , and SYBR Green-based real-time PCR was performed to quantify mRNA expression according to the manufacturer’s protocol . Relative mRNA expression was normalized to GAPDH expression. The primer sequences were as follows (5′-3′): c-myc (forward: CCTGGTGCTCCATGAGGAGAC and reverse: CAGACTCTGACCTTTTGCCAGG), Axin2 (forward: CAAACTTTCGCCAACCGTGGTTG and reverse: GGTGCAAAGACATAGCCAGAACC), LRP6 (forward: CAGTTGGAGTGGTGCTGAAAGG and reverse: CCATCCAAAGCAGCCCGTTCAA), Dvl2 (forward: TCCATACGGACATGGCATCGGT and reverse: CGTGATGGTAGAGCCAGTCAAC), Axin1 (forward: GTATGTGCAGGAGGTTATGCGG and reverse: CACCTTCCTCTGCGATCTTGTC), GSK3β (forward: CCGACTAACACCACTGGAAGCT and reverse: AGGATGGTAGCCAGAGGTGGAT), CDK11 (forward: CCGACTTACAGGACATCAGCGA and reverse: CTCCTCTGATTCTTCACTGGTGC), EEA1 (forward: CTTCTAGCCACCAGGCAAGATC and reverse: CCAATGTAGCCTTGGCAGTCTTC), LAMP1 (forward: CGTGTCACGAAGGCGTTTTCAG and reverse: CTGTTCTCGTCCAGCAGACACT), HRS (forward: GACAGACTCTCAGCCCATTCCT and reverse: TCATGCGGTTCACGAAGGTGGT), TSG101 (forward: TTCTCAGCCTCCTGTGACCACT and reverse: CCATTTCCTCCTTCATCCGCCA), EAP20 (forward: AGAGCAAGTCCAGCTTCCTGATC and reverse: GGTAAAGACGGAGTTGTTCTGGC), and CHMP6 (forward: GACAAGCTGAGGCAGTACCAGA and reverse: CTGCTCCTGGTATCGCTTCTTC).g for 15 min at 4 °C. Protein concentrations were measured with a BCA protein assay kit (Thermo Scientific) according to the manufacturer’s protocol. Twenty micrograms of protein for each sample was separated on 6%–10% polyacrylamide gels after denaturation for 5 min and transferred to polyvinylidene fluoride membranes . After being blocked with 5% nonfat milk in Tris-buffered saline containing Tween 20  for 1 h at room temperature, the membranes were incubated with primary antibody overnight at 4 °C. After being washed with TBST 3 times for 15 min, membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Primary and secondary antibodies were diluted with 5% nonfat milk in TBST. Super Enhanced Chemiluminescence Substrate (Millipore) was used to detect proteins after membranes were washed with TBST 3 times for 15 min.Cells were washed 3 times with cold phosphate-buffered saline (PBS) and lysed on ice with lysis buffer  supplemented with 1% protease inhibitor  and 1% phosphatase inhibitor (Bimake) for 30 min. Cell debris was removed by centrifugation at 14,000 Cells grown on coverslips were rinsed 3 times in PBS, fixed in fresh 4% (w/v) paraformaldehyde (Sigma-Aldrich) in PBS for 15 min, rinsed another 3 times in PBS, and then permeabilized and blocked with 0.3% Triton X-100 and 5% bovine serum albumin (BSA) in PBS for 30 min at room temperature. The cells were then incubated overnight with primary antibodies diluted in 0.15% Triton X-100 and 2.5% BSA in PBS at 4 °C. After being washed with 0.1% Triton X-100 in PBS for 10 min and rinsed twice in PBS, the cells were incubated for 1 h at 37 °C with Alexa Fluor® 488-conjugated secondary antibody (Invitrogen) or Alexa Fluor® 594-conjugated secondary antibody (Invitrogen) diluted in 0.15% Triton X-100 and 2.5% BSA in PBS; the cells were then washed with 0.1% Triton X-100 in PBS for 10 min and rinsed twice in PBS. DNA was stained for 5 min with 1 μg/mL 4′,6-diamidino-2-phenylindole (CST) diluted in PBS. Images were taken with a confocal fluorescence microscope.Protein preparation and quantification were performed as described above. Immunoprecipitation experiments were performed with protein A/G magnetic beads (Bimake) according to the manufacturer’s protocol. In brief, magnetic beads were precleared with lysis buffer and incubated with equal amounts of anti-CDK11 and normal rabbit IgG diluted in 50 μL lysis buffer, for 4 h at 4 °C in a rotator. Then, the beads were washed twice with 150 μL lysis buffer, and 2 mg protein was added to the magnetic beads and incubated for another hour at 4 °C in the rotator. The beads were washed four times with 300 μL lysis buffer and then resuspended in 50 μL protein loading buffer and denatured for 5 min by boiling. Protein analysis was performed by immunoblotting.Cells were seeded in 12-well plates. A scratch was made with a 10 μL pipette tip after cells had reached approximately 90% confluence. PBS was used to wash the cells 3 times to remove floating cells. Serum-free medium was then applied to the cell culture. Images were taken at 0 and 48 h after the scratch assay was performed. t-test, and the differences among 3 or more groups were analyzed with one-way analysis of variance (ANOVA). A Chi-squared (χ2) test was used to analyze the immunofluorescence overlap ratio. The significance level was set at P < 0.05.All data are presented as mean ± standard deviation (SD). The differences between two groups were analyzed with Student’sP < 0.05; Figure 1A). The optimal conditions for Wnt/β-catenin signaling activity in HEK293T cells were 400 ng/mL Wnt3a stimulation for 5 h. To further optimize the transfection time for the large-scale RNAi screen, we used siRNA against β-catenin as a positive control and compared the response of HEK293T cells cotransfected with luciferase reporter plasmids plus nontargeting siRNA or siRNA against β-catenin with or without Wnt3a treatment. The system was shown to be sensitive to β-catenin depletion after 48 h of transfection . Subsequently, a large-scale RNAi screen was performed after the siRNA library was cotransfected with luciferase reporter plasmids for 48 h with 400 ng/mL Wnt3a stimulation for 5 h.To identify kinases involved in Wnt/β-catenin signaling, we designed an approach to recapitulate Wnt/β-catenin signaling. In this assay, recombinant Wnt3a was used to activate Wnt/β-catenin signaling in HEK293T cells cotransfected with TOPFlash plasmid, a classical reporter plasmid for Wnt/β-catenin signaling, and pRL-TK plasmid, an internal control. The ratio of TOPFlash luciferase activity normalized to pRL-TK luciferase activity represented the relative activity of Wnt/β-catenin signaling. To determine the optimized concentration and time of Wnt3a stimulation, we tested different doses and time points of Wnt3a stimulation. Wnt3a strongly increased Wnt/β-catenin signaling activity in a dose- and time-dependent manner . To identify kinases involved in Wnt/β-catenin signaling, we determined the ratio of the relative luciferase activity of the targeting siRNA group to that of the nontargeting siRNA group, both of which received Wnt3a treatment, and converted the results to logarithm scale. Only the kinases that increased or decreased Wnt/β-catenin signaling activity by 2 SD for all siRNAs of the library were considered potentially positive hits . To verify the effectiveness of the primary screen, we chose 8 candidates for secondary screen to confirm their effects on Wnt/β-catenin signaling by the repeating luciferase activity measurements. The results were consistent with those after the first-round screen . Among the positive hits, CDK11 was one of the most effective and reproducible kinases found to regulate Wnt/β-catenin signaling .Under the above optimized conditions, we conducted kinase library screen with a library targeting 720 genes encoding kinases (4 siRNAs per gene as a pool) . Because Wnt/β-catenin signaling activity in untreated HEK293T cells is at a basal level, HeLa cells in which Wnt/β-catenin signaling was active were used in the rest of the assays unless otherwise indicated. As shown in the luciferase assay results, depletion of CDK11 also increased Wnt/β-catenin signaling activity in HeLa cells . In addition, knockdown of CDK11 increased the expression of total β-catenin and decreased the expression of phospho-β-catenin , a hallmark of Wnt/β-catenin signaling activation. Furthermore, we tested the effect of CDK11 depletion on the expression of the downstream target genes of Wnt/β-catenin signaling and found that the mRNA levels of Axin2 and c-myc were up-regulated under Wnt3a stimulation . Thus, our data indicate that CDK11 is a negative regulator of Wnt/β-catenin signaling.To confirm the modulation of CDK11 in Wnt/β-catenin signaling and eliminate possible off-target effects, we performed a CDK11 loss-of-function assay with 2 newly synthesized siRNAs whose target sequences were completely different from that of CDK11 in the kinase RNAi library. Knockdown of CDK11 significantly increased Wnt/β-catenin signaling activity in HEK293T cells , whereas the mRNA levels of LRP6, Dvl2, Axin1, and GSK3β did not change . When CDK11 was overexpressed, LRP6, pLRP6, Dvl2, and GSK3β decreased in a dose-dependent manner , thus further confirming that CDK11 expression affects the protein levels of Wnt/β-catenin signaling receptor complexes.To investigate the mechanism through which CDK11 depletion enhanced Wnt/β-catenin signaling, we assayed the expression of Wnt/β-catenin signaling cascade components after CDK11 depletion. Interestingly, among the signaling cascade components, the protein levels of LRP6, pLRP6, Dvl2, Axin1, and GSK3β, which are components of receptor complexes, were all significantly up-regulated in CDK11-depleted cells . When we measured the main components of the receptor complexes in those two cells, we found that down-regulation of CDK11 led to increased levels of LRP6, pLRP6, Dvl2, Axin1, and GSK3β in HCT116 cells, but we observed no changes in SW480 cells . These data suggest that CDK11 is involved in the regulation of receptor complexes of Wnt/β-catenin signaling in a manner depended on intact APC.In the Wnt/β-catenin signaling destruction complex, the tumor suppressor APC is an essential component. To explore whether APC might participate in the regulation of Wnt/β-catenin signaling after CDK11 depletion, we tested the effect of CDK11 depletion on Wnt/β-catenin signaling in two colorectal cancer cell lines: SW480 cells, whose APC was truncated, and HCT116 cells, whose APC was intact. Intriguingly, CDK11 depletion had no effect on Wnt/β-catenin signaling activity in SW480 cells, but Wnt/β-catenin activity was significantly enhanced in HCT116 cells . A confocal immunofluorescence assay showed that Dvl2 accumulated more in early endosomes in CDK11-depleted cells stimulated with Wnt3a  but less in lysosomes . Figure 4D and 4F shows the overlap coefficient of Dvl2 with EEA1 and LAMP1, respectively, in CDK11-depleted cells under Wnt3a stimulation (P < 0.05). Thus, our data suggest that CDK11 depletion enhances Wnt/β-catenin signaling by retaining the receptor complexes in early endosomes, where Wnt/β-catenin signaling is maintained.When Wnts bind their receptors, the receptor complexes are internalized, trafficked, and degraded through the endolysosomal system39, which in turn is required for transport from early endosomes to late endosomes or lysosomes42. We therefore wondered whether CDK11 may play a role in the intracellular trafficking of the receptor complexes between early endosomes and lysosomes via modulation of microtubule stability. First, we found that the expression of acetyl-α-tubulin, a marker of microtubule stability, decreased in CDK11-depleted cells, whereas total tubulin did not change . Further analysis of protein-protein interactions showed that CDK11 interacted with tubulin deacetylase SIRT2 but not with two other well-known enzymes, the tubulin deacetylase HDAC6 and the tubulin acetylase MEC-17 . Knockdown of SIRT2 reversed the expression of receptor complexes of Wnt/β-catenin signaling in CDK11-depleted cells . These data suggest that CDK11 may affect SIRT2 activity and consequently regulate tubulin stability, thereby playing a role in receptor complex trafficking of Wnt/β-catenin signaling.CDK11 has been suggested to be involved in microtubule stability43. To test whether CDK11 modulation of Wnt/β-catenin signaling might require an ESCRT-mediated mechanism, we knocked down HRS (ESCRT-0), TSG101 (ESCRT-I), EAP20 (ESCRT-II), and CHMP6 (ESCRT-III) individually in CDK11-depleted cells . As shown in Figure 5E to 5H, whereas CDK11 knockdown markedly increased the relative luciferase activity, the suppression of HRS, TSG101, EAP20, or CHMP6 significantly reversed the activity in CDK11-depleted cells (P < 0.05). Furthermore, Western blot analysis showed that expression of LRP6, pLRP6, Dvl2, and Axin1 decreased after TSG101 was perturbed in CDK11-depleted cells , thus suggesting that the negative regulation of CDK11 on Wnt/β-catenin signaling is dependent on the ESCRT machinery.The ESCRT complex comprises four distinct components  and has been reported to be involved in endosomal sortingP < 0.05; Figure 6A-D). The opposite effects were observed in CDK11-overexpressing cells, and overexpression of LRP6 reversed the modulation . We further analyzed the expression of N-cadherin, which is associated with cell migration; in agreement with the results of our scratch wound assays, N-cadherin was up-regulated in CDK11-depleted cells . The above data suggest that CDK11 plays a role in cell migration through the regulation of Wnt/β-catenin signaling.To explore the role of CDK11 in cell biology through modulation of Wnt/β-catenin signaling, we performed scratch wound assays, which indicated that the closure of wound gaps increased in CDK11-depleted cells, but down-regulation of LRP6 reversed the modulation , both of which can bind Wnts, thereby inhibiting interactions between Wnts and Wnt receptors12, whereas other studies have suggested that other receptors in early endosomes can signal17. Here, in CDK11-depleted cells, the receptor complexes of Wnt/β-catenin signaling accumulated in early endosomes and signaled persistently. This result is probably due to either concentration of active receptor complexes in endosomes or sequestration of GSK3β into endosomes, thus allowing more β-catenin to enter the nucleus and subsequently activate Wnt/β-catenin signaling48. Although the inadequacy of current technology precludes accurate measurement of signaling from endosomes, our data suggest a role of CDK11 in the negative regulation of Wnt/β-catenin signaling in the endosomal compartment.A key question regarding Wnt/β-catenin signaling is the location where the signaling of receptor complexes occurs in cells. A previous study has demonstrated that receptor complexes as a whole can signal but do not colocalize with early endosomes49, and microtubule stability is required for transport from early endosomes to late endosomes or lysosomes42. Whether active microtubule-based transport might play a role in Wnt/β-catenin signaling remains a matter of speculation. Here, we showed that CDK11 plays a role in the transport of Wnt/β-catenin signaling receptor complexes through the endolysosomal system, on the basis of the regulation of microtubule stability . Accumulation of receptor complexes in Wnt/β-catenin signaling after CDK11 depletion was not observed in SW480 cells, thus suggesting that intact APC may play an important role in the recruitment of Axin1 and GSK3β as a whole after Wnt3a binds Fzd and LRP6—a possibility requiring further research.Intracellular trafficking of receptors between endosomes has been shown to be dependent on microtubules50, multiple myeloma51, osteosarcoma52, and esophageal squamous cell carcinoma53, and down-regulation of CDK11 arrests growth and induces apoptosis in these cancer cells, thus suggesting that CDK11 acts as an oncogene. However, CDK11 has also been found to be depleted in several cancers, such as neuroblastoma54, melanoma55, and non-Hodgkin’s lymphoma56. These results imply that CDK11 may be a tumor suppressor gene. In this study, our data suggest that CDK11 may function as a tumor suppressor by deregulating Wnt/β-catenin signaling, at least in cervical cancer cells.CDK11’s functions are particularly versatile among those of CDK family members. Previous studies have found that CDK11 is up-regulated in breast cancerIn summary, we conducted a kinase RNAi screen to identify kinases regulating Wnt/β-catenin signaling, from which we identified CDK11 as a negative regulator. In the underlying regulatory mechanism, Wnt/β-catenin signaling receptor complexes are destabilized, and cellular trafficking of signal molecules to lysosomes for degradation is increased via CDK11 regulation. The details of the association of CDK11 with Wnt/β-catenin signaling in the endosome and lysosome trafficking machinery remain to be further elucidated.