Microneedles (MNs) are tiny needle like structures used in drug delivery through layers of the skin. They are non-invasive and are associated with significantly less or no pain at the site of administration to the skin. MNs are excellent in delivering both small and large molecules to the subjects in need thereof. There exist several strategies for drug delivery using MNs, wherein each strategy has its pros and cons. Research in this domain lead to product development and commercialization for clinical use. Additionally, several MN-based products are undergoing clinical trials to evaluate its safety, efficacy, and tolerability. The present review begins by providing bird’s-eye view about the general characteristics of MNs followed by providing recent updates in the treatment of cancer using MNs. Particularly, we provide an overview of various aspects namely: anti-cancerous MNs that work based on sensor technology, MNs for treatment of breast cancer, skin carcinoma, prostate cancer, and MNs fabricated by additive manufacturing or 3 dimensional printing for treatment of cancer. Further, the review also provides limitations, safety concerns, and latest updates about the clinical trials on MNs for the treatment of cancer. Furthermore, we also provide a regulatory overview from the “United States Food and Drug Administration” about MNs. Drug delivery via the oral route is the most preferred route of drug administration due to its simplicity and cost-effectiveness when compared to other routes ,2. NeverCancer is a disease that is resulting due to changes in cellular level leading to uncontrollable growth and division of the cells. Its incidence is augmenting over the last few decades . StandarFor the sake of clarity and brevity, the present review begins by providing general characteristics of MNs, wherein we highlight the information pertinent to conception of MNs, different approaches or strategies employed in drug delivery, the manufacturing methods, materials, clinical applications , design and geometry. The review continues by providing recent updates about the role of MNs as a carrier in delivering anti-cancer drug substances. While there are several developments in the domain of MNs in treatment of cancer, but this review is focused on discussing about sensor technology based MNs, MNs for treatment of cancer  and fabricating MNs by additive manufacturing or 3 dimensional printing (3DP). The review also provides limitations, safety concerns, and updates about the ongoing clinical trials of MNs for the treatment of cancer. Lastly, it discusses regulatory (USFDA) considerations on MNs.Based on our literature review, we found two US patents having inventors ‘Harvey Kravitz’ and ‘Norman Letvinn’ who were the first to conceive and reduce to practice the concept of MNs ,37. ThisThere exist different approaches/strategies for drug delivery using MNs , refer FTurning now to poke and release approach, wherein the MNs tend to dissolve after insertion, refer Turning now to MNs manufacturing methods, the most conventional methods of manufacturing include solvent casting, micro-molding, pulling pipettes, etching, lithography, ceramic sintering, laser cutting/ablation, electropolishing, and micro stereolithography . VariousAs regards the materials employed in fabricating MNs, Guillot and colleagues identified various materials and methods used in the fabrication of MNs, they also provided the advantages and disadvantages of the respective methods . For insBased on our review, it appears that the clinical applications of MNs would not only depend on the ability of MNs array or patch to perform the intended function but also its acceptance across the board by the healthcare professionals. Several exploratory studies about end-user acceptance and views of healthcare professionals were reported in the past. Some of the benefits reported by the end-users about MNs technology include but not limiting to ease in administration (self-administration), less pain and tissue trauma for children. Nonetheless, few healthcare professionals raised concerns via-a-vis the dose, delayed onset, allergic reaction at the site of administration, and its potential for abuse by some subjects ,70. FurtOne of the important clinical benefits of MNs is less or no pain at the site of insertion. For instance, Kaushik et al. carried out early trials to understand the painless insertion of MNs and reported that pain associated with MNs  based drug delivery was statistically less when compared with that of the hypodermic needle . SecondlInsertion of MNs on the surface of skin and formation of pores pose a threat of microbial infection leading to local or systemic infections. Therefore, traversing the pores generated post insertion of MNs on the surface of skin is vital and this aspect was studied by Donnelly and colleagues using porcine skin and reported that pores formed due to MNs are less permeable to microbial penetration when compared with pores formed by hypodermic needle injections . NonetheAnother risk in employing MNs based drug delivery system is skin irritation, which is generally transient in nature. Even repeated application of MNs is found to be safe . Yet anoThe above aspect is explained using ‘Psoriasis’—a skin disease. It is a type of skin disease wherein the subject develops red, itchy, and scaly patches . ExistinDesign and geometry . As regards the colorimetric MNs sensor, the enzyme glucose oxidase is used for analyte recognition and catalysis to release hydrogen peroxide. The hydrogen peroxide induced color change of a substrate is used to determine the concentration of the analyte . SimilarSome of the MNs that work based on sensor technology are discussed below and the same are illustrated in In one of the studies, wearable orthogonal MNs that work based on sensor technology were successfully developed by Goud and colleagues for monitoring levodopa levels in the interstitial fluid of patients with Parkinson’s disease. This helps in proper optimization of the dose of the drug, levodopa . Hahn anTurning now to other sensing mechanisms, ‘The Regents of the University of California’, North Carolina State University along with Sandia Corporation developed MNs that works based on biosensing mechanism to deliver the drug. The disclosed MNs are pictorially depicted in Figure 3b, (adapted from respective reference) wherein the MNs make use of probes that are used for sensing the signal and which in turn are connected to electrically conducting wires for transmitting the detected signal generated by the probes. Interestingly, these MNs could be used for the simultaneous detection of “glucose, lactic acid, and pH”. These parameters are important to determine malignant skin cells because in cancer the lactate levels increase while glucose levels and pH decrease. Therefore, the proposed MNs help in determining malignant skin cells in the body . In anotFor instance, Jain and team reported dip-coated MNs (57 in number) of 5-Aminolevulinic acid (5-ALA) for ‘photodynamic therapy’ (PDT) of various skin related carcinoma . Donnell5-ALA has an inherent natural property wherein it converts to a ‘photosensitizer called protoporphyrin IX (PPIX)’, refer Figure 3c, inside the cells through various transformations in the mitochondria, which undergoes chelation with iron to produce heme in the presence of an enzyme ferrochelatase .Combination cancer therapy  also helps in achieving desired efficacy in treatment or eradication of cancer . PolyvinFrom the above discussion, it can be inferred that different analytes could be targeted for microneedle-based cancer detection. For instance, the target analytes include but not limiting to glucose, glutamate, lactate, hydrogen peroxide, nitric oxide, potassium levels, and T cell. Additionally, determining the pH of the tumor environment is also important in cancer detection using MNs. A table depicting different types of analytes relied on detecting cancer, the principle involved in the detection and the test subjects on which the tests were carried out are provided in After skin cancer, the second and most common cancer that causes death in women is breast cancer . Some ofAs regards the biology, breast cancer originates in the ducts of mammary glands that help in carrying milk from the lobules to the nipple. Drug delivery to the underlying tissues (tumors) in the breast helps in minimizing its blood concentration and thereby protecting the healthy cells and adverse effects . The keyBhatnagar and colleagues reported MNs of zein (prolamine protein from corn) for delivery of tamoxifen and gemcitabine that are used in the treatment of breast cancer. Zein MNs were prepared by micromolding technique through solvent casting, wherein it involved 3D printing of master mold using a polymer, acrylobutyl nitrile styrene, and plasticizers namely glycerol and PEG 400. Both tamoxifen and gemcitabine were encapsulated in zein MNs. Alternately, both the anti-breast cancer drugs were also coated on zein MNs using rhodamine as a dye to examine the quality of coatings. The studies on loading efficiency revealed that more tamoxifen was loaded onto zein MNs when compared to gemcitabine. As regards the release kinetic studies, gemcitabine showed more permeation vis-a-vis tamoxifen. Finally, the study concluded that optimal solubility of a drug in water may exhibit greater permeation when delivered using zein MNs . In anotHonokiol”, refer Magnolia glandiflora.Gao and co-workers reported transdermal delivery of a biphenyl natural anti-cancer compound, “Magnolia glandiflora is indeed associated with various anti-cancer properties; breast [Reports also suggest that ; breast  prostate; breast  liver [1; breast . MNs mad; breast .® 1200) to deliver particulate (1.5 µm size) breast cancer vaccine prepared by freeze-drying method. They used whole-cell lysate as a source of antigens. The micro channels (50 ± 10 µm) formed using MNs were microscopically visualized using methylene blue as a staining agent. Based on the in vivo studies, it is evident that the overall approach was found to be a very promising means of immunization against breast cancer [Chablani and colleagues used metallic MNs  either alone or in combination with anti-CTLA4 antibody (ipilimumab). The proposed MN patch comprises a base end and a tip coated with nanoparticles that encapsulate the immunotherapeutic agent (PD1 antibody and/or anti-CTLA4 antibody) along with glucose oxidase as a pH altering agent. The nanoparticles prepared were acid-degradable and are prepared using dextran and sodium alginate as a surfactant. In some embodiments, Zhen Gu and colleagues used hyaluronic acid in the preparation of MN patches, which helps in lubricating the layers of the skin when the patch is inserted ,159. Zyn15-GRKKRRQRRR”. The invention also disclosed the use of high molecular weight biologically active agents such as infliximab, golimumab, adalimumab, certolizumabpegol, siplizumab, and others either alone or in combination with one another [Eirion therapeutics filed two international patent applications on transdermal delivery of large molecular weight molecules that are used in the treatment of skin cancer. The invention claimed a method for delivering an emulsion formulation of botulinum toxin  with a biologically active agent . The method involved steps of skin conditioning using MNs followed by the application of the formulation. They have basically combined the microneedle technology with emulsion technology (water-in-oil and oil-in-water nanoemulsions) to deliver botulinum toxin, a large molecular agent. The emulsion formulation also comprised non-irritating and penetration enhancing agents such as “cationic peptide” and a positively charged carrier having the sequence “RKKRRQRRRG-(K) another ,162.N-methylsulfamoylphenyl bacteriochlorin”, a preformed photosensitizer (near-IR). The studies revealed that the MNs had good mechanical strength for insertion into the layers of the skin, 5mm down the skin for treatment of nodular skin carcinoma. Thus, the study suggested usage of polymeric  MNs in photodynamic therapy of skin lesions that are deep [Lu and co-workers reported dacarbazine loaded MNs for skin carcinoma. The MNs were prepared by micro stereolithography using poly (propylene Fumarate) as a polymer and diethyl fumarate was added to control the viscosity of the polymer. The prepared MNs have a cylindrical base and a conical tip with excellent skin insertion force. Dacarbazine was released in a controlled fashion for the above five weeks . Hamdan are deep . Al-mayaare deep . In anotare deep .Inventors from the University of Pittsburgh and Carnegie Mellon University disclosed carboxymethyl cellulose (CMC) MNs that can deliver various chemotherapeutic and immune-stimulating agents (Toll-like Receptor (TLR) antagonist) either alone or in combination. For instance, the chemotherapeutic agent was selected from a group comprising of doxorubicin, valrubicin, epirubicin, idarubicin, and other known anthracycline agents for treatment of skin cancer. The MNs were prepared using CMC, wherein the drugs are deposited by micro milling method in the form of drug cargo layers, as shown in The second most common cancer in men after lung cancer is Prostate Cancer (PC) . It is cAnti-cancer vaccines, such as PC DNA vaccines, are other alternatives that can be employed to target cells that are expressing immunogenic tumor-associated antigens (TAAs). Nonetheless, recent clinical trial results on PROSTVAC raised concerns about the efficacy of DNA vaccine alone . The effFor ADT, Chen and colleagues reported transdermal delivery of goserelin (luteinizing hormone-releasing hormone (LHRH) analogue) using dissolvable polymeric MNs of Chitosan and polyvinyl alcohol/polyvinyl pyrrolidone base as a support. The LHRH per each microneedle was 73.3 ± 2.8 µg . The porVery recently, Cole and colleagues reported for the first time a robust two-tier microneedle-based platform for PC DNA vaccine delivery. In its study, RALA/pDNA nanoparticle based dissolving polymeric microneedle system was developed to immunize mice encoded with mPSCA (Prostate Stem Cell Antigen). The results of the study showed production of encoded PSCA. Additionally, immunization using RALA/pPSCA based MNs exhibited anti-tumor activity (inhibited TRAMP-C1 tumors) in the PC models .The traditional manufacturing methods and materials employed in fabrication of MNs pose different challenges that hamper its adoption for TDDS. For instance, the micro-molding method used for fabricating gel-forming and dissolving MNs is a time consuming and tedious process that often poses challenges with respect production scale-up. Additionally, the MNs drug coating technologies suffer with non-uniformity, lack of accuracy and uncertainty about the amount of drug that is loaded or coated on the MNs. Further, the materials used in fabrication of MNs are basically approved for oral use but majority of them are being utilized to fabricate MNs for drug delivery via the transdermal route. This leads to the problem of biocompatibility leading to irritation at the site of administration. One alternate and upcoming method of manufacturing MNs is additive manufacturing or 3-dimensional printing (3DP). The first three dimensional printed MNs for wound healing were reported by Gittard and colleagues, wherein they employed 3DP technique called steriolithography (SLA) to fabricate MNs followed by coating them with zinc and silver oxide . In anot® was used as a hydrophilic graft copolymer [Uddin and colleagues reported coated MNs of 5-fluorouracil, curcumin, and cisplatin, prepared by inkjet printing technology. The formulation used for coating comprised of a drug-polymer at different ratios and Soluplusopolymer . In anotopolymer .In short, employing 3DP technologies in fabricating MNs has a strong potential in printing complex MNs that need to be organized in a systematic fashion. The beauty with this technique is that the product is printed layer-by-layer . Such prMany CTs on MN-based drug delivery systems are carried out across the globe. The present review is focused on providing details about MN-based drugs that are used in the treatment of cancer. Reported below are studies that are recruiting patients/subjects and active studies but not recruiting subjects.The University of Pittsburgh is recruiting subjects for its ongoing study to test the safety and effectiveness of microneedle applicator of doxorubicin, a chemotherapeutic agent . DoxorubTM. It makes use of electrically conducting microneedles to apply nano electric pulses at targeted tissue and is aimed to study the effect in benign skin tumors [Pulse biosciences, a US based company has an ongoing active clinical study on its proprietary platform technology branded as Nano-Pulse Simulationn tumors ,183. Nonn tumors .Despite significant research in the domain of MNs, it still suffers from different challenges that are to be considered while deciding to use them as carriers for anti-cancer drug delivery. They are preferred for anti-cancer drugs that have low dose. Nonetheless, hydrogel-forming and hollow MNs could be used to deliver small or large molecules with high dose MNs that are to be used repeatedly are generally restricted to avoid safety concerns ,184. TheGenerally speaking, depending on the duration of use of MNs they are associated with three safety concerns namely pain at the site of administration when used repeatedly for a long time, infection and reaction of the skin after inserting MNs. Pain depends on the duration of MNs use and also the number of needles in the MNs patch. For instance, Gill et al. reported less pain than the pain caused by a hypodermic needle with a gauge of 26, this study also provided correlation between the level of pain and number of needles and its length ,187.Infections could be due to entry of microorganisms via the micro-channels after insertion of MNs. The probability of such entry is less but it cannot be ruled out. Therefore, administration of MNs shall be done carefully to the subject to prevent such infections. Additionally, the length of the needle also plays a pivotal role in preventing/controlling the infections, if any, after inserting MNs .Skin reactions such as erythema (redness of skin), inflammation, and irritation at the site of administration are observed with MNs made of materials such as glass and ceramics ,189,190.MNs are emerging drug delivery systems that are associated with unique advantages as depicted in


Here, we found that STK4 was significantly downregulated in colon cancer and was associated with distal metastasis and poor survival. STK4 colocalized with β‐catenin and directly phosphorylated β‐catenin resulting in its degradation via the ubiquitin‐mediated pathway. This may suggest that STK4 knockdown causes β‐catenin phosphorylation failure and subsequently β‐catenin accumulation, consequently leading to anchorage‐independent growth and metastasis in colon cancer. Drosophila Hippo. STK4 plays an important role in controlling cell growth, apoptosis, and organ size. STK4 has been studied in many cancers with previous studies indicating an involvement in colon cancer lymph node metastasis and highlighting its potential as a diagnostic marker for colon cancer. However, the role of STK4 defect in promoting colon cancer progression is still understudied. Here, we found that STK4 was significantly downregulated in colon cancer and was associated with distal metastasis and poor survival. Furthermore, STK4 knockdown enhanced sphere formation and metastasis in vitro and promoted tumor development in vivo. We found that STK4 colocalized with β‐catenin and directly phosphorylated β‐catenin resulting in its degradation via the ubiquitin‐mediated pathway. This may suggest that STK4 knockdown causes β‐catenin phosphorylation failure and subsequently β‐catenin accumulation, consequently leading to anchorage‐independent growth and metastasis in colon cancer. Our results support that STK4 may act as a potential candidate for the assessment of β‐catenin‐mediated colon cancer prognosis.Mammalian STE20‐like kinase 1 (MST1/STK4/KRS2) encodes a serine/threonine kinase that is the mammalian homolog of  GAPDHglyceraldehyde 3‐phosphate dehydrogenaseH&Ehematoxylin and eosinHCChepatic cell carcinomaIHCimmunohistochemistryPLAproximity ligation assayqRT–PCRquantitative real‐time polymerase chain reactionScrscramble shRNASTK4serine/threonine kinase 4TMAtissue microarray1Colon cancer is the fourth common cancer in the world . Colon cDrosophila Hippo  ATP. Reactions were stopped by the addition of 2× sample buffer and heating to 100 °C. Reaction products were visualized by SDS/PAGE followed by autoradiography or subjected to western blotting analysis.Cells were transfected with wild‐type STK4 or STK4 mutant plasmids for 24 h to collect total cell lysate. Twenty microlitre of immobilized β‐catenin antibody bead slurry was added to 200 μL cell lysate, incubated with gentle rocking overnight at 4 °C, and then the pellet was washed two times with cell lysis buffer. 2.15The luciferase activity was measured as described previously . Cells w2.16prism5 and spss17 software . Disease‐free survival rate and overall survival rate were estimated using Kaplan–Meier methods and compared among age groups using log‐ranking tests. Cox proportional hazards regression models with univariate and multivariate analyses were summarized with hazard ratios and 95% confidence intervals. Data from all three independent experiments were presented as the mean ± SD and analyzed by unpaired two‐tailed Student's t‐test. For all tests, P < 0.05 was used to define statistical significance .The statistical analysis was performed using 33.1P < 0.01) , distal metastasis , and disease recurrence . To validate whether the expression level of STK4 in colon can use as an early diagnosis marker, tissues of 93 patients with stage I colon cancer were collected and IHC was performed. The results indicated that STK4 highly expressed in nontumor sections, but low or no expression of STK4 was found in the benign or tumor tissues (P < 0.001)  Fig. . Taken t3.2STK4‐shRNAs were established. STK4 knockdown in H3347 cells significantly increased migration (P < 0.001) and invasion (P < 0.01)  and invasion (P < 0.05) . Hematoxylin and eosin (H&E) staining and IHC were performed to confirm the distal metastatic cells derived from human colon cancer cells. The expression of human mitochondria was detected in cecum, liver, and lung tissue sections in the STK4‐shRNA group. In the scramble control group, the tumor cells were only observed in the orthotopic tissues . There was no significant difference in cell proliferation rate after manipulating STK4 expression levels in colon cancer cells , and ues Fig. . Intrasplls Fig. , which w3.3STK4‐shRNAs were used to evaluate tumor initiation ability. Figure 3 cells injected mice was increased in the STK4‐shRNA group (90%) compared with the control group (40%). STK4 downregulation significantly increased sphere‐forming ability in HT29 and CX‐1 cells  and Nestin in the STK4‐shRNA group were increased compared with the control group. The drug‐resistant related genes (ABCB1 and ABCG2) showed no difference after STK4 downregulation. Two ways of demonstrations by overexpression and knockdown of STK4 in colon cancer cells can conclude that STK4 levels were inversely correlated with CD133 expression , and immunoprecipitation assay were performed in colon cancer cells expressing Flag (vector control) and Flag‐STK4. Figure To investigate whether STK4 interacted with β‐catenin in vitro kinase assay was performed in cells expressing STK4 or mutant‐STK4 (K59R). The results showed that phosphorylated β‐catenin signal in the mutant‐STK4 group was decreased . STK4 overexpression in LS174t cells reduced β‐catenin transcriptional activity (P < 0.01). Furthermore, STK4‐K59R significantly increased β‐catenin transcriptional activity compared with the control group (P < 0.01). Thus, STK4 enhanced β‐catenin/TCF‐mediated transcriptional activity.To examine whether STK4 knockdown affected β‐catenin‐mediated transcriptional activity, the TOP luciferin activity assay was performed in colon cancer cells. Figure 3.5in vivo, mice colons were injected with human colon cancer cells expressing Scr or STK4‐shRNA followed by immunofluorescence analysis to detect the expression of β‐catenin. Figure In vitro and in vivo studies revealed that STK4 downregulation enhanced metastasis ability and increased β‐catenin expression. In addition, STK4 interacted with β‐catenin and directly phosphorylated β‐catenin that led to its degradation via ubiquitin‐mediated pathway (Fig. To evaluate the expression of β‐catenin in colon cancer patients, IHC was performed. Figure way Fig. .4Previous studies have reported that STK4 has tumor‐suppressive ability , 25, 27.Metastasis remains the main factor of mortality in patients with colon cancer , 3 and iPrevious studies have reported that β‐catenin overexpression or downregulation regulates anchorage‐independent cell growth , 41. OurGene therapy is an experimental strategy for colon cancer therapy with high efficiency . STK4 ac5In conclusion, STK4 plays an important role through interaction with β‐catenin in colon cancer disease models. Our study has provided insights into the role of STK4 in β‐catenin‐mediated colon cancer. STK4 downregulation enhances tumor growth of colon cancer through blocking β‐catenin degradation. Thus, we propose a mechanism how STK4 regulates β‐catenin degradation between normal cells and cancer cells.The authors declare no conflict of interest.C‐HL, T‐IH, P‐YC, and P‐JL designed experiments. C‐HL, T‐IH, and P‐YC performed experiments and data analyses. C‐HL, T‐IH, MH, and P‐JL interpreted the data. W‐CW, Y‐CC, J‐TL, J‐YW, P‐CL, F‐CL, Y‐KT, and C‐LC collected clinical samples and analyzed the clinical data with software. H‐CC assisted with some of the experiments. C‐HL, T‐IH, P‐YC, and P‐JL wrote the manuscript. All authors discussed the results and contributed to the manuscript.Fig. S1. Quantification of STK4 expression in non‐tumor and tumor areas of patients with different cancers.Click here for additional data file.Fig. S2. STK4 is highly expressed in the normal tissue but show lower or no expression in tumor tissue in early stage I colon cancer patients.Click here for additional data file.Fig. S3. The effect of STK4 expression in cell proliferation of colon cancer cells.Click here for additional data file.Table S1. List of antibodies used in the study.Table S2. Sequences of primers sets.Click here for additional data file.


Angelica gigas Nakai (AGN) has been reported to possess anti-obesity effects and higher amounts of coumarin compounds are present in AGN. However, the active compounds suppressing adipogenesis in AGN and mechanisms of action have not been investigated in adipose-derived stem cells (ASCs) isolated from visceral adipose tissue (VAT). Among four coumarin compounds of AGN, decursin (D) and decursinol angelate (DA) significantly inhibited adipocyte differentiation from ASCs. D and DA downregulated CCAAT/enhancer binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid binding protein (aP2), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) at both mRNA and protein levels. Next, treatment with adipogenic differentiation medium (ADM) on ASCs downregulated β-catenin expression at protein level, while addition of D and DA could restore protein expression and nuclear translocation of β-catenin suppressed by ADM. D and DA treatment on ADM treated ASCs increased inhibitory phosphorylation of Glycogen synthase kinase (GSK)-3β, thereby preventing β-catenin from degradation. Additionally, si-β-catenin transfection significantly upregulated protein expression of C/EBPα and PPARγ, alleviating the anti-adipogenic effect of D and DA on ADM treated ASCs. Overall, D and DA, active compounds from AGN, suppressed adipogenesis through activation of β-catenin signaling pathway in ASCs derived from human VAT, possibly using as natural anti-visceral adiposity agents.Visceral adiposity is closely associated with metabolic disorders and cardiovascular diseases.  Obesity is excessive fat deposition and has a profound impact on quality of life. Specifically, visceral obesity characterized by high visceral adipose tissue (VAT) distribution in intra-abdominal, associated with a high risk of metabolic disorders including type 2 diabetes, hypertension, and atherosclerosis ,2. ObesiAdipocyte differentiation from ASCs is regulated by key adipogenic transcription factors, including members of the CCAAT/enhancer binding protein (C/EBP) family and peroxisome proliferator-activated receptor γ (PPAR-γ) ,6. TheseAngelica gigas Nakai (AGN) is also known as ‘Korean dang-gui’. Higher amounts of coumarin compounds such as decursin (D), decursinol angelate (DA), nodakenin (ND) and 7-dimethyl suberosin (DS) are present in AGN than in other Angelica species such as Angelica sinesis (Chinese dang-gui) and Aneglica acutiloba (Japanese dang-gui) [ang-gui) . HoweverHerein, we aimed to investigate anti-adipogenic effect of the active compounds present in AGN and their mechanisms of action using ASCs isolated from human VAT. Our findings show that D and DA, the active compounds of AGN, suppress adipogenesis of ASCs through activation of β-catenin signaling pathway.D and DA were purchased from Biopurify Phytochemicals Ltd.  with 98% purity. The chemical structure of D and DA is shown in n = 5) receiving gynecologic surgery. Clinical information regarding these patients is shown in g for 4 min. After centrifugation, stromal vascular fraction (SVF) pellet was collected. The SVF was filtered using MACS through negative selection of CD31  and CD45 (hematopoietic stem cell marker). The CD31 negative and CD45 negative SVF was plated onto 100 mm culture dish in MesenPRO RS medium containing 1% glutaMAX and 1% penicillin-streptomycin. After 3 days, non-adherent cells were removed by washing with PBS. ASCs were obtained by expansion of the adherent cells and they were passaged at least two times prior to experimental use. ASCs at passages 3–6 were used for experiments.VAT  samples were obtained from retroperitoneal fat tissue of human donors  which is DMEM F-12 supplemented with 10% FBS, 1% PS, 10 μg/mL insulin, 0.5 mM IBMX, 50 μM indomethacin, and 1 μM dexamethasone. The medium was changed every second day and the cells were cultured for 14 days. Differentiation of ASCs into adipogenic lineage was examined by Oil Red O (ORO) staining. Briefly, differentiated adipocytes were washed with PBS, and then fixed in 4% PFA for 30 min. The fixed cells were then washed with deionized water, and 60% saturated ORO staining was carried out for 1 h. For ORO quantification, isopropanol was added to each well. Light absorbance was measured at 495 nm.ASCs were seeded onto 24-well plates at 0.015 × 10To analyze the intracellular content of cholesterol, the cells were washed with PBS, the neutral lipids were extracted by 1 mL of mixture of chloroform and methanol (3:2) for 30 min. The lipids were dried in the fume hood. Cholesterol content was determined using Amplex red cholesterol assay kit according to the manufacturer’s protocol. Intracellular triglyceride was measured in cell lysates by a colorimetric triglyceride quantification assay kit according to the manufacturer’s instruction.Total RNA was extracted from ASCs using Trizol reagent and RNA concentration was determined by Nanodrop . The cDNA was obtained by reverse transcription containing 1 μg of total RNA, oligo (dT), and reverse transcription premix. The qRT-PCR reactions were performed with the SYBR green PCR system  in a thermal cycler . The cycling conditions were as follows: 95°C for 10 min; followed by 40 cycles involving denaturing at 95 °C for 5 s, annealing at 60 °C for 15 s, and extension at 72 °C for 10 s. Expression of mRNAs was normalized by the mRNA levels of GAPDH. The following forward and reverse primer sequences were used: C/EBPα, 5′-GCAAACTCACCGCTCCAATG-3′,5′-CTTCTCTCATGGGGGTCTGC-3′; PPARγ:5′-AGGTCAGCGGACTCTGGATTC-3′,5′-AGTGGGGATGTCTCATAATG-3′; aP2, 5′-ATGGGGGTGTCCTGGTACAT-3′, 5′-ACGTCCCTTGGCTTATGCTC-3′; LPL: 5′-CGAGCGCTCCATTCATCTCT-3′, 5′-CCAGATTGTTGCAGCGGTTC-3′; FAS, 5′-AAGGACCTGTCTAGGTTTGATGC-3′, 5′-TGGCTTCATAGGTGACTTCCA-3′, ACC, 5′-CTTGAGGGCTAGGTCTTTCTGG-3′, 5′-CTGGTTCAGCTCCAGAGGTT-3′, SREBP-1c, 5′-GCCCCTGTAACGACCACTG-3′, 5′-CAGCGAGTCTGCCTTGATG-3′, GAPDH, 5′-GAGTCAACGGATTTGGTCGT-3′,5′-TTGATTTTGGAGGGATCTCG-3′.ASCs were lysed with lysis buffer supplemented with 1% triton X-100, EDTA free protease inhibitor cocktail, Na3VO4, phenyl methyl sulfonyl fluoride, and sodium deoxycholate. Nuclear and cytoplasmic fraction were prepared according to the manufacturer procedure of NE-PER nuclear and cytoplasmic extraction reagents. Protein concentrations were measured using BCA protein assay kit. Protein samples (5 μg per well) in each sample were separated by 9% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and incubated with primary antibodies (1:1000 dilution) overnight at 4 °C. Immunoblot analyses were performed using the antibodies. The membrane was incubated with peroxidase-conjugated secondary antibody (1:5000 dilution). Target proteins were detected using a western blot detection kit.6 cells per well. After 3 days incubation, ASCs were cultured in ADM with or without D and DA treatment for additional 2 days. The cells were subsequently washed and fixed with 4% PFA solution for 20 min. Cells were permeabilized with 0.25% Triton X-100 for 20 min and blocked with 10% normal goat serum control in PBS/T for 1 h. The cells were incubated with β-catenin antibody overnight at 4 °C and then washed with 1% goat serum in PBS/T three times. Cells were then incubated with Alexa Fluor 488-conjugated IgG secondary antibody in 10% goat serum with PBS/T for 1 h and washed with 1% goat serum in PBS/T three times at dark. A drop of mounting solution with DAPI was added to each slide. After mounting, the fluorescence signal was captured under confocal microscopy . To quantify nuclear β-catenin signals per cell, the number of cells with β-catenin signal in nucleus (n = 90–100) in each group of samples in two independent experiments was measured.ASCs cells were seeded on four-well chamber slides at a density of 0.01 × 106 cells per well. The transfection was conducted in Opti-MEM and performed as described in instruction of lipofectamineTM 2000 transfection reagent. ASCs were transfected with 50 nM of β-catenin or negative control siRNAs. siRNAs were synthesized by Genolution (Korea). The following forward and reverse primer sequence were used: 5′-GUGCUAUCUGUCUGCUCUADTDT-3′,5′-UAGAGCAGACAGAUAGCACDTDT -3′. After 8 h of transfection, the medium was changed and the cells were incubated for another 2 days to reach at confluence. The transfected cells were cultured in ADM with or without D and DA treatment.ASCs were seeded in 6-well plates at a density of 0.04 × 10p < 0.05.Values are expressed as mean ± SD of three independent experiments. For multiple comparisons, analysis of variance (ANOVA) was used followed by Tuckey’s test. Statistical analysis was performed with SPSS (version 23) program. Differences are regarded significant if the value of To evaluate the anti-adipogenic effect of active compounds from AGN, ASCs were differentiated with ADM in the presence of coumarin compounds  from AGN at 40 μM for 14 days a. CytotoNext, we conducted further experiments with D and DA concentration fixed at 40 μM to find molecular signaling responsible for anti-adipogenic effect. To determine whether D and DA affect the expression of adipogenic transcription factors, we performed qRT-PCR 3 days after adipogenic induction with ADM treatment. The mRNA levels of adipogenic genes, such as C/EBPα, PPARγ, aP2, and lipoprotein lipase (LPL) were significantly increased by ADM exposure. However, upregulated the adipogenic genes with ADM treatment were significantly suppressed by addition of D and DA to ADM a–d. To eWe explored the mechanism of anti-adipogenesis by D and DA. Recently, reduction in β-catenin content in ASCs has been shown to facilitate adipocyte differentiation . So, we To investigate the upstream regulator of β-catenin during adipogenesis of ASCs, ERK 1/2 expressions were analyzed. Treatment with ADM on ASCs increased inhibitory phosphorylation of GSK-3β at S9 residue . In ASCsTo further confirm whether inhibition of adipogenesis by D and DA is mediated via activation of β-catenin, we conducted knockdown of β-catenin using si-RNA in ASCs. Transfection with si-β-catenin significantly downregulated β-catenin expression. Transfection with si-β-catenin into ASCs suppressed β-catenin expression induced by treatment with D and DA. In addition, C/EBPα and PPARγ have been reported as the downstream targets of β-catenin signaling in 3T3-L1 murine preadipocytes ,15. TherWe demonstrate for the first time anti-adipogenic effect of D and DA, active compounds from AGN, through activation of β-catenin signaling pathway in human visceral ASCs. AGN has been shown to reduce body weight significantly in the high-fat diet (HFD) mouse model . AlthougThe formation of adipose tissue from adipocytes in our body is important in maintaining metabolism. However, excessive accumulation of VAT is associated with incidence of chronic non-communicable diseases such as cardiovascular disease and cancer and contributes to induction of inflammation, hypoxia, fibrosis, and necrosis ,18,19. Sp > 0.05). Interestingly, the other ASCs showed significantly higher ORO-staining intensity than ASCs #20 (ASCs used in this study had a different adipogenic potential. ADM-treated ASC #20 showed 1.32-fold higher ORO-staining intensity than the undifferentiated parental ASCs (ASCs #20 . TherefoD and DA inhibited differentiation of ASCs into adipocytes in our study, verified by ORO-staining of lipid contents in adipocytes. However, there have been contradictory results about the effect of AGN on adipogenesis. Previous study showed that AGN promoted adipocyte differentiation through activation of insulin signaling pathway in 3T3-L1 murine preadipocytes . This coD and DA increased nuclear translocation of β-catenin, together with inhibition of adipogenic differentiation of ASCs. Recent studies highlighted the involvement of β-catenin signaling during adipogenesis ,24. ThesIn the absence of Wnt signal, β-catenin phosphorylated by GSK-3β undergoes proteasomal degradation. Activation of Wnt signaling leads to inhibitory phosphorylation of GSK-3β at S9 followed by increased nuclear translocation of non-phosphorylated β-catenin . ConsistNext, we explored signal upstream of GSK-3β. Activation of ERK and AKT could affect activation status of GSK-3β . During We then conducted experiments to see if inhibition of adipogenesis by D and DA is mediated through activation of β-catenin in ASCs. D and DA induced upregulation of C/EBPα and PPARγ was suppressed by si-β-catenin, suggesting that β-catenin signaling could play a critical role in the anti-adipogenic effect of D and DA . Our resAlso, microenvironmental factors of ASCs may affect the adipogenesis. Specially, inflammatory microenvironment from adipocytes have emerged as an important contributor to nutrient overload in adipocytes of adipose tissue . In our In summary, our study shows that D and DA inhibit adipocyte differentiation of ASCs from human VAT through the activation of GSK-3β/β-catenin signaling pathway .Prevalence of visceral obesity has been gradually increasing in older people and especially in postmenopausal women ,33. Visc


Correct bioriented attachment of sister chromatids to the mitotic spindle is essential for chromosome segregation. In budding yeast, the conserved protein shugoshin (Sgo1) contributes to biorientation by recruiting the protein phosphatase PP2A-Rts1 and the condensin complex to centromeres. Using peptide prints, we identified a Serine-Rich Motif (SRM) of Sgo1 that mediates the interaction with condensin and is essential for centromeric condensin recruitment and the establishment of biorientation. We show that the interaction is regulated via phosphorylation within the SRM and we determined the phospho-sites using mass spectrometry. Analysis of the phosphomimic and phosphoresistant mutants revealed that SRM phosphorylation disrupts the shugoshin–condensin interaction. We present evidence that Mps1, a central kinase in the spindle assembly checkpoint, directly phosphorylates Sgo1 within the SRM to regulate the interaction with condensin and thereby condensin localization to centromeres. Our findings identify novel mechanisms that control shugoshin activity at the centromere in budding yeast. Proper chromosome segregation in eukaryotes is ensured through correct attachment of the spindle microtubules to the centromeric chromosomal regions. The attachment is mediated via the multimolecular proteinaceous complex called the kinetochore. This enables the establishment of bioirentation, when each sister chromatid is attached to microtubules emanating from opposite spindle poles. Shugoshin (Sgo1) is a conserved centromeric protein that facilitates biorientation through its interactions with the protein phosphatase PP2A-Rts1, chromosome passenger complex and centromeric condensin. Here, we identified a serine-rich motif that is required for the interaction of shugoshin with the condensin complex. We show that loss of this region impairs condensin enrichment at the centromere, chromosome biorientation, segregation as well as the function of the chromosome passenger complex in the error correction. Moreover, the interaction is phosphoregulated, as phosphorylation of the serine-rich motif on Sgo1 disrupts its interaction with condensin. Finally, we show that the conserved spindle assembly checkpoint kinase Mps1 is responsible for this phosphorylation. Our findings uncover novel regulatory mechanisms that facilitate proper chromosome segregation. Biorientation of sister chromatids relies on two major processes. First, spindle and kinetochore geometry facilitates the capture of sister kinetochores (KT) by microtubules (MTs) emanating from the opposite spindle poles ,2. SeconSaccharomyces cerevisiae encodes one Sgo1 variant. As in other species, Sgo1 localizes to the centromeric chromatin by binding to histone H2A phosphorylated on serine 121 (T120 in human) by the SAC kinase Bub1 [Budding yeast ase Bub1 . This inase Bub1 . The intase Bub1 ,11– 14. ase Bub1 . Anotherase Bub1 ,12,14,15ase Bub1 . There iase Bub1 . Therefoin vitro. We demonstrate that a loss of the interaction motif impairs localization of condensin to the centromeres in vivo. The Sgo1 mutations that fail to recruit condensin to the centromeres also negatively affect sister chromatid biorientation and segregation. Finally, we determine that the interaction of Sgo1 with condensin is phosphoregulated. Based on our results we propose that, in budding yeast, tightly regulated presence of condensin on centromeres is essential for correct chromosome segregation and is mediated via its interaction with Sgo1.To uncover how Sgo1 recruits condensin to the centromeric region, we identified a region within Sgo1 that is essential for interaction with condensin 137KRTSSRSRSCSLSSPTYSKSYTRLSN163)  . To reflRLSN163) .E. coli (137LKRTSSRSRSCSLSSPTYSKSYTRLSN163 that is required for condensin binding. The identified serine-rich motif (SRM) is essential, but not sufficient for the interaction. The N-terminus of Sgo1 contains an extensive coiled-coil domain (aa 40–84) that is required for Sgo1 dimerization [in vitro. Thus, the serine-rich motif (SRM) in the N-terminal region of Sgo1 presents a novel functional domain of Sgo1 protein.To further corroborate the interaction, GST-fused Sgo1 and its mutants purified from E. coli  were useE. coli . DeletioE. coli . The SerE. coli . We founE. coli . Taken trization . We founSGO1 allele in haploid yeasts , the SRM important for the recruitment of condensin (aa 137–163) and the H2A-binding motif within the basic region that ensures localization of Sgo1 to the centromere (aa 364–391). The functional motifs were joined with linkers of 10 amino acids of random sequence. Additionally, we added the C-terminal SV40 nuclear localization signal (NLS) that was previously used to guide Sgo1 to the nucleus  did not affect the sensitivity of yeast cells to benomyl, the strain carrying phosphomimetic mutations (sgo1-13E) was highly sensitive to benomyl ; α-HA, 1:1000, Santa Cruz Biotechnology (F-7: sc-7392); α-FLAG, 1:1000, SIGMA ; α-myc, 1:1000, Santa Cruz Biotechnology ; α-GFP, 1:1000, Santa Cruz Biotechnology (B-2: sc-9996); α-Pgk1, 1:10000, Santa Cruz Biotechnology (F-7: sc-7392); α-PAP, HRP-conjugated, 1:2000 Sigma (1291); Secondary α-mouse Polyclonal Goat IgG, 1:5000, R&D SYSTEMS (FAH007).All yeast strains were derived from the genetic background of W303 or BY4743 and are listed in All plasmids used in this study are listed in Exponentially growing yeast cells were harvested, resuspended in 100 μl lysis buffer and incubated 10 min on ice. 40 μl 100% TCA was added and incubated for 10 min on ice. Precipitated proteins were spun down for 10 min at 4°C, 13 000. The pellet was washed with 1 ml ice-cold acetone, dried at 50°C for 5–20 min and resuspended in 50 μl 2x Laemmli buffer. Protein lysates were boiled for 5 min at 96°C.Exponentially growing yeast cells were harvested, washed with cold PBS, and 300 μl yeast lysis buffer was added and resuspended completely. The tube was filled with 0.5 mm ZIRCONIA/SILICA beads (Biospec) and processed in a bead-beater 6x for 1 min and 30 Hz, with a 5 min break in between each beating. The tubes were pierced, placed in a falcon tube and spun down for 2 min at 1000 rpm. The lysate was transferred to a fresh tube, 30 μl NP-40 (10% stock) was added, rotated for 30 min at 4°C and centrifuged for 5 min at 4°C and 5000 rpm. The supernatant was transferred to a fresh tube.E. coli cells were resuspended in 40 ml GST lysis buffer, lysed with high pressure (40 mBar) using the Emulsiflex-C3 (Avestin). 50 μl lysate was mixed with 50 μl 4x Laemmli buffer as a control sample. 2 mM MgCl2, 40 μl Pefabloc (100x) and 4 μl Benzonase were added to the lysate and incubated on ice for 15 min. The lysate was then centrifuged 20 min at 15 000 rpm, 4°C. 1ml pre-washed GST beads (Glutathione-Sepharose 4 Fast Flow HE) were added to the cleaned supernatant and incubated for 2 hours at 4°C on a rotator wheel. The mixture was washed 20x with 20 ml GST washing buffer on a disposable column (QIAGEN). The GST-fused protein was eluted with 1 ml GST elution buffer and the eluted sample was dialyzed overnight at 4°C in 2 l 1x PBS in a Slide-A-Lyzer Dialysis Cassette G2 10 000 MWCO (Thermo Scientific).IPTG-induced 100 μl PierceTM Glutathione Magnetic Beads (Thermo Scientific) and 100 μg protein in 500 μl 1x PBS were mixed and rotated for 1 h at 4°C, followed with 3x washing with 600 μl GST washing/binding buffer 10 min and 2x with yeast lysis buffer (5 min). Yeast lysate was added to the beads and rotated for 2 h at 4°C. The protein lysate was removed and the beads were washed 2x for 5 min with Washing buffer 1 and 1x for 5 min with Washing buffer 2. The proteins were eluted with 50 μl elution buffer and mixed with 50 μl 4x Laemmli buffer. Samples and the beads were boiled for 5 min at 96°C.The experiments were performed as previously described .600 of log-phase cells grown exponentially in YPD were harvested and washed twice with water then lysed by beating with 0.5-mm-diameter glass beads (Sigma-Aldrich) and ice cold, freshly prepared native lysis buffer . To pulldown GFP-tagged proteins, lysates were incubated for 2 hours at 4°C with 25 μL GFP-Trap coupled agarose beads (Chromotek). After incubation, the agarose beads were washed three times in lysis buffer before samples were eluted with 2X SDS sample buffer. Nitrocellulose membranes were probed with rat anti-GFP , PAP , and mouse anti-HA  primary antibodies.Approximately 100 ODFUJIFILM Wako Pure Chemical Corporation) and 100 μM MnCl2. Gel running and transfer conditions were optimized according to the manufacturer's protocol.Phospho-affinity gel electrophoresis was performed using Phos-tag acrylamide 7.5% (w/v) running gels polymerized with 50 μM Phos-tag acrylamide  equipped with a MS-2000 stage , the CSU-X1 spinning disk confocal head , LaserStack Launch with selectable laser lines  and an X-CITE Fluorescent Illumination System. Images were captured using a CoolSnap HQ camera  under the control of the Slidebook software . The fluorescence signal was imaged with a 63x oil objective by using a 473 nm or 561 nm laser. A total of 10 z-stacks were collected at 0.5 μm optical sections. Visual inspection was performed for considering “positive” centromeric signal. Intensity of the centromeric signal was evaluated by signal intensity on line connecting the two SPBs normalized to the the intensity of the average nuclear signal. At least 100 cells were evaluated in each experiments; three independent biological experiments were performed. The V plots showing signal localization at increasing interkinetochore distance were generated from line scans across preanaphase spindles of single cells assembled from 100 images as in .a, Smc2-TEVs-HaloTag-TwinStrep::HIS3, Smc4-V5-His6::TRP1). An over-night culture in YPAD medium was inoculated from a single colony. Cells were diluted into 2 L of fresh medium to an OD600 of 0.2. Nocodazole  was added at an OD600 of 0.8. After 1.5 hours of incubation, cells (~12 g) were harvested, washed with PBS twice, resuspended in 12 ml of lysis buffer ), and flash frozen in liquid nitrogen. Frozen cell suspension was crushed in a swing-mill set to 30 Hz for 3 minutes. After thawing the samples on ice, the cleared supernatant was loaded on 2x 400 μl StrepTactin Sepharose (IBA). After washing three times with 2.5 mL of wash buffer . Protein was eluted with 3.2 ml PBS containing 2 mM biotin and 1x PIC.Condensin was purified from yeast strain YSG0168 . Reactions were incubated at 30° C for 30 min and terminated by addition of Laemmli SDS sample dilution buffer and boiling the samples. Proteins were separated on the pre-cast 12.5% SDS-PAGE (Invitrogen), and phosphorylation was visualized by autoradiography.Sgo1 peptide arrays were synthesized automatically using SPOT technology with the MultiPep instrument from INTAVIS Bioanalytical Instruments AG as previously described . To idenTo identify the phosphosites on Sgo1, the TAP-tagged protein was overexpressed from a GAL1 promoter for 180 min followed by treatment with nocodazole for 120 min to arrest the overexpressing cells in mitosis. The protein was purified in presence of phosphatases by calmodulin pulldown, eluted with EGTA and separated by PAGE followed by Coomassie staining. The band corresponding to the expected size of Sgo1-TAP was cut from the gel and subjected to the analysis. Mass spectrometry identification of phosphosites on the isolated protein was performed by Nagarunja Nagaraj at the Mass Spectrometry Core Facility at the MPI Biochemistry, Martinsried, Germany, as previously described . The datS1 FigA) Far western was performed on a peptide print of Sgo1 using tagged condensin purified from budding yeasts (upper blot) or untagged control. The nonspecific binding is marked in black. Positions A1-A15: peptides covering the Sgo1 sequence from aa 37 to 60 (A9-A13 in the primary screen) and its various mutations. Positions A17- C15: peptides covering the Sgo1 sequence from aa 137 to aa 167 and its various mutations (B15 -B20 in the primary screen). D1 to G20: additional individual peptides that showed week positivity in the primary screen. Positions H17—H20: tags for antibody controls. (B) Comparison of the primary and secondary peptide plot of the putative interacting region aa 37–60. Upper blot—primary screen, lower blot—secondary screen. Several mutant peptides were added to evaluate the specificity of the putative binding. (C) GST-tagged Sgo1 purified from E. coli, wt and the mutant variants.((TIF)Click here for additional data file.S2 FigSGO1, sgo1Δ101–310 and sgo1Δ. Cells carrying Gal10-Mcd1 allele were arrested in G1 by α-factor and released into medium with and without glucose. Samples were collected at indicated time points. Pgk1 –phosphoglycerate kinase (loading control). (C) Examples of Ipl1-GFP localization in sgo1Δ, wild type SGO1, sgo1Δ137–163 and sgo1Δ101–310. (D) Quantification of Ipl1-GFP mean intensity for control condition in C. (E) Purified GST-tagged Sgo1 wild type and Sgo1-mini. Unpaired t test was used for statistical analysis in D. ****, p<0.0001; ns (not significant).(A) Chromosome segregation (centromeres of chromosome 4 labeled by tetO/TetR-GFP) in cells released from an arrest in medium containing nocodazole (2 h treatment). Cells were collected 75 min after the release from nocodazole, fixed and imaged. (B) Cell cycle arrest in response to loss of sister chromatid cohesion monitored by immunoblotting of Pds1-9myc degradation in cell lysates of wild type (TIF)Click here for additional data file.S3 Fig(A) Migration of Sgo1-TAP in Phostag-PAGE. Cells were collected at specified time points after a release from a G1 arrest by α-factor. After 45 min, α-factor was again added to limit the cells to one cell cycle. (B) Migration of Sgo1-TAP in Phostag-PAGE after release from α-factor. The sample 75/l was treated with l-phosphatase for 60 min. (C) Levels of phosphomimic (13E) and phosphoresistant (13A) mutants of Sgo1—immunoblot and the quantification of three biological replicates. (D) Localization of Sgo1-GFP-DB3A and DB3E to preanaphase spindle. (E) Localization of condensin in Sgo1-DB3A and Sgo1-DB3E mutants. (F) Sensitivity of sgo1-DB3A and sgo1-DB3E mutants to benomyl.  Protein levels of Sgo1-DB3A and Sgo1-DB3E - immunoblot and quantification. (I) Localization of the Sgo1-T159 and Sgo1-S151 mutants to the preanaphase spindle.(TIF)Click here for additional data file.S4 Fig(A) Migration of Sgo1-TAP in Phostag-PAGE after release from α-factor showed similar pattern in the presence or absence of Bub1. (B) Migration of Sgo1 in Phostag-PAGE was not affected by the presence or absence of Ipl1 kinase activity; (C) in contrast, H3 phosphorylation was almost completely abolished in cells expressing ipl1-321, a temperature-sensitive mutant that impairs the Ipl1 kinase activity at 37°C. (D) Migration of Sgo1-TAP extracted from cells harboring an analog sensitive allele of MPS1 (mps1-as1) in Phostag-PAGE in presence or absence of the ATP analog 1NM-PP1. (E) Autophosphorylation of Mps1 was monitored in the same experiment as an internal control for kinase activity. (F) Immunoblots showing that total protein level of Sgo1, mps1-as1 and Pgk1 were not affected by the prolonged 1NM-PP1 treatment. * marks the phosphorylated form of the indicated proteins.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLS)Click here for additional data file.


Yet fundamental knowledge regarding its protein expression and localization remains poorly described. Thus, we sought to define whether there was a temporal and cell-specific regulation of β-catenin activities that correlate with distinct cardiac morphological events. Our findings indicate that activated nuclear β-catenin is primarily evident early in gestation. As development proceeds, nuclear β-catenin is down-regulated and becomes restricted to the membrane in a subset of cardiac progenitor cells. After birth, little β-catenin is detected in the heart. The co-expression of β-catenin with its main transcriptional co-factor, Lef1, revealed that Lef1 and β-catenin expression domains do not extensively overlap in the cardiac valves. These data indicate mutually exclusive roles for Lef1 and β-catenin in most cardiac cell types during development. Additionally, these data indicate diverse functions for β-catenin within the nucleus and membrane depending on cell type and gestational timing. Cardiovascular studies should take into careful consideration both nuclear and membrane β-catenin functions and their potential contributions to cardiac development and disease.  Tdhesions ,25,26. Udhesions ,28,29. Tdhesions ,31. Althdhesions ,33,34. Wdhesions ,36,37. Ldhesions ,40,41,42dhesions ,44. DCHS1, and how it can impact valve structure and function through stabilizing adherens junctions [The fundamental aspects of valve development can be broadly characterized into two major morphogenetic events: Endothelial to Mesenchyme Transformation (EMT) and those events that follow EMT . The molecular and cellular processes that are defining characteristics of each of these two phases have been covered extensively in many reviews ,54,55,56unctions , we wishAlthough heavily studied, a more thorough characterization of the expression and localization of either nuclear or membrane β-catenin throughout cardiac development has yet to be reported. Additionally, scant data have been shown that clarify the correlation of Lef1 and nuclear β-catenin at different gestation stages. This information is needed to inform various cardiovascular phenotypes due to its genetic perturbation. Our detailed analysis of β-catenin’s temporal, cell-specific subcellular protein expression pattern is the focus of this report. Herein we report a detailed β-catenin expression map within the heart, which will facilitate the proper interpretation of the gain and loss of functional data, as well as provide new insight into fundamental biological processes that are regulated by this important protein. Mouse husbandry and genotyping: Animals were kept in a 12-h light–dark cycle with food and water ad libitum. For all murine studies, a minimum of 3 mice were used at each stage of development. Each mouse was from a separate litter to account for any background variability. Data shown are representative images and stainings were consistent among each mouse analyzed at each particular timepoint. pS552. Both Akt and PKA were shown to phosphorylate β-catenin at Ser552 [Immunohistochemistry (IHC) and fluorescence imaging: Immunohistochemical and fluorescence stains were performed on 5 μm paraffin-embedded sections from embryonic timepoints , fetal gestation (E17.5), neonatal (P0), and adult (6-month). Human mitral valve samples were processed and sectioned, as previously described . Primaryt Ser552 ,59. Phost Ser552 ,61. The t Ser552 ,63,64,65pS552 and membrane β-catenin. Phosphorylation at Ser552 induces β-catenin accumulation in the nucleus and increases its transcriptional activity [pS552) and membrane β-catenin revealed dynamic staining patterns that are spatially and temporally regulated during embryonic gestation. At E11.5, activated β-catenin is ubiquitously expressed in the nuclei of virtually all cardiac cells, including myocardium, epicardium, endothelium and aortic and atrioventricular valve progenitor cells  and non-phosphorylated β-catenin during fetal cardiac morphogenesis at E17.5. Within the mitral valves, activated β-catenin was observed in the nuclei of some cells in a spatial pattern similar to what was observed at E13.5 (IHC was performed for both activated (β-cateninat E13.5 A–D with at E13.5 B,D. Thisat E13.5 . Within at E13.5 F—arrows.Regions outside of the mitral or aortic valves showed prominent nuclear β-catenin staining, including the primary atrial septum, the myocardial rim lining the mitral–aortic continuity and the inter ventricular septum A–G. The The staining for membrane bound, non-phosphorylated β-catenin at E17.5 exhibited a much different pattern than observed for the nuclear form, especially within the aortic valves . Within As either loss of or gain of function of β-catenin has been shown to contribute to cardiac valvular diseases and postnatal cardiac regenerative processes, we sought to evaluate the expression of β-catenin after birth. As shown in Activated β-catenin can enter the nucleus and interact with members of the TCF/LEF transcription factor gene family to activate the transcription of downstream target genes ,35,66. TTo determine the importance of β catenin signaling in human myxomatous valves, activated and membrane bound β-catenin antibodies were used to stain human valve tissues. Similar to what was observed in murine adult tissues C,F, unafβ-catenin is a member of the armadillo family of proteins, which plays a significant role in cadherin-based cell–cell adhesion and is an indispensable co-activator of Wnt-mediated gene expression ,73,74,752+-dependent cell–cell adhesion event is fundamental for regulating morphogenetic processes, such as endocardial stabilization post-EMT [Within the membrane, β-catenin is a critical component of the adherens junction, which helps stabilize intercellular interactions . This Capost-EMT ,78. The post-EMT . Of notepost-EMT . Future post-EMT . Previoupost-EMT ,82. Herepost-EMT  and CAD-post-EMT ,84,85. T


Autophagy is an important factor in liver ischemia-reperfusion injury. In the current study we investigate the function of interferon regulatory factor-1 (IRF1) in regulating autophagy to promote hepatic ischemia reperfusion injury (IR). The high expression of IRF1 during hepatic IR exhibited increased liver damage and was associated with activation of autophagy shown by Western blot markers, as well as immunofluorescent staining for autophagosomes. These effects were diminished by IRF1 deficiency in IRF1 knock out (KO) mice. Moreover, the autophagy inhibitor 3-MA decreased IR-induced liver necrosis and markedly abrogated the rise in liver injury tests (AST/ALT). β-catenin expression decreased during liver IR and was increased in the IRF1 KO mice. Immunoprecipitation assay showed the binding between IRF1 and β-catenin. Overexpression of IRF1 induced autophagy and also inhibited the expression of β-catenin. β-catenin inhibitor increased autophagy while β-catenin agonist suppressed autophagy in primary mouse hepatocytes. These results indicate that IRF1 induced autophagy aggravates hepatic IR injury in part by inhibiting β-catenin and suggests that targeting IRF1 may be an effective strategy in reducing hepatic IR injury. Liver ischemia-reperfusion injury (IR) is involved in hypoxic liver organ stress followed by reperfusion injury after re-oxygenation , 2. HepaAutophagy is an intracellular process where impaired proteins or organelles are phagocytosed and transported to lysosomes for degradation . Low levIRF1 is a master transcription factor triggered by type I and II IFNs to activate gene expression of many IFN-responsive genes. IRF-1 plays a harmful role in liver IR , 13, as β-catenin has a vital signaling function in many cancer types including hepatocellular carcinoma and colon cancer . HoweverMouse warm IR and hepatocytes isolation were performed using protocols approved by University of Pittsburgh Institutional Animal Care and Use Committee (IACUC).Male C57BL/6J wild type (WT) and IRF-1 knock out (KO)  mice were obtained from Jackson Laboratory, 8–12 weeks old and weighed 22-28g. Animals were maintained under strict guidelines of the University of Pittsburgh. The protocol was approved by the University of Pittsburgh Institutional Animal Care and Use Committee (Protocol Number: 17019893). The mice were housed in a constant temperature, laminar-flow, specific pathogen-free environment and had access to food and water ad libitum.Hepatic parenchymal cells were isolated from murine liver using collagenase digestion, as previously described . The livA non-lethal model of segmental (70%) hepatic warm I/R was used as previously described . The mouLiver function tests following IR were performed using a veterinary chemistry analyzer  to measure serum alanine aminotransferase (ALT).2 and 0.1% Triton X-100. Following a centrifugation step at 13,000g for 15 min at 4°C, nuclear proteins were obtained. Protein concentration was quantitated with BCA assay reagent (Thermo).Frozen liver tissues or cells were suspended in cold PBS containing 0.1% Triton X-100 and lysed by homogenization. The mixture was centrifuged at 1000g for 5 min at 4°C and the supernatant stored at -80°C for analysis of cytoplasmic proteins. Nuclear proteins were extracted using a premade buffer containing 20 mM Tris (pH 7.5), 20% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl4 cells/well in 96 well plate and cultured overnight. CCK-8 solution was added for 1h at 37°C. The absorbance was measured at 450 nm using a plate reader .Hepatocyte function was determined using a commercially available CCK-8(MCE) kit. Cells were plated at a density of 10Adenovirus was transfected in vitro using the protocol as follows: cells were plated in 10 cm plates and recovered overnight. Culture media was removed and 4ml media that contains adenovirus was added with adenoviral concentration of 50 MOI. Six hours later, another 2 ml of culturing media was added. Two days after transfection, cells were used for testing. Adenoviral IRF-1 (AdIRF-1) is used as previously described , 20. TheBafilomycin A1 (Autophagy inhibitor), XAV939 (β-catenin inhibitor) and SKL2001(β-catenin agonist) were from MedChemExpress.tgttaccaactgggacgaca3`5`, Reverseggggtgttgaaggtctcaaa3`5`; β-catenin Forward 5`TGGACCCTATGATGGAGCATGA3`, Reverse5`GGTCAGTATCAAACCAGGCCAG3`Total RNA from liver and hepatocytes was extracted by Trizol reagent (Invitrogen). 2ug of total RNA was reversely transcribed into cDNA with RNA to cDNA Ecodry Premix Kit (Takara). Then, PCR was performed using SYBR Premix Kit (Takara) on an ABI Stepone PCRSystem (Applied Biosystems). The relative expression of mRNA was quantitated using the 2-ΔΔCt method and normalized to β-actin. Primers used were: β-actin Forward A commercially available lysis buffer was used to extract cellular proteins . β-actin or Histone H3 protein was detected as standardization of whole cell protein or nuclear protein respectively. The membrane was scanned by Li-Cor Odyssey. Antibodies used were as follows: antibodies against IRF1, LC3A/B, Beclin1and P62 ; β-actin and Histone H3 (Abcam); β-catenin (BD biosciences). The results of Western Blot was quantified by Image Studio (Ver 5.2) software (Li-Cor). β-actin and Histone H3 were used for standardization.Hepatocytes cells were infected with IRF1-adenovirus. After 48 h, cells were incubated on ice and lysed in cold lysis buffer  adding Protease Inhibitor Cocktail (Med Chem Express). Lysates were centrifuged at 12000g at 4°C for 10 min and incubated with anti-IRF1 antibody or anti-β-catenin antibody attached to Protein A/G(Santa Cruz) agarose overnight at 4°C on a shaker platform. Beads were collected by centrifugation at 3000g for 5 min at 4°C, washed in lysis buffer and resuspended in SDS gel loading buffer. The proteins were analyzed by Western blot.Cells were treated with IRF1-adenovirus and LacZ-adenovirus (as control) and were fixed with 2% paraformaldehyde for 15 min. The cells were incubated with LC3 antibody (Novus) and Cy3-conjugated goat anti-rabbit IgG (Jackson Immunoresearch). F-actin phalloidin (Invitrogen) and Hoechst nuclear stain was also applied. Imaging conditions were maintained at identical settings within each antibody-labeling experiment with original gating performed using the negative control. Large area images in X and Y were obtained using a Nikon A1 confocal microscope.P < 0.05 was considered statistically significant.All experiments were performed in triplicates. Mean standard deviation was calculated for all experiments and shown within the results. Using Student t test or one-way analysis of variance (ANOVA) was used to determine significance. A level of To determine the effect of IRF1 on warm hepatic IR, we used IRF1-KO or wild-type B6 mice to evaluate the changes in liver damage. Serum ALT levels increased significantly (p < 0.05) in wild-type mice and peaked at 6 hr after reperfusion . CompareTo determine the role of IRF1 in autophagy in warm IR, we evaluate the changes in autophagic signaling. Autophagy proteins p62 and beclin1 are decreased at 6 and 12 hrs after warm IR in the IRF1 KO mice, but not in the WT mice . IRF1 prThe Wnt/β-catenin pathway is an important regulator of autophagy. To examine the effect of IRF1 on β-catenin during hepatic warm IR, β-catenin gene expression was measured by Western blot 6–24 hrs after warm IR. Compared to WT mice, the IRF-1 KO mice had markedly increased β-catenin mRNA levels at 6 and 12 hrs after warm IR . LikewisNext, to determine if IRF1 can directly bind β-catenin protein, we used co-immunoprecipitation assay to test the binding between IRF1 and β-catenin proteins. IRF1 was overexpressed in normal mouse hepatocytes using adenoviral-IRF1 transduction, and co-immunoprecipitation pull-down assay showed that antibody to IRF1 bound β-catenin . ControlTo explore the effect of IRF1, we transduced mouse hepatocytes with AdIRF1. As expected, AdIRF1 transduction resulted in strong cytoplasmic  and nuclTo examine the role of hepatocyte IRF1 in autophagy, we transduced mouse hepatocytes with AdIRF1. This resulted in high levels of IRF1 protein and was associated with decreased β-catenin protein levels on Western blot . We thenTo determine the role of β-catenin in autophagy, we used the β-catenin inhibitor XAV939 and β-catenin agonist SKL2001 in hepatocytes. In primary liver cells, XAV939 inhibited β-catenin protein levels, and this was associated with increased autophagy with elevated LC3-II levels compared to control . The β-cThe major findings of this study are: (i) IRF1 was induced in hepatic IR injury; (ii) IRF1 activity induced autophagy during liver warm IR; (iii) IRF1 bound β-catenin protein and IRF1 expression decreased β-catenin in vitro and in vivo with IRF1 KO mice exhibiting increased hepatic β-catenin levels; (iv) Expression of IRF1 induced autophagy by inhibiting β-catenin. (v) β-catenin expression is inversely correlated with autophagy. Taken together, these findings demonstrate that IRF1 is induced in hepatic IR and inhibits β-catenin and is associated with increased autophagy. In IRF1 KO mice, β-catenin expression is increased and is associated with decreased autophagy and less liver damage .Transcription factors within the IRF family play an important role in a variety of cellular responses including hematopoietic development, immune response and oncogenesis . IRF1, iβ-catenin also plays a key role in liver ischemia reperfusion injury , 28 and In summary, this study provides novel evidence that IRF1 induces autophagy in part by inhibiting the β-catenin pathway during liver IR. Our observations have significant clinical relevance as liver IR is present during the majority of surgical procedures involving the liver. Future studies are needed to evaluate the role of IRF1 manipulation or other modulators of IRF1 signaling in clinically relevant settings involving a hepatic oxidative stress response.S1 FigP< 0.05.(A and B) hematoxylin-eosin (HE) staining of liver tissue following warm I/R in 6h with treated 3-MA (15mg/kg i.p.) (A) or PBS (control) (B). (C and D) The serum ALT and AST levels were significantly increased in 3-MA treated mice after exposure to IR 6 hrs as compared with the control mice (n = 3). data are presented as mean±SD, *(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Accumulating evidence strongly implicates iron in the pathogenesis of aging and disease. Iron levels have been found to increase with age in both the human and mouse retinas. We and others have shown that retinal diseases such as age-related macular degeneration and diabetic retinopathy are associated with disrupted iron homeostasis, resulting in retinal iron accumulation. In addition, hereditary disorders due to mutation in one of the iron regulatory genes lead to age dependent retinal iron overload and degeneration. However, our knowledge on whether iron toxicity contributes to the retinopathy is limited. Recently, we reported that iron accumulation is associated with the upregulation of retinal and renal renin–angiotensin system (RAS). Evidences indicate that multiple genes/components of the RAS are targets of Wnt/β-catenin signaling. Interestingly, aberrant activation of Wnt/β-catenin signaling is observed in several degenerative diseases. In the present study, we explored whether iron accumulation regulates canonical Wnt signaling in the retina. We found that in vitro and in vivo iron treatment resulted in the upregulation of Wnt/β-catenin signaling and its downstream target genes including renin–angiotensin system in the retina. We confirmed further that iron activates canonical Wnt signaling in the retina using TOPFlash T-cell factor/lymphoid enhancer factor promoter assay and Axin2-LacZ reporter mouse. The presence of an iron chelator or an antioxidant reversed the iron-mediated upregulation of Wnt/β-catenin signaling in retinal pigment epithelial (RPE) cells. In addition, treatment of RPE cells with peroxisome proliferator-activated receptor (PPAR) α-agonist fenofibrate prevented iron-induced activation of oxidative stress and Wnt/β-catenin signaling by chelating the iron. The role of fenofibrate, an FDA-approved drug for hyperlipidemia, as an iron chelator has potentially significant therapeutic impact on iron associated degenerative diseases. A stringent balance during iron uptake, transport, storage and utilization is required to maintain iron homeostasis2. However, excess iron promotes the generation of reactive oxygen species (ROS) through Fenton reaction causing molecular and cellular dysfunctions3. Age-related increases in iron levels have been found in the human and mouse retinas5. In addition, disrupted iron homeostasis in hereditary and acquired diseases such as aceruloplasminemia6, hemochromatosis8, Friedreich’s ataxia9, diabetic retinopathy (DR)10, age-related macular degeneration (AMD)11, glaucoma12, ocular siderosis13, multiple blood transfusions14, and excess dietary iron supplementation15 have been reported to cause retinal iron accumulation and degeneration.Iron plays a vital role in the retina with many iron containing proteins involved in the phototransduction cascade10. RAS plays an important role in regulating vasoconstriction and electrolyte balance. Angiotensin II (Ang II) is the primary molecule of RAS produced as a result of cleavage of angiotensinogen (AGT) by renin and angiotensin-converting enzyme (ACE) sequentially. Ang II then binds to Ang II type 1 (AT1) and Ang II type 2 (AT2) receptors wielding diverse pathophysiological effects. Activation of retinal RAS due to concurrent upregulation of multiple RAS genes is associated with many ocular diseases including DR, AMD, uveitis, and glaucoma16. Recent studies established that all components of the RAS, including AGT, renin, ACE, AT1, and AT2 are downstream targets of Wingless-related integration site (Wnt)/β-catenin signaling17. However, it is not known whether iron can regulate retinal Wnt/β-catenin signaling. The Wnt signaling is a group of signal transduction pathways with diverse role during embryonic growth and development. In the canonical Wnt pathway, secreted Wnt ligands bind to the Wnt receptor Frizzled (Fzd) and co-receptor low-density lipoprotein receptor-related protein (LRP5/6) leading to its phosphorylation and activation18. This leads to inactivation of the “destruction complex” consisting of GSK-3β (glycogen synthase kinase-3β), Axin, and APC (adenomatous polyposis coli). This prevents the proteasomal degradation of β-catenin and promotes its accumulation and nuclear translocation. In the nucleus, β-catenin associates with T-cell factor (TCF) and regulates the expression of many target genes, including VEGF . Altered Wnt signaling has been reported as a contributing factor in various ocular disorders and malignancies21. Wnt activation may contribute to the angiogenesis, inflammation, and fibrosis22. In addition, the Wnt pathway affects cell adhesion through the binding of β-catenin and E-cadherin23. Wnt signaling has antagonistic pleiotropic effects, because they are quintessential earlier in life but deleterious later in life19. Downregulation of Wnt/β-catenin signaling and RAS blockade have been shown to protect against aging24. However, current therapy with RAS inhibitors have limited efficacy because of compensatory upregulation of renin expression25 necessitating the need for a new strategy to simultaneously target multiple RAS genes for a more effective treatment.Our recent study showed that retinal iron accumulation exacerbates cell death through oxidative stress, inflammasome activation, and enhanced renin–angiotensin system (RAS) activity26. PPARα, PPARβ/δ, and PPARγ are the three isoforms, of which PPARα is crucial in the regulation of fatty acid oxidation, inflammation and angiogenesis26. Fenofibrate, a PPARα agonist, has been found to be effective in the amelioration of microvascular complications of diabetes in the FIELD and ACCORD trials29. Also, fenofibrate has been shown to potently block the activation of Wnt signaling in the kidney by destabilizing LRP6 mediated through inhibiting the renal ROS production30. In the present study, we determined if iron accumulation regulates canonical Wnt signaling in the retina and the therapeutic role of PPARα agonist fenofibrate in preventing iron-induced Wnt signaling.Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that play an important role as transcription factors in regulating the expression of genes involved in the lipid metabolismAxin2, cMYC, CCND (cyclin D), and VEGF were significantly upregulated in FAC-treated human RPE cells in a dose dependent manner  cell line ARPE19 was treated with different concentrations of ferric ammonium citrate (FAC) for 24 h. FAC treatment increased the expression of total and non-phospho (active) β-catenin levels in a dose dependent manner Fig. . The expner Fig. . Similarner Fig. . These rAxin2lacZ /+ was used to confirm if iron regulates retinal Wnt signaling in vivo. The Axin2 gene is a known target of the canonical Wnt signaling and Axin2LacZ mice were generated by a stable knock in of LacZ in frame with the start codon of the endogenous Axin2 to visually localize the Wnt signaling activation32. X-Gal  staining of retinal sections from intravitreal holo-transferrin (iron)-treated eyes of Axin2lacZ/+ mouse showed upregulated Wnt signaling activity, mainly in the ganglion cell layer, inner nuclear layer, and, to a lesser extent, in the outer nuclear layer and RPE compared to the apo-transferrin (control)-treated eyes . RP is the leading cause of inherited retinal blindness in the United States and is characterized by progressive degeneration of rod photoreceptors with no effective treatments currently availableted Fig. . Bindingice Fig. . Similarice Fig. . Labile P16 Fig. . 4-hydroP16 Fig. . In postP16 Fig. , indicat36. RPE cells treated with FAC in vitro and mice treated with iron dextran in vivo upregulated the expression of Src collagen homolog (p66 Shc1)  or iron chelator deferiprone  in the presence of FAC. Treatment with NAC or DFP significantly reduced the FAC-induced upregulation of LRP6 and active β-catenin  Fig. , an indinin Fig. . Furthernin Fig. . In summ30. To investigate whether the iron-induced activation of canonical Wnt signaling in the retina is abrogated by fenofibrate, ARPE19 cells were treated with fenofibrate in the presence of 100 µg/mL FAC for 24 h. Treatment with fenofibrate significantly reversed the upregulation of FAC-induced active β-catenin and total β-catenin levels , a three-arm parallel-group open-label randomized trial, reported that 52 patients undergoing fenofibrate monotherapy alone had a significant reduction in the red blood cell count and hemoglobin starting from 4 weeks after treatment was initiated until 52 weeks for the entire duration of the study68. Our present finding that fenofibrate is an iron chelator provides a plausible explanation for the results seen in EFECTL trial. In summary, our study demonstrates that iron accumulation in the retina inhibits GSK-3β and activates LRP6 and β-catenin signaling in an oxidative stress-dependent mechanism. In addition, we report a previously unidentified pharmacological effect of fenofibrate as an iron chelator reducing intracellular iron accumulation. This work has significant translational potential as fenofibrate could be an attractive therapeutic drug for the treatment of many chronic diseases associated with iron overload.PPARα downregulation has been implicated in DRLacZ/+ mice, and C57BL/6Jrd1/le (rd1 mice) breeders were purchased from Jackson Laboratory . All the mice were housed at the animal facility of Saint Louis University School of Medicine. Gender, age and weight matched animals were randomly divided into different groups as indicated in the results. All procedures involving mice were approved by the Saint Louis University Institutional Committee on Animal Use for Research and Education and were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.C57BL/6J mice, Axin2Considering the toxicity of FAC to the retina, holo-transferrin was injected intravitreally in mice to simulate iron overload. Briefly, animals were anesthetized with 1 : 1 mix of ketamine (8 mg/ml) and xylazine (1.2 mg/ml). Pupils were dilated with 1% atropine sulfate ophthalmic solution. Animals were injected with 1 μL of 2.4 mM holo-transferrin (Holo-Tf) or 1 μL of 2.4 mM apo-Tf as control. Twenty-four hours post injection, mice were killed and retinas were collected. For systemic iron overload, mice were administered with iron-dextran (1 g/kg body weight) intraperitoneally once a week for a duration of 8 weeks. PBS was administered intraperitoneally to the control group.2. For biochemical experiments, cells were seeded in a six-well plate (unless otherwise mentioned) and were utilized after reaching 90% confluence followed by a 5–6 h serum starvation before any treatment was done.The human RPE cell line, ARPE19 was procured from American Type Culture Collection  and maintained in 1 : 1 Dulbecco’s modified Eagle’s medium/F12  supplemented with 10% fetal bovine serum (FBS)  and 100 IU/ml penicillin and 100 μg/ml streptomycin  at 37 °C in a humidified air chamber with 5% CO7. Cells were grown in 1 : 1 DMEM/F12  supplemented with 25% FBS  and 5% penicillin/streptomycin  at 37 °C cell culture incubator with 5% CO2. Purity of the cultures was verified as described previously by immunodetection of RPE65, a known marker for RPE cells 7.Three-week-old C57BL/6 pups were used to isolate primary RPE cells as described previouslyCells were treated with the experimental conditions and total RNA was extracted using Trizol  and the quantity of the extracted RNA was assessed by measuring the absorbance at 260 nm. RNA (500 ng) was reverse transcribed to synthesize the cDNA using iScript™ cDNA Synthesis Kit  according to the manufacturer’s protocol. Then, cDNA was diluted 1 : 5) and utilized to assess the gene expression by ABI Quant Studio3 real-time PCR system using iTaq™ Universal SYBR® Green Supermix . The list and sequence of specific primers used are provided in Supplementary Table  : 5 and Protein was extracted from the cells and the tissues using a lysis buffer  supplemented with protease inhibitors  and 1% NP-40. The concentration of the protein was estimated by bicinchoninic acid assay . Protein 20 µg) was subjected to SDS-polyacrylamide gel electrophoresis and transferred onto the nitrocellulose membrane. The membrane was then blocked for 1 h with 3% bovine serum albumin (fatty acid free) and incubated with the primary antibodies overnight at 4 °C. Then, the blots were washed and incubated with secondary antibodies for 90 min at room temperature (RT). Blottings were again washed and the proteins were visualized using enhanced chemiluminescence western blotting detection system . Antibody sources and dilutions used are listed in Supplementary Table 0 µg was Cells with or without treatment were washed with PBS at 4 °C and the cytosolic and nuclear fractions of cells lysates were purified using the NE-PER™ nuclear and cytoplasmic extraction reagent  following the manufacturer’s guidelines.Renilla, and calculated according to the TCF/LEF Reporter Kit data sheet.Wnt activity was measured by using the TCF/LEF Reporter Kit . Briefly, cells were seeded at a density of 10,000 cells per well in a 96-well plate, transfected with either negative control or reporter plasmid as mentioned in the kit. Wnt pathway activity was detected using the Dual Luciferase Assay System, normalized with 69. Labile iron (Fe2+) in retinal sections were detected using FeRhoNox-1 fluorescent imaging probe as mentioned in our recent publications70. RhoNox-1 was dissolved in dimethyl sulfoxide to a final concentration of 5 μM, added to the retinal sections, and incubated for 30 min at 37 °C in a dark chamber. The sections were then counterstained with Hoechst nuclear stain and examined by laser-scanning confocal microscopy .Eyecups were fixed in 4% paraformaldehyde and embedded in OCT  compound for cryosectioning as previously describedRetinal sections were fixed, blocked with 1× power block and incubated overnight at 4 °C with rabbit anti-HNE. Negative control sections were incubated without the primary antibody. Sections were rinsed and incubated for 1 h with goat anti-rabbit IgG coupled to Alexa Fluor 488. Coverslips were mounted after staining with Hoechst nuclear stain and sections were observed using laser-scanning confocal microscopy. Immunohistochemistry studies were repeated twice, and the results from these two experiments were similar.LacZ/+ mice according to the manufacturer’s protocol.β-Galactosidase reporter gene staining kit  was used to stain retinal sections from Axin2Intracellular ROS generation was monitored by CM-H2DCFDA staining according to the manufacturer’s protocol .g for 10 min at 4 °C and the resultant pellet was stored at −80 °C until ICP-MS analysis. On the day of analysis, frozen cell pellets were thawed and thermally digested in 1 ml nitric acid (HNO3) using Mars 6 Microwave Digestion System (CEM Corporation). Digested samples were diluted for mass spectrometric analysis and total iron content of the samples was estimated using PerkinElmer NexION 2000 ICP-MS in Washington University, St. Louis. A standard calibration curve was performed prior to sample analysis. The total iron content per cell was calculated, accounting for the number of cells present, as well as dilution factors, as the mean value of three individual experiments groups.Intracellular iron levels were estimated by using ICP-MS. ARPE19 cells were cultured in T-75 tissue culture flask and treated according to the experimental conditions. At the end of the treatment, the flask was rinsed with PBS for three times and cells were detached by 0.25% trypsin-EDTA. After centrifugation, the cells were suspended in 1 ml PBS and counted. Then the cells were centrifuged at 16,100 × 71. Briefly, FeCl2  was mixed with different concentrations of fenofibrate. Then, 200 µl of 5 mM ferrozine solution was added to initiate the reaction followed by vigorous shaking. After incubating the reaction for 10 min at RT, the absorbance of the solution was recorded at 562 nm. The percentage inhibition of the ferrozine-Fe2+ complex formation was calculated as (A0 − As/As) × 100. Here, A0 and As represents the absorbance of the control and fenofibrate/fenofibric acid/DFP, respectively.The chelation of ferrous iron by fenofibrate was determined by method described previouslyt-test. All the experiments were performed in triplicates. The values of p < 0.05 were considered significant 48.Data are presented as mean ± SEM. The fold change for bar graphs were calculated by normalizing the sample values with the respective control values. Statistical significance was determined with the Student’s Further information on research design is available in the Supplementary FiguresReporting Summary