APC mutation modulated the wild-type allele to mimic the properties of the mutant β-catenin allele. The one-step tagging strategy demonstrates how genome engineering can be employed for the parallel functional analysis of different genetic variants.Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in β-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of endogenous β-catenin in a colorectal cancer cell line. Wild-type and oncogenic mutant alleles were tagged with different fluorescent proteins, enabling the analysis of both variants in the same cell. We analyzed the properties of both β-catenin alleles using immunoprecipitation, immunofluorescence, and fluorescence correlation spectroscopy approaches, revealing distinctly different biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3β inhibitor or a truncating  CTNNB1). When Wnt signaling is inactive, β-catenin forms part of the destruction complex, which consists of adenomatous polyposis coli (APC), axis inhibition protein 1 (Axin1)  , casein  (Axin1)  and then (Axin1) . Subsequ complex .via its DEP domain, leading to polymerization of Dvl  protein family and the transmembrane LRP5/6 (low-density lipoprotein receptor-related protein) co-receptor . Fzd pron of Dvl . Dvl facn of Dvl , which in of Dvl . As a con of Dvl .The human β-catenin protein consists of 781 amino acids and contains three distinct structural domains. Crystal structure and NMR analysis of β-catenin have revealed that both N- and C-terminal domains are structurally flexible, whereas the central armadillo repeat has a rather rigid scaffold structure . The N-tCTNNB1/β-catenin in colorectal cancer are typically found in its N-terminal domain, particularly in exon 3 that carries multiple phosphorylation sites for CK1 and GSK3β, including amino acids Ser33, Ser37, Thr41, and Ser45  and Cas9 protein from pyogenes  and the pyogenes . Transfepyogenes . Infrequpyogenes .CTNNB1 locus. In addition, 7 clones showed a heterozygous integration of the Cherry fluorophore in the β-catenin/CTNNB1 locus, and 24 clones were heterozygous for the Clover fluorophore-tagged allele. Moreover, six clones had an insertion of the donor cassette after the stop codon of β-catenin/CTNNB1. Two isogenic cell lines had partial integration of the Clover tag and four clones were wild-type indicating no editing events in the CTNNB1 locus  cell line and to an HCT116 isogenic cell line in which the ∆Ser45 allele was removed (β-cateninWT+/−)  . To analinWT+/−) . While Pe allele . Since gcassette . Separatcassette . These PB1 locus . FinallyB1 locus . Taken tCTNNB1 allele remained untagged in clones #24, #33, and #45. While the untagged allele was not altered at the sgRNA target site in clones #24 and #45, we detected a 40-bp mutation in clone #33, presumably from error-prone non-homologous end joining of a Cas9-induced double-strand break. This mutation results in four additional amino acids at the end of β-catenin. Importantly, sequencing of the bi-allelically tagged clone #37 revealed that both alleles had been modified without unintended mutations  was tagged with Clover and which with Cherry using cDNA from reverse-transcribed mRNA of each isogenic cell line and specific reverse primers to Clover or Cherry. Sanger sequencing of cDNA templates revealed that both clones #37 and #45 carried the Cherry fluorophore in the ∆Ser45 allele . In contNext, total cell lysates of parental and isogenic knock-in cell lines were analyzed for β-catenin fusion proteins by Western blot . As showWild-type β-catenin-Clover protein runs higher than 97 kDa and it is recognized by both GFP and Flag antibodies . Mutant WT/∆45 and the β-cateninWT+/− cell line, which only had a wild-type but not a mutant β-catenin allele. All three clones #33, #37, and #45 showed a similar growth behavior as the parental β-cateninWT/∆45 cells (WT+/−) displayed impaired growth. Next, we tested the functionality and localization of the endogenously tagged β-catenin variants. We performed siRNA-mediated silencing of β-catenin and analyzed gene expression by RT-qPCR  is a powerful quantitative microscopy method for measuring local concentrations and diffusional mobilities of fluorescent molecules in cells and tissues, for example, to determine Wnt ligand-receptor interactions in the cell membrane . To simueff, from which the fluorescence emanates, so that the molecule concentration, c, in the observation volume can simply be calculated as c=N/Veff. In addition to autocorrelation analysis, we calculated cross-correlation functions to identify correlated intensity fluctuations in the two color channels. These will appear if the Clover and Cherry labeled β-catenin molecules co-diffuse, that is, bind to each other and migrate in concert through the observation volume. Cross-correlation amplitudes between wild-type Clover-tagged and mutant Cherry-tagged β-catenin were always found to be zero within the experimental error, implying that these two β-catenin variants diffuse independently and do not associate to any significant degree , we obtain an estimated D=26 ± 5 µm² s–1 for the tagged β-catenin, which is on the high end of the distribution of D values of the respective ensemble  cells. In the cytosol  was designed by E-CRISP to target the mutation cluster region (MCR) in exon 15 of APC , introduAPC . Edited mutation . Subsequ cytosol  and in t cytosol  of APCLOTo investigate whether mutant β-catenin can still be found in the destruction complex, we performed immunoprecipitations with an APC antibody and with GFP/Clover and RFP/Cherry beads. Both wild-type β-catenin-Clover and mutant β-catenin-Cherry bind to APC, Axin1, and GSK3β , indicatHow disease-causing genetic alleles affect protein function has remained a subject of intense research . MutatioWhile tagging of proteins can interfere with protein functions, it can also lead to the accumulation of the protein and its potential overactivation. In line with our study, previous overexpression studies showed that C-terminal tagging of β-catenin did not affect its functions . We alsoUsing FCS, we analyzed mutant and wild-type proteins in the same cell. These measurements revealed that wild-type β-catenin diffuses faster than the mutant isoform in the cytoplasmic and the nuclear compartments, suggesting that wild-type protein is present as a monomer or as part of a smaller complex in the cell. In support of our data, a similar diffusion coefficient was recently determined by FCS for β-catenin endogenously tagged with monomeric SGFP2 in HAP1 cells . It appeActivation of Wnt signaling by inhibition of GSK3β or truncation of APC results in slower diffusion and higher abundance of wild-type β-catenin, indicating that β-catenin now becomes part of larger multi-protein complexes. This might—at least in part—be due to enhanced shuttling into the nucleus, where β-catenin interacts with the Wnt enhanceosome, a large transcriptional complex including TCF/LEF, PYGO, BCL9, and other proteins to activate transcription of Wnt target genes . Along tIn the present work, we have used FCS to quantify the concentration and diffusional mobility of β-catenin in the nuclear and cytosolic compartments for the first time. The observed small diffusion coefficients indicate that β-catenin migrates as part of multi-protein complexes. Unraveling the protein composition of these complexes will require dual- or multicolor FCS experiments, which allow us to analyze co-diffusion of proteins via cross-correlation analysis . Furtherβ-catenin plays an important role not only in Wnt signaling but also in cell adhesion, in which it links the adhesion complex to the actin cytoskeleton via its interaction with the actin-binding protein α-catenin . Since tIn summary, we engineered, to our knowledge, the first biallelically tagged cell model carrying a different fluorescent fusion protein on each allele, demonstrating the feasibility of our one-step tagging strategy to analyze the consequences of genetic variants. β-catenin knock-in cell lines provide a powerful tool for investigating β-catenin’s role in the canonical Wnt signaling, and also in adhesion. Due to these properties of β-catenin, these cell models can serve to evaluate both functions in parallel on the endogenous level. Cell models of wild-type and mutant variants can also be used for drug discovery, identifying small molecules that only interfere with mutant, but not wild-type β-catenin, thereby circumventing side effects that are often associated with interfering with homeostatic levels of Wnt signaling. In general, bi-allelic tagging of genetic variants in their endogenous locus can open new avenues toward understanding and possibly targeting disease-causing variants in a broad spectrum of cellular and organismal phenotypes.WT/∆45) cell line was purchased from the American Type Culture Collection. HCT116 β-cateninWT+/− cells were obtained from Horizon Discovery. Cells were culture in McCoy’s medium (Gibco) supplemented with 10% fetal bovine serum (Biochrom GmbH). Cells were grown at 37°C and 10% CO2 in a humidified atmosphere. Cells were tested for cross-contamination using SNP profiling by a Multiplex human Cell Authentication (MCA) assay (Multiplexion) and regularly checked for Mycoplasma contamination.The parental colon cancer HCT116  for DNA plasmids and Lipofectamine RNAiMax (Thermo Fisher Scientific) for siRNAs according to the manufacturer’s protocol. 15–25 nM Dharmacon siRNAs (Horizon Discovery) and 5–10 nM Ambion siRNAs (Thermo Fisher Scientific) were used for reverse siRNA transfection .GSK-3β inhibitor CHIR99021 (Cat. no. 361571) was obtained from Merck Millipore. ICG001 (Cat. no. Cay16257-1) was obtained from Biomol. DMSO was used as the vehicle control. LGK974 (TRC-L397640-50mg) was synthesized by Hölzel Diagnostika.Whole-cell lysates were extracted using a buffer containing non-ionic detergents such as Triton X-100 or NP-40, supplemented with protease inhibitors (Roche). Protein concentration was determined using BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Samples were loaded on 4–12% NuPAGE Bis-Tris gels or 3–8% NuPAGE Tris acetate gels (Thermo Fisher Scientific) and transferred to the nitrocellulose membranes  following standard Western blotting procedure. Antibodies and their dilutions are listed in CTNNB1 and APC genes were designed using E-CRISP  according to manufacturer’s instructions. As controls, each donor template was also transfected alone together with px459sgCTNNB1 to generate single-tagged β-catenin cells and to adjust gates for FACS sorting. After 72 hr, cells were selected with 2 µg/ml puromycin . After 48 h, 100 µg/ml hygromycin 10687010 (Gibco/Thermo Fisher Scientific) and 10 µg/ml blasticidin  were added to select edited cells for 5 days. Subsequently, surviving cells were sorted as single-cell clones by FACS into 96-well plates for cultivating. To validate correctly endogenously tagged β-catenin clones genotyping was performed. Genomic DNA was extracted with phenol-chloroform or with DNeasy Blood & Tissue Kit . Different primer pair combinations were designed for genotyping . Primer To determine Wnt signaling activity, the luciferase-based dual Wnt reporter assay was performed as described previously . BrieflyTotal RNA from cell pellets was isolated with the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. RNA was reverse-transcribed into cDNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). qPCR was performed using the Universal probe library (Roche) with the LightCycler 480 (Roche) instrument according to the manufacturer’s instructions. A list of primers used in this paper for qPCR is shown in Sterile coverslips (Thermo Fisher Scientific) were placed into 12-well plates and cell suspension was added. After 1 day, cells were treated with the indicated drugs for 20–24 hr. Cells were fixed using 4% PFA (VWR) for 20 min at room temperature. For antibody staining, cells were permeabilized with 0.25% Triton/phosphate-buffered saline (PBS). After several washing steps, cells were blocked for 1 hr with 5% goat serum (v/v) and 5% BSA (v/v) in PBS at room temperature. After overnight incubation with the primary antibody at 4°C, cells were incubated with fluorescently labeled secondary antibodies (1:250) for 1 hr at room temperature in the dark. Antibodies were diluted in PBS with 5% goat serum (v/v) and 5% BSA (v/v). Finally, cover slips were gently transferred onto microscope slides (Carl Roth GmbH) and fixed with Vectashield mounting media containing DAPI solution (Biozol Diagnostica). Fluorescence images were acquired with a Leica TCS SP5 confocal microscope. For live-cell imaging, cells were added into μ-slide with a glass bottom (Ibidi) and staining was performed as described. For automated microscopy, cells were seeded into 384-well plates and subjected to fixation using the CybiWell Vario robotic system. Fluorescence images were acquired using InCell Analyzer 2200 microscope  at 20× magnification in three channels  with four sites per well.For LGK974 treatment, cells were seeded into wells of a six-well plate and treated with Control/DMSO or 5 µM LGK974 for 48/72 hr, then washed several times with PBS and split into the Ibidi µ-Slide (80826) and wells of a six-well plate. After an additional treatment with LGK974/Ctrl for 32 hr, cells were stimulated with 200 ng/ml of recombinant Wnt3a for 16 hr. Cells were fixed with 4% PFA for 10 min and stained with the Hoechst solution (1:2000 in PBS) for 30 min. Fluorescence images were acquired with a Leica TCS SP8 confocal microscope. 5–20 slides per condition were taken. The fluorescence intensities of Clover, Cherry, or Hoechst were analyzed with ImageJ without any additional manipulation of the images. The Clover and Cherry intensities were normalized to Hoechst and further to control.In the silencing experiment, CTNNB1, CLOVER, and CHERRY were downregulated by applying siRNA directly on the Ibidi µ-Slide for 72 hr. Subsequently, the staining and analysis were performed similar to the LGK974 experiment.Image analysis of automatic microscopy was performed using R package EBImage  and adap–1 in Dulbecco’s phosphate buffered saline . Each well of a eight-well Nunc Lab-Tek chambered cover glass (Thermo Fisher Scientific) was incubated with 200 µl of the solution for at least 1 hr at room temperature. Afterward, the solution was removed by aspiration, and the wells were allowed to dry. These chambers were either used immediately or stored at 4°C for a maximum of 1–2 weeks and washed with DPBS directly before use. For FCS measurements, HCT116 cells were cultured in McCoy’s medium without phenol red in the chambers. For drug treatment, the cell culture medium was replaced with a medium containing 10 µM CHIR99021 ~16‒26 hr before the FCS measurements. For LGK974 treatment, cells were seeded in six-well plates and incubated with 5 µM LGK974 for 5 days. During this period, the drug-containing medium was renewed and, if necessary, cells were split. On day 6, cells were transferred into fibronectin-coated eight-well chambered cover glass, where they were left to adhere for ~4‒6 hr before adding 100 ng ml–1 of recombinant Wnt3a. FCS measurements were started at ~16‒26 hr after Wnt treatment. All FCS measurements were carried out at 37°C and 5% CO2.To prepare fibronectin-coated sample chambers, fibronectin was diluted to 50 µg ml−2) and 5 µW (2.8 kW cm−2), respectively. The emission was filtered by 525/50 nm and 607/37 nm (center/width) band-pass filters . For data acquisition, the observation volume of the microscope was first positioned into the cytoplasm and directly afterward into the nucleus. The intensity decrease during the measurement due to photobleaching was compensated as described As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:The authors describe a fluorescent tagging strategy that allows them to knock in one copy each of differently tagged wild-type and a non-destructible β-catenin mutant, so that the behavior of the mutant can be directly compared to the wild-type protein under different activities of the β-catenin destruction complex. The differential tagging of a wild-type and mutant Wnt components offers the opportunity to compare directly their properties in a living cell, and the approach is likely to be useful for future mechanistic investigations by the Wnt and cancer communities.Essential revisions:The authors show that wt and mutant β-catenins both diffuse in larger complexes that differ between wt and mutant, possibly destruction complexes vs. transcriptional partners. These are interesting observations that may ultimately provide insights into the mechanism of β-catenin destruction. There is concern regarding the apparent degradation of the cherry fluorescent protein, and although the authors rationalize what is happening based on prior literature, there is no experimental validation of these ideas. Nonetheless, the ability to IP the cherry tag and the observation of fluorescence from this tagged version is consistent with their interpretation. There are, however, a number of technical issues that require attention, specifically a more rigorous validation that the targeting is correct, and better support for the conclusions regarding nuclear β-catenin translocation.1. The genotyping is not adequate to prove both alleles have been correctly targeted in the double targeted clone.In the case of the double targeted cells, specific genotyping is required for the 5' and 3' ends of both alleles. As the 3' genotyping only is the same primer set for both the Cherry and Clover cassettes it is possible that only one is targeted. 3' genotyping should be shown both for the Cherry and Clover alleles separately. Especially as CRISPR was used to generate these, it is quite possible the other allele has an NHEJ generated mutation which does not amplify by PCR, meaning that the positive band is derived from just one targeted allele  .2. Some of the clones are apparently positive in the wrong genotyping reactions.Clone 33 and 24 are supposedly heterozygous for the Clover allele but also appear to be positive for the specific Cherry allele . This may indicate that the clones are carrying extra integrated copies of the targeting vectors or that the clones analysed contain a mixture of cells both of which may impact the downstream analysis.Clone 37 also has an apparent 3rd band when PCR is performed with the internal primer set . This may indicate a duplication of the cassette at the targeted locus. This is especially important to resolve given that the data presented does not prove both alleles are correctly targeted at the 3' end.3. Sequencing of the targeted alleles had not been done or has not been adequately detailedThere is no real description of the sequencing done on the targeting alleles. It is not uncommon for CRISPR generated alleles to have rearrangements including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated :70.). Consequently it is important that these alleles should be properly characterised.4. Although the quantification  shows increase in nuclear/cytoplasmic ratio of β-catenin, the images presented do not seem to support this. Can the authors present zoomed-in images of different cells to demonstrate that this is the case?5. There seems to be more perinuclear puncta for Cherry-β-catenin in general compared to Clover-β-catenin, and they increase in the presence of CHIR99021 compared to DMSO control. The authors do not comment on this, and how it might possibly impact the quantification of the images, given that these puncta are outside the nuclear area.6. Consistent with point #2 above, siCLOVER experiments  demonstrate that a pool of Cherry-β-catenin seems to localize at the perinuclear region in cells. The authors do not comment on this and also do not provide quantification of the fluorescence imaging experiments to support their conclusion.7. How do clover-WT and Cherry-mut-β-catenin diffuse in response to treatment with exogenous Wnt ligand, given that Wnt secretion is still required for Wnt signaling activity in HCT116 cells? Is there a difference in their nuclear translocation?8. Why is there a band corresponding to Cherry β-catenin in the western blot of clone #33 ? The authors do not comment on this. Also, were all lanes loaded with equivalent amounts of total protein? The β-catenin expression levels in knock-ins are clearly higher than in the parental line.eLife. Your article has been reviewed by 2 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Marianne Bronner as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Owen J Sansom (Reviewer #3).Thank you for submitting your article "Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. Regarding the new experiments looking at diffusion after treatment with porcupine inhibitor, which show some level of Wnt regulation of the Δ45 mutant, have the authors considered that GSK3 inhibition produced by Wnt treatment will also reduce the association of β-catenin with APC; perhaps this is the origin of their observation.2. The discussion starting at line 430 could use some clarification. Can the authors be more specific as to what they think are the causes of the difference in diffusivity of the WT and mutant alleles? One can imagine that the mutant is bound to Axin and APC in the destruction complex and not turned over as fast as WT. The authors also mention association with transcription factors, and also cytoplasmic-nuclear shuttling of destruction complex components. Perhaps a cartoon or at least a clear delineation of the possible reasons for these behaviors would help.3. Figure S1d is frequently referred to but is not clearly indicated. I assume it's part of S1B but not clear. These supplemental figures are also called out in the rebuttal but don't exactly correspond to what is in the manuscript. Lowercase vs. uppercase? Labeling of the individual FACS plots is needed. Essential revisions:The authors show that wt and mutant β-catenins both diffuse in larger complexes that differ between wt and mutant, possibly destruction complexes vs. transcriptional partners. These are interesting observations that may ultimately provide insights into the mechanism of β-catenin destruction. There is concern regarding the apparent degradation of the cherry fluorescent protein, and although the authors rationalize what is happening based on prior literature, there is no experimental validation of these ideas. Nonetheless, the ability to IP the cherry tag and the observation of fluorescence from this tagged version is consistent with their interpretation. There are, however, a number of technical issues that require attention, specifically a more rigorous validation that the targeting is correct, and better support for the conclusions regarding nuclear β-catenin translocation.1. The genotyping is not adequate to prove both alleles have been correctly targeted in the double targeted clone.In the case of the double targeted cells, specific genotyping is required for the 5' and 3' ends of both alleles. As the 3' genotyping only is the same primer set for both the Cherry and Clover cassettes it is possible that only one is targeted. 3' genotyping should be shown both for the Cherry and Clover alleles separately. Especially as CRISPR was used to generate these, it is quite possible the other allele has an NHEJ generated mutation which does not amplify by PCR, meaning that the positive band is derived from just one targeted allele  .To validate the correct integration of Cherry and Clover in each allele, we performed the additional experiments suggested by the reviewers . We now show 3’ genotyping with three different primer pairs . While primer pair e selectively amplifies the Cherry-tagged allele and e’ selectively amplifies the Clover-tagged allele, primer set c amplifies both alleles, which can be distinguished by their size, due to the different lengths of the antibiotic resistance markers . All three PCRs support the correct integration of each cassette in the different clones. To make these results more accessible, we now show all primer pairs separately in Modified Figure 1—figure supplement 2A.Furthermore, we have also performed additional genotyping of the double targeted clone 37 with primers annealing 5’ and 3’ of the integration cassettes outside of the homology arms . This further supports the correct integration and does not indicate that multiple copies of the cassette are present in the target locus. Moreover, we have used the PCR amplicons spanning the modified locus for Sanger sequencing , which further supports successful gene targeting. We have revised the text to better explain these results.Specifically, we have made the following changes to the text:WT/∆45) cell line and to an HCT116 isogenic cell line in which the ∆Ser45 allele was removed (β-cateninWT+/-) . First, to analyze them in depth, we used primers annealing upstream and downstream of the fluorophore tags (primers (a) in Figure 1—figure supplement 2A). While PCR amplicons of successfully tagged CTNNB1/β-catenin alleles are expected to be more than 3 kb in length, amplicons from the untagged locus would be approximately 1.2 kb. PCR from genomic DNA from clone #37 resulted in a single band of 3kb, indicating that both alleles of CTNNB1 were edited. In contrast, genotyping of clones #33, #45 and #24 resulted in 3 kb and 1.2 kb amplicons, suggesting that only a single donor integrated in one allele . Since genotyping with primers annealing within the donor construct cannot distinguish between homology-mediated integration at the target locus and non-targeted integration elsewhere in the genome, we next performed genotyping PCRs with one primer annealing in the CTNNB1 locus and the other inside the integration cassette  .....”“In the following we focus on clones #24, #33, #37, #45, as they were found to represent different integration events. To confirm the editing events, genomic DNA, mRNA and protein lysates were analyzed and compared to the parental HCT116 . This may indicate that the clones are carrying extra integrated copies of the targeting vectors or that the clones analysed contain a mixture of cells both of which may impact the downstream analysis.We thank the reviewers for this comment. To further verify the genotype of the indicated clones, we performed additional PCRs . These include primer sets, which distinguish the Cherry and Clover alleles by their BRS/blasticidin  and HGR/hygromycin  resistance genes. Clones #24 and #33 result in bands with primer pair e’, but no amplification with primer pair e, indicating that they exclusively harbor a Clover-tagged allele . Importantly, we also performed PCR with primer pair c, where both primers anneal inside the donor plasmid and would therefore also amplify integrations elsewhere in the genome . Due to the size difference of the blasticidin and hygromycin resistance genes, this PCR can differentiate between both donor templates. In clones #24 and #33, we exclusively detect a larger PCR amplicon expected from the Clover donor. Together, these assays support that clones #24 and #33 only contain a single integration of the Clover donor template in one β-catenin allele.Clone 37 also has an apparent 3rd band when PCR is performed with the internal primer set . This may indicate a duplication of the cassette at the targeted locus. This is especially important to resolve given that the data presented does not prove both alleles are correctly targeted at the 3' end.As described above, we performed genotyping with a number of additional primer pairs . We would expect to detect larger bands in the case of full or partial cassette duplication by most of the primers . We do not detect such bands, however, indicating single copy integration of our donor sequences. Occasionally, a weak and often fuzzy DNA signal appears above the main bands, which we interpret as an unspecific amplification event or artefact of the agarose gels.3. Sequencing of the targeted alleles had not been done or has not been adequately detailed.There is no real description of the sequencing done on the targeting alleles. It is not uncommon for CRISPR generated alleles to have rearrangements including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated :70.). Consequently it is important that these alleles should be properly characterised.We thank the reviewers for highlighting that the sequencing results were not adequately discussed in the initial submission. We sequenced the whole set of clones and the results are summarized in the table presented as Figure S1C. As expected, we found that several clones carried mutations and/or rearrangements . Sequencing also confirmed that only clone #37, which has been used for the bi-allelic comparison presented in this manuscript, harbors an integration cassette in both alleles. Importantly, extended sequence analysis of clone #37 covered both integrated regions completely and confirmed correct integration .4. Although the quantification  shows increase in nuclear/cytoplasmic ratio of β-catenin, the images presented do not seem to support this. Can the authors present zoomed-in images of different cells to demonstrate that this is the case?To clarify this point we have added enlarged images and show them in the New Figure 4—figure supplement 2.5. There seems to be more perinuclear puncta for Cherry-β-catenin in general compared to Clover-β-catenin, and they increase in the presence of CHIR99021 compared to DMSO control. The authors do not comment on this, and how it might possibly impact the quantification of the images, given that these puncta are outside the nuclear area.As suggested by the reviewer, we looked specifically for perinuclear puncta of both alleles of βcatenin and detected them for both WT and mutated alleles. We did not quantify them, since this was not the focus of our study, but we did not notice a significant difference. We could detect cells without these puncta, just puncta with one color  or yellow . Similar structures were described upon overexpression of the amino-terminal part of β-catenin by Giannini et al. (2000).6. Consistent with point #2 above, siCLOVER experiments  demonstrate that a pool of Cherry-β-catenin seems to localize at the perinuclear region in cells. The authors do not comment on this and also do not provide quantification of the fluorescence imaging experiments to support their conclusion.As suggested by the reviewer, we quantified immunofluorescence as shown in New Figure 5B, New Figure 5—figure supplement 1. Since β-catenin is difficult to detect on an endogenous level, its quantification is challenging, especially upon downregulation, and interpretation of the results is limited. New Figure 5B represents one of the four biologically independent experiments and normalized results of four independent ones are provided in New Figure 5—figure supplement 1.7. How do clover-WT and Cherry-mut-β-catenin diffuse in response to treatment with exogenous Wnt ligand, given that Wnt secretion is still required for Wnt signaling activity in HCT116 cells? Is there a difference in their nuclear translocation?As indicated by the reviewer, HCT116 cells express canonical Wnt ligands, resulting in strong Wnt pathway activity. Therefore, it is not surprising that no changes were detected by IF upon addition of recombinant Wnt3a . Presumably, the cells reached maximal Wnt activity even without Wnt ligand stimulation, as shown previously . Hence, to address the role of Wnt secretion, we inhibited Wnt protein secretion using the porcupine inhibitor LGK974. First, we treated HCT116 #37 cells with the porcupine inhibitor LGK974 for five to six days to inhibit endogenous Wnt ligand secretion, then added 200 ng/ml of recombinant Wnt3a to induce Wnt signaling and performed immunofluorescence analysis 16 h later. Upon inhibition of Wnt secretion from HCT116 #37 cells, we observed a strong decrease of β-catenin that was rescued upon addition of recombinant Wnt3a . Surprisingly, not only β-catenin-Clover WT protein but also β-catenin-Cherry ∆Ser45 protein was regulated by LGK974. As we saw striking differences in the signal of β-catenin, we cannot measure translocation from the cytoplasm to the nucleus of the protein.WT,LGK = 36 ± 10 nM to CWT,LGK = 7.2 ± 2.6 nM. Additional treatment with recombinant Wnt3a raised the concentration again and almost reversed the effects of the LGK treatment . The concentration of the mutant β-catenin-Cherry decreased from CΔ45,DMSO = 101 ± 18 nM to CΔ45,LGK = 79 ± 13 nM upon LGK974 treatment and rose again to CΔ45,LGK+Wnt3a = 94 ± 22 nM by adding Wnt3a .The quantification of IF data of endogenous β-catenin especially upon signal reduction could lead to an over-interpretation of the data as the signal intensity is not linear. To confirm this result, we performed FCS measurements of β-catenin alleles after LGK794 treatment. Indeed, concentrations of both alleles of β-catenin are regulated by LGK974 treatment. β-catenin-Clover WT protein concentration in the cytoplasm is strongly dependent on Wnt secretion, for which the concentration in the cytosol was reduced from C8. Why is there a band corresponding to Cherry β-catenin in the western blot of clone #33 ? The authors do not comment on this. Also, were all lanes loaded with equivalent amounts of total protein? The β-catenin expression levels in knock-ins are clearly higher than in the parental line.To improve the presentation of Figure 2B, we now indicated the different isoforms of β-catenin in the Western blot. Regarding clone #33, the lower band corresponds to the WT allele of β-catenin indicating the heterozygosity of clone #33. β-actin serves as a loading control in Figure 2B. Since less total protein was detected in the lane of the parental cell line, this might explain the lower β-catenin levels. Comparison of qPCR  and CTNNB1/β-catenin mRNA expression levels indicated no differences between parental cells and clones. To clarify this point, we made additional Western blots to compare the parental cells and clone #37 and added them as New Figure 2—figure supplement 1A.Essential revisions:1. Regarding the new experiments looking at diffusion after treatment with porcupine inhibitor, which show some level of Wnt regulation of the Δ45 mutant, have the authors considered that GSK3 inhibition produced by Wnt treatment will also reduce the association of β-catenin with APC; perhaps this is the origin of their observation.Our results indicate that the mutant form of β-catenin can also (in part) be regulated by Wnt signaling, likely involving the regulation of its interaction with APC. In line with our report, previous studies have indicated that β-catenin mutants with deletion or a mutation of Ser 45 retain signaling activity and are still responsive towards Wnt activation (PMID: 12060769 and PMID: 32751567). Together, these data suggest that, despite the absence of Ser45 phosphorylation, interactions of β-catenin with the destruction complex and its proteasomal degradation still occur. Further studies will be required to test if inhibition of GSK3 after Wnt stimulation is an essential mechanism that controls the cellular behavior of mutant β-catenin, including its interaction with APC.2. The discussion starting at line 430 could use some clarification. Can the authors be more specific as to what they think are the causes of the difference in diffusivity of the WT and mutant alleles? One can imagine that the mutant is bound to Axin and APC in the destruction complex and not turned over as fast as WT. The authors also mention association with transcription factors, and also cytoplasmic-nuclear shuttling of destruction complex components. Perhaps a cartoon or at least a clear delineation of the possible reasons for these behaviors would help.As suggested by the reviewers, we have rephrased and expanded this part of the discussion to indicate the different possibilities. In short, wt β-catenin protein diffuses faster than mutant β-catenin, suggesting that wt protein is present as individual molecules or as part of smaller complexes, whereas the mutant is part of larger, more slowly diffusing complexes. FCS analysis only reveals the size but not the composition of the complexes. Hence, we can only speculate about the different possibilities. For example, in the nucleus, the mutant form presumably already interacts with the Wnt enhanceosome, in the absence of stimulation. We agree with the reviewer that, in the cytoplasm, the mutant form is likely to interact with the destruction complex, but might not be turned over as fast as the wt isoform. In the revised manuscript, we discuss these possibilities in much more detail.3. Figure S1d is frequently referred to but is not clearly indicated. I assume it's part of S1B but not clear. These supplemental figures are also called out in the rebuttal but don't exactly correspond to what is in the manuscript. Lowercase vs. uppercase? Labeling of the individual FACS plots is needed.We would like to thank the reviewer for pointing out the inconsistency in the figure labels, which we have corrected in the revised manuscript. We have added the labeling of the FACS plots, which was accidentally removed from the Figure 1—figure supplement 1B. Now every individual plot is labeled as (i), (ii), (iii) and (iv). We have also corrected the inconsistencies in the figure labeling.


Pyrvinium pamoate (PP), the FDA-approved anthelminthic drug, has been reported to inhibit the Wnt/β-catenin pathway within numerous cancer types, and Wnt/β-catenin signaling pathway can modulate the expression of MGMT gene. However, whether PP affects the expression of MGMT and enhances TMZ sensitivity in GBM cells remains unclear. In the present study, we found that PP and TMZ had synergistic effect on inhibiting the viability of GBM cells, and PP induced inhibition of MGMT and enhanced the TMZ chemosensitivity of GBM cells through down-regulating Wnt/β-catenin pathway. Moreover, the overexpression of MGMT or β-catenin weakened the synergy between PP and TMZ. The mechanism of PP in inhibiting the Wnt pathway was indicated that PP resulted in the degradation of β-catenin via the AKT/GSK3β/β-catenin signaling axis. Moreover, Ser552 phosphorylation in β-catenin, which promotes its nuclear accumulation and transcriptional activity, is blocked by PP that also inhibits the Wnt pathway to some extent. The intracranial GBM mouse model also demonstrated that the synergy between PP and TMZ could be achieved through down-regulating β-catenin and MGMT, which prolonged the survival time of tumor-bearing mice. Taken together, our data suggest that PP may serve as the prospect medicine to improve the chemotherapeutic effect on GBM, especially for chemoresistant to TMZ induced by MGMT overexpression.Temozolomide (TMZ) is the mainstream chemotherapeutic drug for treating glioblastoma multiforme (GBM), but the intrinsic or acquired chemoresistance to TMZ has become the leading clinical concern, which is related to the repair of DNA alkylation sites by O Currll death . As a DNA injury . The higA injury , 9. TherA injury , 11.Pyrvinium pamoate (PP) Fig.  is a claβ-catenin is the pivotal downstream effector in the canonical Wnt signaling pathway, which combines with the LEF/TCF transcription factors (TFs) complex in cell nucleus to activate the expression of target gene , 18. BesTo explore the PP role in GBM cell proliferation in vitro, MTT assay was performed to assess the viability rates of LN18, T98G, LN229, and U87MG cells. According to Figs. Cell apoptosis was examined through flow cytometry with annexin V/ PI analysis following 48 h of PP treatments. According to Fig. For exploring whether PP affected the cell cycle, flow cytometry was carried out to analyze the DNA content of LN18 and T98G cell. Briefly, the LN18 and T98G cell lines were subjected to 48 h of PP treatments at different doses. As shown in Fig. MGMT, one of the DNA repair proteins, removes the cytotoxic lesions caused by TMZ ; besidesFor better exploring the MGMT effect on the PP-induced enhanced anti-tumor effect of TMZ chemotherapy, the scrambled control or MGMT cDNA plasmid was transfected into LN18 and T98G cells. MGMT protein expression increased with the MGMT overexpression within both cell lines either in the presence or absence of PP treatment Fig. . FurtherPP is suggested to inhibit the Wnt/β-catenin signaling pathway within certain cancer types , 34–36. For exploring the role of β-catenin in regulating MGMT level as well as the GBM cell chemosensitivity to TMZ, both LN18 and T98G cells were transfected with scrambled or β-catenin shRNA. According to Figs. To examine the β-catenin effect on the PP-caused MGMT suppression and the enhancement in TMZ chemosensitivity, scrambled control or β-catenin cDNA was transfected into LN18 and T98G cells. β-catenin overexpression partially restored MGMT and β-catenin down-regulation induced by PP Fig. . In addiβ-catenin metabolism is mainly controlled by ubiquitination and proteasomal degradation –40. TypiPrevious studies show that Ser552 of β-catenin can be phosphorylated by AKT, and this results in β-catenin disassociation from the contacts between cells, increasing the transcriptional activity of β-catenin and promoting tumor cell invasion . TherefoThe intracranial orthotopic GBM xenograft mouse model was established for evaluating the effect and tolerability of PP in vivo. U87MG-Luc cells were injected into brain hippocampus. One week later, the mice were randomized as four groups, including control (vehicle), intraperitoneal injection of PP at 0.5 mg/kg/day for 3 consecutive weeks, gavage of TMZ at 20 mg/kg/day for 3 consecutive weeks, and PP at 0.5 mg/kg combined with 20 mg/kg TMZ for 3 consecutive weeks. Bioluminescence imaging (BLI) and hematoxylin-eosin (HE) staining of brain tumor sections revealed the markedly reduced tumor volume in PP treatment group compared with control group Fig. . In addiAt present, TMZ is verified as the only chemotherapeutic that remarkably extend the overall survival (OS) for GBM , 11. HowThis study first examined the individual drug PP effect on GBM. The results suggested that, PP obviously inhibited GBM cell proliferation but induced apoptosis through up-regulating pro-apoptotic proteins cleaved caspase-3, cleaved PARP-1 and Bax, while down-regulating the anti-apoptotic protein survivin. Besides, expression of Bcl-2, the anti-apoptotic protein, was not affected. On the other hand, the Bax/Bcl-2 ratio may determine the susceptibility of cells to apoptosis following an apoptotic stimulus . TherefoAberrant activation of the Wnt/β-catenin signaling pathway frequently occurs among various type of cancers, including GBM –53. The Accumulating evidences suggests that, PP inhibits the Wnt signaling pathway and decreases β-catenin level in some cancer types –36, 57, β-catenin is a dual function protein that has at least two distinct pools, among which, one binds with E-cadherin and is located in cytoplasm, while the other one participates in the Wnt signaling and shuttles between cytoplasm and nucleus , 61. AKTTo sum up, this work has first demonstrated that, PP enhances the GBM chemosensitivity to TMZ both in vivo and in vitro. Further, PP mediates MGMT suppression through β-catenin to achieve TMZ sensitization. Additionally, it should be noted that, the effect of PP on suppressing the Wnt signaling pathway may be mediated through β-catenin degradation and anchoring with cell membranes. The above observations suggest the potential of using PP as a TMZ chemotherapy sensitizer in GBM treatment.PP was provided by MedChem Express . CHIR-99021 was provided by Selleckchem Company . TMZ and lithium chloride were provided by Sigma . The anti-Cleaved Caspase-3, anti-Caspase 9, anti-Bcl-2, anti-Bax, anti-Survivin, anti-cyclin B1, anti-cyclin D1, anti-β-catenin, anti-p-β-catenin (S552), anti-p-β-catenin (S33/37/T41), anti-GSK-3β, anti-p-GSK-3β (Ser9), anti-AKT, anti-p-AKT, anti-TBP, anti-GAPDH, and anti-histone H3 antibodies were provided by Cell Signaling Technology . The anti-MGMT and anti-β-actin antibodies were provided by Abcam . The anti-p-GSK3β (Y216) antibody was provided by Thermo . The anti-PARP-1 antibody was provided by Santa Cruz Biotechnology .3 cells/well were planted into 96-well plates to culture for a period of 12 h. Then, the selected medicine at specific contents was used to treat cells for specific times. Later, the MTT assay was carried out to measure cell viability, which was presented in the form of absorbance (%) in untreated cells at the wavelength of 570 nm.The human LN18, T98G, LN229, and U87MG GBM cells, provided by ATCC, were cultured in DMEM containing 100 μg/mL streptomycin, 100 U/mL penicillin, and 10% fetal bovine serum (FBS) under 5% CO2 and 37 °C conditions. STR profiling was used to authenticate GBM cells and all the cells were verified none mycoplasma contamination. In all, 5 × 10The LN18 and T98G cells (1000 cells/well) were inoculated into six-well plates for 24 h and then treated with PP at indicated contents for 10 days. Later, these cells were subjected to 4% formaldehyde fixation and 0.2% crystal violet staining. Each well in the six-well plates was photographed and colonies that contained ≥50 cells were counted microscopically under ×4 magnification.LN18 and T98G cells were treated with the indicated concentration of PP for 48 h and then collected. For cell apoptosis analysis, cells were stained with Annexin V and PI according to the manufacturer’s instructions of the Annexin V/PI Apoptosis Detection kit . Flow cytometry was performed and the data was analyzed using FlowJo Version 10.0 software. For the cell cycle analysis, those collected cells were subjected to 70% ethanol treatment under the temperature of −20 °C overnight, followed by 25 min of PI (BD) and RNase (BD) treatment in dark. The flow cytometry was conducted and ModFit LT 3.3 was adopted for data analysis.The RIPA buffer  containing the phosphatase and protease inhibitors was employed to lyse the frozen GBM xenograft tissues and cultured GBM cells. Then BCA protein detection kit  was adopted to determine protein content. After 10 min of heating in the 100 °C water, the protein samples were isolated through SDS-PAGE, followed by transfer to the PVDF films . Afterwards, the films were blocked using 5% skim milk for 30 min under ambient temperature, incubated using appropriate primary antibodies under the temperature of 4 °C overnight, and then incubated using the secondary peroxidase-conjugated goat anti-rabbit antibody for 2 h under ambient temperature. Finally, the Odyssey Infrared Imaging System (LI-COR) was used to analyze the immunoblots.The TRIzol  was utilized to isolate the total RNA based on GBM cells in accordance with manufacturer protocols. Later, the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit  was used to prepare cDNA from the extracted RNA. Later, the SYBR green PCR master mix  was utilized to perform qRT-PCR onto the MxPro-Mx3005 P Real-time PCR system . All primers used in this assay were prepared via Sangon Biotech , including:GAPDH: 5′-ATCATCCCTGCCTCTACTGG-3′ (forward),GAPDH: 5′-GTCAGGTCCACCACTGACAC-3′(reverse);MGMT: 5′- GTTTTCCAGCAAGAGTCGTTC -3′ (forward),MGMT: 5′- GCTGCTAATTGCTGGTAAGAAA-3′ (reverse).GAPDH served as the internal reference.The type of interaction produced by TMZ and PP combination was performed using two methods, the isobologram analysis using CalcuSyn software version 2  and the dose-matrix approach using Combenefit software  according to the instructions , 64. ComLipofectamine 2000  was used to transfect scrambled shRNA, β-catenin shRNA, scrambled cDNA, β-catenin cDNA, or MGMT cDNA  into the LN18 and T98G cell lines following manufacturer protocols. Thereafter, the original medium was discarded, while complete DMEM medium was added after 6 h of transfection. Later, cells were incubated with selected drug after transfection for 24 h, and they were then used in later analysis.LN18 cells were subjected to 24 h of cultivation onto the uncoated glass slides, followed by another 24 h of PP treatment. The cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at 37 °C, permeabilized with 0.1% Triton X-100 for 10 min, and blocked in 5% BSA (Sigma) for 1 h at room temperature. The cells were immunostained with an antibody to β catenin  at 4 °C overnight and subsequently incubated with FITC conjugated secondary antibodies  for 0.5 h at room temperature in darkness. Cell nuclei was stained with DAPI , and the LSM 880 confocal laser scanning microscope  was performed to visualize cells.LN18 and T98G cells cultured in 24-well plates were co-transfected with 20 ng pRL-TK  and 500 ng of M51 Super 8 × FOPflash or M50 Super 8 × TOPflash (TOPflash mutant) plasmids for 24 h before another 24 h of PP treatment. Subsequently, dual-luciferase reporter assay was carried out in accordance with manufacturer protocols by the use of the assay kit (Promega).http://jaspar.genereg.net/).MGMT gene sequence was searched using NCBI Genome Database. The 717 bp, 1308 bp and 1951 bp fragments of MGMT promoter region were cloned to the pEASY-T5 Zero cloning vector , then cut with HindIII/XholI and subcloned at the HindIII/XholI site of PGL 4.1 basic vector to generate p-755/−39 ML, p-1346/−39 ML and p-1989/−39 ML, respectively. These above-mentioned 3 MGMT promoter constructs possessed 2, 4 and 8 candidate binding sites of TCF/LEF Fig. , respectLN18 and T98G cell cultured in 24-wells plate were co-transfected with 500 ng/well MGMT promoter reporter plasmids and 20 ng/well pRL-TK , followed by 24 h of PP or LiCl treatment. After 48 h of transfection, cells were harvested, and the detection kit (Promega) was used to measure the luciferase activity of the cells.5) were suspended in 2 μl PBS and injected into brain hippocampus. Tumor formation was verified by bioluminescence imaging (BLI) of all transplanted mice.The 8-week-old female BALB/c nude mice, provided by Charles River , were kept under the specific pathogen-free environment with free access to food and water. The animal procedure conformed to the stipulations outlined in Guide for the Care and Use of Laboratory Animals. The Jilin University Animal Care and Use Committee had approved our study protocol. For establishing an intracranial brain tumor-bearing mouse model, U87MG-Luc cells (2 × 10n = 5 mice per group). BLI was carried out to detect the intracranial tumor growth with IVIS Spectrum Live Imaging System . The survival rate of mice in every group was observed.One week after tumor cells inoculation, the mice were randomized as four groups, including control (vehicle), intraperitoneal injection of PP at 0.5 mg/kg/day for 3 consecutive weeks, gavage of TMZ at 20 mg/kg/day for 3 consecutive weeks, and 0.5 mg/kg PP combined with 20 mg/kg TMZ (n = 3 mice per group). All mice from the two groups were sacrificed 48 h after the last treatment (at day 30 after tumor implantation), and the brains or tumors of the mice in both groups were harvested for histopathology, TUNEL staining and Western blot analysis.In order to explore the mechanisms of PP on GBM in vivo, U87MG-Luc tumor-bearing mice were divided into two groups and intraperitoneally injected of PP at 0.5 mg/kg/day, or PBS for 3 consecutive weeks  and photographed.The tumor slides were subjected to xylene deparaffinage and gradient alcohol hydration, 4% paraformaldehyde fixation, and Proteinase K solution permeabilization in succession. Then, the in situ cell apoptosis detection kit  was utilized according to the manufacturer’s instructions to detect the stained slides using CLSM.t test and differences between more than two groups were compared by one-way ANOVA with Tukey’s multiple comparison. For analyzing the Kaplan–Meier survival curves, the Mantel–Cox test was used. Statistical analyses were carried out with GraphPad Prism 8.0 and P-values of <0.05 indicated that difference was statistically significant.All data obtained from at least three independent experiments and were expressed as mean ± standard deviation (SD). The differences between two groups were conducted using two-tailed unpaired Student’s Figure S1Figure S2Figure S3Figure S4Supplementary information


F = 0.745, p = 0.54. Thus, we did not find that different images had a significant influence on donations to conservation. This may be due to our choice of visual frames or to the use of a donation box, which is a passive fundraising channel. Future research should examine how visual framing influences donations in other public settings and should test the influence of other visual frames on philanthropic behavior.Many environmental organizations use photographic images to engage donors and supporters. While images play a role in fundraising, visual framing remains understudied in the environmental field. Few real-world experiments have examined which types of images result in higher donations to biodiversity conservation. We examined the role of images in conservation fundraising through a public experiment at Zoomarine, a marine park located in southern Portugal. Zoomarine runs a program called Dolphin Emotions where visitors pay to learn about dolphin biology and to interact with dolphins. We placed a donation box and a large informational poster about the Marine Megafauna Foundation, a conservation partner, in the lounge of the Dolphin Emotions program, which is open to participants and their families. The text on the poster, which solicited donations for the Marine Megafauna Foundation, was held constant, while four different image conditions were tested: dolphins, ocean wildlife, children, and people staring out from the poster . Each image condition was displayed for three days at a time and was on display for at least seven randomly assigned three-day periods over the course of 91 days. 20,944 visitors passed the donation box and the four poster conditions during this time and a total of € 952.40 was collected. The differences in mean donations in € per visitor per 3-day period were not statistically significant,  Many environmental non-governmental organizations rely on public donations to fund their conservation efforts , 2. To ePhotographic images are frequently used in fundraising campaigns for environmental causes and are generally regarded as having an impact on donor behavior –11. HoweFraming theory, which helps explain how the way an issue is presented influences how people respond to that issue, can be used to study people’s responses to different types of images , 16. By It is possible that certain visual frames could increase pro-social behaviors, including monetary donations to conservation. Certain visual cues may influence charitable giving and pro-social behavior, including images of charismatic flagship species , images In this study, we sought to address these gaps by running a real-world experiment at a marine park in Portugal to test the influence of different visual frames on charitable donations to a conservation project. We used a randomized controlled trial to test four different visual framing conditions paired with a donation box. We measured actual donations and compared the donations received for each visual framing condition.The experiment took place at Zoomarine, a marine park located in southern Portugal . ZoomariFor this study, a transparent donation box and large informational poster (207 x 90 cm) were placed in the public lounge of the Dolphin Emotions program, which only program participants and their families are able to access . The posThe experiment ran for a total of 91 days between June 28 and September 26, 2019, although data were only collected for 87 days due to park closures on four days. The text displayed on the poster (which was in both English and Portuguese) and the video playing on the screen related to the ocean conservation work of the Marine Megafauna Foundation, a partner of Zoomarine, and was held constant throughout the experiment. The short video played on a loop on the screen and highlighted the positive efforts of the organization to conserve ocean wildlife. The board clearly stated that all collected funds would be given to the Marine Megafauna Foundation. Four poster conditions were tested, differing only in terms of the images presented . Each poThe first condition displayed images of dolphins, the flagship species of the Dolphin Emotions program and a species that is thought to be highly charismatic . We usedThe second condition used images of multiple charismatic flagship species from the ocean  to test whether the use of a flagship fleet, or a group of flagship species, was more effective than a single flagship species . This coThe third condition included images of happy children to test whether framing the issue in terms of its influence on humans would be more effective. Altruism is a strong motivator for charitable giving , 28 and The fourth condition included images of people staring out from the poster to test the “watching eyes” hypothesis, which suggests the feeling of being watched by eyes that are directly gazing at the viewer may increase the likelihood that individuals engage in pro-social behaviors, such as charitable giving , 30. FieAt the beginning of each three-day period, the same amount of seed money (€ 100) was put in the box before visitors arrived to encourage further donations. At the end of each three-day treatment period, the amount of money collected was withdrawn from the donation box and counted.n = 7), three-day periods with images of ocean wildlife condition (n = 7), three-day periods with images of children condition (n = 8), and three-day periods with images of watching eyes condition (n = 7). The experiment ran for 91 days and data were collected on 87 of those days. A total of 20,944 visitors passed by the donation box and the different poster conditions during this time. In total, € 952.40 were collected in the donation box with 84% of that amount being in coins with a value of €1 or less. The mean and median number of visitors per 3-day period for each poster condition were somewhat different: dolphin condition , watching eyes condition , ocean wildlife condition , and children condition . However, while there are differences between conditions, we have adjusted for this difference by taking the mean donation per visitor for each three-day period.There were four conditions, each in place for at least seven three-day periods: three-day periods with images of dolphins condition (p = 0.187). There were three outliers in the data (3-day periods that had higher donations per visitor than other periods for a condition), as assessed by boxplots, but the test results were not materially affected by them, as determined by comparing the result of a one-way ANOVA with and without the outliers (p = 0.141), children (p = 0.469), and ocean wildlife conditions (p = 0.392), the data for the dolphin condition was not normally distributed (p = 0.037). Mean donations in € per visitor per 3-day period for each condition increased from the watching eyes condition , to the children condition , to the dolphins condition , to the ocean wildlife condition , in that order, but the differences between the different conditions were not statistically significant, F = 0.745, p = 0.54 (2(3) = 1.576, p = 0.665.We found that neither a one-way ANOVA (parametric) nor a Kruskall Wallis H test (nonparametric) showed significant differences between treatments in terms of the mean donation per visitor per 3-day period. We first used a one-way ANOVA, a more conservative test, to determine if the mean donation per visitor per 3-day period (in €) was different for different poster conditions. There was homogeneity of variances, as assessed by Levene’s test for equality of variances  were in coins of €1 or less. The use of a cash-based donation box is another limitation. Other visitors may have wanted to donate (or to donate larger amounts) but may not have had cash with them. Future experiments could include a card terminal allowing for contactless payments or a text-to-give option to ensure that all visitors have the opportunity to contribute. Additionally, many visitors were likely in family units and may have made a donation as a group. This suggests that it might be interesting to calculate donation per group instead of donation per individual visitor in future studies. Finally, the video in the poster likely created tradeoffs. On the one hand, the video may have attracted people who would not have otherwise looked at the display. On the other hand, it might have competed for the attention of visitors, obscuring the effect of different poster conditions . The latIt is clear that much remains to be understood when it comes to the relationship between visual marketing and charitable giving. Future research should examine how visual framing influences donation behavior in public settings and should test these effects outside of animal-oriented settings, such as zoos and marine parks, to see if the other public audiences respond differently to visual frames. These insights may well reveal themselves to be critical to the effectiveness and efficiency of fundraising for biodiversity conservation.S1 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


Recently, phenylboronic acid (PBA) gel containing microneedle (MN) technology with acute and sustained glucose-sensitive functionality has attracted significant research attention. Herein, we report a polyvinyl alcohol(PVA)-coated MNs patch with an interconnected porous gel drug reservoir for enhanced skin penetration efficiency and mechanical strength. The hybrid MNs patch fabricated with a novel, efficient method displayed a “cake-like” two-layer structure, with the tip part being composed of boronate-containing smart gel attached to a porous gel layer as a drug reservoir. The porous structure provides the necessary structural support for skin insertion and space for insulin loading. The mechanical strength of the hybrid MNs patch was further enhanced by surface coating with crystallized PVA. Compared with MNs patches attached to hollow drug reservoirs, this hybrid MNs patch with a porous gel reservoir was shown to be able to penetrate the skin more effectively, and is promising for on-demand, long-acting transdermal insulin delivery with increased patient compliance. Thus, barrier . To over barrier ,8,9. Amo barrier . MN patc barrier . Therefo barrier .x) [x polymeric nanoparticles embedded smart insulin MN patches are capable of providing on-demand insulin release; this is essential to achieve continuous glycemic control in a manner which is minimally dependent on patient compliance [x, such as intolerance of long-term use, protein-denaturing issues during storage, the creation of toxic byproducts via reactions with glucose, and “burst-like” release behavior in response to hyperglycemia conditions, may affect its wide application [Another concern associated with insulin therapy is inconsistent glycemia control. Overdoses of insulin can result in hypoglycemia that may lead to behavioral and cognitive disturbance, coma, brain damage, and even death . Smart ix) , glucosex)  phenylbox) , negate x) . For exampliance ,18,19. Hlication ,20,21,22lication . In addilication .mECFPBA), achieving a pKa value of about 7.2, i.e., close to physiological pH [mECFPBA undergoes a glucose-dependent change in anionic boronate fraction, combining it with acrylamide hydrogel generates a surface localized, microscopically dehydrated “skin layer” in response to glucose. This “skin layer” is able to control the diffusion of the loaded insulin, and disappears upon hydration with an increase of glucose concentration. Thus, a “skin layer” driven insulin release control capacity allows the synthetic PBA gel to function as a self-regulated insulin delivery system [These features underline the potential of PBA in glucose sensing and self-regulated insulin delivery applications. Our group has designed a derivative of PBA bearing optimally electron-withdrawing para-carbamoyl and meta-fluoro substituents (4-(2- acrylamidoethylcarbamoyl)-3-fluorophenylboronic acid, Agical pH ,26. Sincy system . We havey system ,29,30.Aspects which require further research are as follows: (i) the hollow structure of the drug reservoir attached to the boronate-containing smart hydrogel MNs considerably impacts their skin insertion efficiency; and (ii) limited drug loading capacity remains an issue common to all MNs . The strHence, here, we report a hybrid glucose-responsive insulin MNs patch attached to a porous gel drug reservoir for enhanced skin penetration efficiency. Three fabrication methods were investigated, and ultimately, a two-step photopolymerization method was selected to fabricate the MNs patch. The PBA-containing hydrogel constitutes the needle tips only, while the porous gel layer attached to the back serves as the drug reservoir. The interconnected porous structure of the porous gel reservoir provide space for insulin loading and ensure physical support, especially in the middle part of the MNs, where force is applied during use. Furthermore, the loaded liquid insulin solution could be transported to the needle tip by capillary action. To further improve the mechanical properties of the proposed device, the MNs patch was coated with a crystalized PVA layer by the dipping-drying method. This MNs patch, with a “cake-like”, two-layer structure, displayed acute and sustained self-regulated insulin delivery with significantly improved mechanical strength and skin insertion efficiency, which suggests its potential for diabetes management applications.Porous gel was synthesized according to a method described in a previous publication . A crossThe MNs patch was fabricated with widely-used micromolding technology . The depType 1 hybrid MNs were fabricated by immersing the porous gel MNs in a glucose-responsive pregel solution A. The poThe type 2 hybrid MNs patch was fabricated by one-step photopolymerization. The polymerization of glucose-responsive gel was initiated by photoinitiator Irgacure 184 instead of APS/TEMED. As shown in The hybrid MNs fabrication method was further optimized. Two-step photopolymerization was applied to avoid inhomogeneous distribution of the two gels. Relatively low mechanical strength limits the wide application of hydrogel MNs . To incrBesides mechanical strength enhancement due to the optimized gel formulation and PVA coating, the porous drug reservoir design also increased skin insertion efficiency. The increased monomer concentration and crosslinking density, different polymerization methods and surface coating with a PVA layer would obviously affect the functionality of the gel. Hence, it was important to verify the glucose-responsive insulin release capability of the type 3 hybrid MNs. Insulin was added to the porous drug reservoir of the PVA-coated MNs patch, and the back and side parts of the porous reservoir were sealed with water-proof epoxy resin (silicone one-component room temperature vulcanizing rubber) to avoid leakage. This insulin-loaded type 3 hybrid MNs patch was challenged with various glucose patterns , 2-methoxyethanol, glycidylmethacrylate, Irgacure 184, N-Isopropylacrylamide (NIPAAm), ammonium persulfate (APS), tetramethyl-ethylenediamine (TEMED), 4-(2-acrylamidoethylcarbamoyl)-3-fluorophenylboronic acid (AmECFPBA) and methanol, N,N′-methylenebis- (acrylamide) (MBAAm) were purchased from Wako Pure Chemical Industries . Poly  and fluorescein isothiocyanate (FITC)-labeled bovine insulin were purchased from Sigma-Aldrich . Mouse skin was purchased from Hoshino Laboratory Animals, Inc., Bandou-shi, Japan. Waterproof epoxy resin KE-3424-G was obtained from Shin-Etsu Chemical Co. Ltd., Tokyo, Japan.Trimethylolpropane trimethacrylate (TRIM), triethyleneglycol dimethacrylate, polyethylene glycol  with a fixed molar ratio of 92.5:7.5 and a total monomer concentration of 3 M. The cross-linker, MBAAm was added to the pregel solution with 20 mol% monomer concentration. After the addition of 12 μL of 10 wt% APS and 12 μL of TEMED into 1 mL of reaction solution, the polymerization was initiated  several times. The internal structure of the hydrogels was visualized by scanning electron microscopy .The glucose-responsive PBA gel reaction solution was prepared by dissolving NIPAAnitiated . To syntType 1 hybrid MNs were fabricated by micromolding technology with conical shape needle template . Pregel solution of the porous gel was added on the MNs template, vacuumed for 5 min to ensure that the reaction solution perfectly filled the MNs tip and base layer region, and photopolymerized under 365 nm UV light for 15 min. Once the porogen was removed and the MNs were totally dried, the MNs were immersed in the glucose-responsive PBA pregel solution containing TEMED/APS as an accelerator and initiator, and vacuumed for 2 min. Radical copolymerization of the glucose-responsive PBA gel was carried out at room temperature as described before. The unreacted monomers were removed by washing.Type 2 hybrid MNs were fabricated by combining the porous gel and the glucose-responsive PBA gel. To ensure gelation under UV light, Irgacure 184 was added to the glucose-responsive PBA gel solution as an initiator, instead of APS and TEMED. The porous gel reaction solution was mixed with the glucose-responsive PBA reaction gel at a certain ratio, and photopolymerization was carried out under 365 nm UV light for 15 min. The unreacted monomers were removed by washing.Type 3 hybrid MNs were fabricated in two steps. Firstly, the glucose-responsive PBA reaction gel with Irgacure 184 as a photoinitiator was added to the MNs template, vacuumed for 5 min, and photopolymerized under 365 nm UV light for 90 s. Then, the porous gel solution was added to the MNs template and photopolymerization continued for 15 min. The unreacted monomers were removed by washing.The tip part of the type 3 hybrid MNs was coated with PVA by the tipping-drying method. The MNs needles were tipped with 10 wt% PVA solution, with residual solution being carefully removed by tissue paper. The samples were then left to dry overnight at room temperature. The following day, the PVA-coated MNs were placed in an oven at 130 °C for 1 h to induce crystallization of PVA. This coating and heating step was repeated several times, as needed.The morphology of the MNs was observed by optical microscope  and SEM. The dried MNs sample was coated with a thin layer of gold (around 150 nm in thickness) to ensure sufficient conductivity. The gold-coated sample was titled at a certain angle as needed and visualized by SEM .The mechanical strength of the MNs was evaluated by a bond tester . The MNs were fixed to the test platform by vacuum with a stainless steel testing probe placed 200 μm above the MNs base layer. During the test, the testing probe pressed horizontally against the needles with increasing force. The mechanical strength was recorded as the maximum loading force before needle fracture.The micropores resulting from MNs skin penetration were observed by staining MNs administrated (5 min) mouse skin with trypan blue for 30 min. The skin penetration efficiency was calculated by the following equation:x 490 nm, Em 520 nm). The in situ glucose concentration was determined by the refractive index detector.The glucose-responsive release profile of the MNs was investigated by high-performance liquid chromatography  with a Tricorn empty high-performance column . Insulin was loaded into the porous drug reservoir of the MNs patch coated with crystallized PVA by immersing the MNs in 130 mg/L FITC-insulin solutions and vacuuming for 5 min. The drug reservoir was sealed by a waterproof epoxy resin, i.e., KE-3424-G. The drug-loaded MNs patch was challenged with glucose at various concentrations (between 100 and 500 mg/dL) by passing a mixture of PBS and 100 mg/dL glucose solution through the two pumps of the HPLC system, controlled by the ChromNAV software . The flow rate was kept at 1 mL/min, and the MNs patch was equilibrated with PBS until no leakage was observed. The in vitro release of FITC-labeled insulin was monitored by determining the real-time fluorescence of FITC (En = 3). Reported results and graphical data are mean values with a standard deviation encompassing a 95% confidence interval.All measurements were taken in triplicate (


Current approaches to cancer immunotherapy include immune checkpoint inhibitors, cancer vaccines, and adoptive cellular therapy. These therapies have produced significant clinical success for specific cancers, but their efficacy has been limited. Oncolytic virotherapy (OVT) has emerged as a promising immunotherapy for a variety of cancers. Furthermore, the unique characteristics of OVs make them a good choice for delivering tumor peptides/antigens to induce enhanced tumor-specific immune responses. The first oncolytic virus (OV) approved for human use is the attenuated herpes simplex virus type 1 (HSV-1), Talimogene laherparepvec (T-VEC) which has been FDA approved for the treatment of melanoma in humans. In this study, we engineered the recombinant oncolytic HSV-1 (oHSV) VC2-OVA expressing a fragment of ovalbumin (OVA) as a fusion protein with VP26 virion capsid protein. We tested the ability of VC2-OVA to act as a vector capable of stimulating strong, specific antitumor immunity in a syngeneic murine melanoma model. Therapeutic vaccination with VC2-OVA led to a significant reduction in colonization of tumor cells in the lungs of mice intravenously challenged B16cOVA cells. In addition, VC2-OVA induced a potent prophylactic antitumor response and extended survival of mice that were intradermally engrafted with B16cOVA tumors compared with mice immunized with control virus. It is currently understood that cancers result from individual cellular transformation events resulting in genetically and phenotypically unique tumors even within the same tissue environment . This isCurrent molecular diagnostics, including genomic and proteomic tools, allow us to employ greater precision in the design and delivery of anti-cancer treatments and therapies . These tde novo TAA specific immune responses via mRNA and vaccines, peptide vaccines and viral vectored TAAs . All mice were maintained in pathogen-free facilities. Protocols involving animals were reviewed and approved by the Louisiana State University Institutional Animal Care and Use Committee (IACUC), and all animal experiments were performed in accordance with the protocols.The bacterial artificial chromosome (BAC) plasmid VC2 was used to construct VC2-OVA as previously described . High-efβ-mercaptoethanol to a final 1x concentration. These mixtures were then boiled at 100°C for 10 min and cooled on ice before loading into a 12% Mini-PROTEAN TGX precast gel (Bio-Rad) and separated for 1 h at 100 V in 1x Tris-Glysine-SDS buffer (Bio-Rad). Separated protein was then transferred to a nitrocellulose membrane in 1x Tris-Glysine + 20% methanol (Bio-Rad). The membrane was then blocked for 30 min in 5% BSA in PBS-T. Rabbit anti-VP26 , was diluted 1:1,000 in 5% BSA PBS-T and applied to the membrane and incubated overnight at 4°C while rocking. The next day, the membrane was then washed 3x with PBS-T and secondary goat anti-Rabbit IgG (Abcam: ab6721) diluted 1:1,000 in 5% BSA PBS-T applied to the membrane and incubated at room temperature for 1 h. The membrane was then washed 3x in PBS-T and visualized using ECL Western Blot Substrate (Pierce) and exposure film.Vero cells were uninfected or infected at an MOI 1 with either VC2 or VC2-OVA for 24 and 48 h in a six well plate. Adherent cells were washed 3x in PBS followed by lysis in 200 μl of NP40 lysis buffer with protease/phosphatase inhibitors. Twenty microliters of whole cell lysate were then mixed with Laemmli sample buffer (Bio-Rad) and 1 μl of The ovalbumin-expressing B16 melanoma cell line (B16cOVA) was a kind gift from Dr. Timothy N.J. Bullock . B16cOVA cells were grown in RRPMI 1640 medium  supplemented with 10% filtered, heat inactivated fetal bovine serum , 100 μg/ml Primocin , plus 10 μg/ml Blastocydin . African green monkey kidney (Vero) cells were cultured in DMEM containing 10% FBS and 100 μg/ml Primocin.6 pfu of VC2 or 1 × 106 pfu of VC2-OVA in volumes of 100 ul. Fourteen days after prime immunization, booster immunizations were administered. Six days post-boost, mice were engrafted with 5 × 105 B16cOVA cells in 100 μl PBS orthotopically in the dermis of the dorsal left dorsal pinna. Tumors were measured approximately every 2,3 days by using a digital caliper when tumors reached 50 to 100 mm3. Tumor volumes were calculated by using formula 1/2 (length × width2). Tumor bearing mice were euthanized when tumors reached greater than 1000 mm3 or when mice were excessively moribund. To assess the therapeutic effect, mice were injected intravenously with 5 × 105 B16cOVA cells in 100 μl PBS, and then intramuscularly; intradermally; or subcutaneously vaccinated the next day for two consecutive days. Mice were sacrificed 3 weeks post engraftment, and lungs were removed and the tumor colonies on the lung surface were counted.For prophylactic assessment, mice were not treated; intramuscularly; intradermally; or subcutaneously vaccinated with 1 × 10One day after boost vaccination, mice were sacrificed, and spleens were removed.5) were isolated and cultured overnight with either gB peptide (1 μg/ml) or ovalbumin [OVA257–264 (SIINFEKL)] peptide (1 μg/ml). IFN-γ-producing splenocytes were quantified according to the manufacturer’s instructions using an Immunospot  murine IFN-γ single-color ELISPOT assay.Splenocytes . Analysis of data between three or more groups was performed by using one-way ANOVA. Survival data were presented using Kaplan–Meier survival curves and differences among groups were analyzed by the log rank test. A 6 pfu we can deliver nearly 109 OVA-VP26 antigens. However, this extrapolation is likely an underestimation due to a particle to pfu ratio for tissue culture-derived HSV-1 reported to be 100:1  was fused to the amino terminus of VP26 to generate VC2-OVA.We wished to fully exploit the potential of viruses to deliver antigen and promote strong, broad, and effective anti-immunogen responses in the host. To this end, we fused the immunogenic portion of chicken egg ovalbumin to VP26, the minor capsid protein of HSV-1 . Ovalbumbe 100:1 . FurtherTo confirm expression of the fusion protein in recovered VC2-OVA, Vero cells were infected at a multiplicity of infection of 1. Twenty-four and 48 hours post-infection, protein lysates were prepared, and a western blot was performed. Using an antibody to detect VP26 we readily observed a protein of the expected size (12kDA ) in lysaTo determine any effect of fusing ovalbumin to VP26 on viral replication we performed a multi-step growth curve comparing parental VC2 virus and VC2-OVA. Vero cells were infected at an MOI of .01 and cells were harvested at 0, 4, 12, 24 and 48 h post infection. Standard plaque assays were performed to quantify virus in cell lysates. We were unable to identify any difference in viral replication between parental and VC2-OVA viruses .+ T-cell epitope  with VC2-OVA or the parental virus. 72 hours post boost vaccination, mice were sacrificed and splenocytes were harvested . Splenoc epitope  and serv epitope . However epitope . InteresWe have previously shown the efficacy of parental VC2 in intratumoral treatment of mice engrafted with modified B16F10 melanoma . In thosNext, we investigated the efficacy of VC2-OVA when used in a therapeutic context, where engraftment preceded treatment. In these experiments, B16cOVA cells were inoculated intravenously. The introduction of these cells intravenously leads to colonization of the lungs by the B16F10 cells resulting in tumors that can be enumerated approximately 3 weeks post engraftment. This approach is a commonly used approach to test intervention strategies for metastasis and the development of systemic anti-tumor immunity. B16cOVA cells were administered intravenously, and mice were treated with either VC2 or VC2-OVA IM, SC, or ID 2 days post tumor administration . Twenty-The identification of safe, and immunogenic vaccine vectors capable of inducing potent immune responses is critical to the development of anti-infectious disease and anti-cancer intervention strategies . PreviouOf particular interest is our finding that the route of vaccination was a large factor in the efficacy of treatment. Intradermal route of vaccination proved best in our B16cOVA melanoma model in a prophylactic context. Intramuscular route of vaccination proved to be the least effective in both extending survival and reducing tumor growth rates. It is unclear why intradermal delivery of the virus produced a more efficient vaccination approach. It has been documented that immune responses are affected by the route of vaccination . There aIt is important to point out that route of administration is not a one size fits all problem. Likely each route of administration induces specific types of immunity that may be individually suited to protect against different infection and tumor types. Along these lines we note that our studies used two different engraftment sites: intradermal and intravenous. While we saw large differences in route of administration for the intradermally engrafted tumors we didn’t find any difference for the route of administration when tumors were engrafted intravenously. These findings suggest that the route of administration may be an important consideration for infections and tumor types at some sites but not others.In these experiments we have used an experimental immunogen, OVA, to evaluate the utility of HSV-1 in general, and VC2 specifically, as a vector to deliver tumor associated antigens for treatment of cancer. It is important to note that the clinical utility of our approach will depend on the identification of similarly immunogenic tumor associated antigens in human patients. The identification of such antigens in human tumors is an active area of investigation with encouraging results . The ideIn summary, we find that there is significant evidence to pursue viral vectored TAA delivery in general and VC2-derived TAA vaccines specifically. VC2 has proven safe and efficacious as an HSV vaccine in a variety of animal models and preparations are ongoing for a pilot in-human trial. As we have shown that VC2 works very well as an oncolytic virotherapy, we are excited about the prospect of using VC2 as a combination OVT and personalized vaccine for the treatment of human and animal cancers.


Approximately 25% of patients with colorectal cancer will present with or develop colorectal liver metastasis (CRLM). Surgical resection of CRLM offers these patients the best chance of a cure. However, liver resection and the subsequent regenerative response has been linked to tumor recurrence in the liver remnant. The Wnt/β-catenin pathway is one of many pathways common to both post-hepatectomy liver regeneration and tumorigenesis. Wnt signaling modulates multiple genes of the renin-angiotensin system (RAS), and Wnt inhibition can attenuate fibrotic responses and improve cancer outcomes via diverse mechanisms. In this study, we investigate the effects of captopril, a RAS inhibitor (RASi), on the Wnt/β-catenin pathway and phenotypic changes associated with tumor progression in the context of the regenerating liver. We show that RASi induced increased Wnt signaling whilst downregulating features of epithelial-to-mesenchymal transition (EMT). Furthermore, RASi induced significant down-regulation of Wnt target genes, c-myc and cyclin D1, indicating that expression of these genes can be down-regulated by RASi despite the accumulation of stabilized β-catenin.(1) Background: Recent clinical and experimental data suggests that the liver’s regenerative response following partial hepatectomy can stimulate tumor recurrence in the liver remnant. The Wnt/β-catenin pathway plays important roles in both colorectal cancer carcinogenesis and liver regeneration. Studies have shown that the Wnt/β-catenin pathway regulates multiple renin-angiotensin system (RAS) genes, whilst RAS inhibition (RASi) reduces tumor burden and progression. This study explores whether RASi attenuates features of tumor progression in the regenerating liver post-hepatectomy by modulating Wnt/β-catenin signaling. (2) Methods: Male CBA mice underwent CRLM induction, followed one week later by 70% partial hepatectomy. Mice were treated daily with captopril, a RASi, at 250 mg/kg/day or vehicle control from experimental Day 4. Tumor and liver samples were analyzed for RAS and Wnt signaling markers using qRT-PCR and immunohistochemistry. (3) Results: Treatment with captopril reduced the expression of down-stream Wnt target genes, including a significant reduction in both c-myc and cyclin-D1, despite activating Wnt signaling. This was a tumor-specific response that was not elicited in corresponding liver samples. (4) Conclusions: We report for the first time decreased c-myc expression in colorectal tumors following RASi treatment in vivo. Decreased c-myc expression was accompanied by an attenuated invasive phenotype, despite increased Wnt signaling. Around 25% of patients with colorectal cancer (CRC) will present with or develop colorectal liver metastasis . SurgicaThe Wnt/β-catenin pathway is one of many pathways common to both post-hepatectomy liver regeneration and tumorigenesis. Wnt signaling is an evolutionary conserved pathway which regulates cell fate, migration, polarity and organogenesis. In health, the canonical Wnt pathway controls the degradation of β-catenin, which in turn influences the transcription of Wnt target genes in a context-dependent manner ,7. Wnt sAdenoma Polyposis Coli (APC) gene leads to the formation of a faulty degradation complex, which inadequately degrades β-catenin. Subsequently, the transcriptional effects of β-catenin remain unchecked and Wnt/β-catenin signaling is constitutively turned on  and Wnt pathway  signaling was analyzed by quantitative (q) reverse transcriptase (RT)-PCR on tissues harvested from captopril- and saline-treated mice . The exp 0.1465) . Captopril treatment was also associated with a significant reduction in cyclin D1 gene expression in mouse tumors following liver resection, compared to the control. Treatment was associated with a more than two-fold reduction in cyclin D1 expression by qRT-PCR E. This rAssessment of Ki67 staining, a measure of cells that are in any of the active cell cycle phases , revealeIHC demonstrated that β-catenin staining in the tumors of treated mice, was significantly higher than the control . FurtherRecently, inhibition of BRD4, the chromatin reader, has been shown to specifically reduce the expression of myc in many different cancer types ,36,37,38However, qRT-PCR revealed that captopril treatment was not associated with a significant change in BRD4 expression . AlthougCRC frequently metastasizes to the liver, and hepatic metastases may be present at the time of diagnosis or develop metachronously . The maiAPC gene, leading to pathogenic expression of Wnt target genes, including c-myc and cyclin D1, as a result of failure to successfully degrade cytoplasmic β-catenin , at 99 °C for 30 min. Peroxidase-blocking solution was applied for 30 min (DAKO Peroxidase-Blocking solution S2023). Slides were incubated with 10% normal goat serum. Primary antibodies included vimentin , Ki67 (Thermo-Scientific SP6), Cyclin D1 , c-myc , E-Cadherin , β-Catenin  and active β-catenin . Control sections were incubated with rabbit control antibody. Anti-rabbit horseradish peroxidase secondary antibody was applied for 1 h to all sections (Dako EnVision + System-HRP K4003) and the final detection step was performed using 3,3 diaminobenzidine . Antibodies used are listed in t test or one-way ANOVA and Tukey post hoc test as indicated in the legends; p values less than 0.05 were considered significant.Statistical analysis was performed using GraphPad Prism using the unpaired student-In conclusion, tumor recurrence following hepatic resection for CRLM and other primary liver cancers remains a major clinical problem for hepatobiliary surgeons. Treatments that can be safely administered alongside surgery to negate this risk may prolong patient survival. The micro-environment of the regenerating liver following partial hepatectomy is unique and many factors may promote tumor growth. The Wnt/β-catenin pathway is just one signaling pathway involved in both tumorigenesis and liver regeneration. Inhibition of this pathway has been shown to modulate EMT and provide a mechanism for reversing both fibrosis and improving cancer outcomes. Here, we have shown that captopril appears to increase Wnt signaling whilst reducing phenotypic features of EMT, providing a mechanism for tumor control in the regenerating liver. We also demonstrated a significant reduction in both c-myc and cyclin D1, although the mechanism is unclear. Whilst we have not elucidated the precise mechanism for c-myc or cyclin D1 down-regulation, these findings have important clinical ramifications. C-myc has been difficult to target therapeutically, so the finding that captopril down-regulates this important proto-oncogene deserves further attention and may provide an alternative therapy for patients with myc addicted cancers.


Reactive microglia are suggested to be involved in neurological disorders, and the mechanisms underlying microglial activity may provide insights into therapeutic strategies for neurological diseases. Microglia produce immunological responses to various stimuli, which include fractalkine (FKN or CX3CL1). CX3CR1, a FKN receptor, is present in microglial cells, and when FKN is applied before lipopolysaccharide (LPS) administration, LPS-induced inflammatory responses are inhibited, suggesting that the activation of the FKN signal is beneficial. Considering the practical administration for treatment, we investigated the influence of FKN on immunoreactive microglia using murine primary microglia and BV-2, a microglial cell line. The administration of LPS leads to nitric oxide (NO) production. NO was reduced when FKN was administered 4 h after LPS administration without a change in inducible nitric oxide synthase expression. In contrast, morphological changes, migratory activity, and proliferation were not altered by delayed FKN treatment. LPS decreases the CX3CR1 mRNA concentration, and the overexpression of CX3CR1 restores the FKN-mediated decrease in NO. CX3CR1 overexpression decreased the NO production that is mediated by LPS even without the application of FKN. ATP and ethanol also reduced CX3CR1 mRNA concentrations. In conclusion, the delayed FKN administration modified the LPS-induced microglial activation. The FKN signals were attenuated by a reduction in CX3CR1 by some inflammatory stimuli, and this modulated the inflammatory response of microglial cells, at least partially. Thus, the FKN-mediated reduction in NO appeared to be greater compared with the mock-transfected cells.To examine whether the downregulation of CX3CR1 impairs the anti-inflammatory response to FKN, the BV-2 cells were transfected with CX3CR1. Two days after the transfection, LPS was added, followed by FKN application 4 h later. Following the transfection, CX3CR1 was overexpressed, and its exogenous form was detected immunohistochemically  . A two-wIf FKN is released constantly from microglial cells, it may be reasonable that exogenous CX3CR1 receives endogenous FKN, which results in the inhibition of NO production, regardless of FKN administration. To address this possibility, the extracellular FKN concentration was investigated using ELISA. As shown in The overexpression of CX3CR1 did not alter iNOS gene expression , 1.00 ± 0.57; CX3CR1-HA(+), 1.84 ± 0.37) ; this ruTo investigate whether FKN signaling is also responsible for other stimulations, the primary microglial cells were treated with some irritants. ATP and ethanol, as well as LPS, lowered CX3CR1 gene expression significantly  . Taken tSince the discovery of FKN, intensive efforts have resulted in the recognition of the significant roles of FKN in cellular survival or activity. Its benefits are still being explored in immune cells and other cell types, including endothelial and neuronal cells. Similarly, the roles of FKN have also been reported in microglia, but they were not determined clearly in already activated microglia. Because the attenuation of microglial activity may provide insights into the FKN-related therapeutic strategy, we investigated these aspects.First, we showed that the NO production triggered by LPS was ameliorated by administering FKN after LPS in microglia. Although the impact of these reagents on NO production varies between primary microglia and BV-2 cells, some studies also show similar trends. For example, primary microglia and BV-2 cells produce NO by LPS/IFN-γ treatment, but the amount of NO in BV-2 cells are about 10-fold greater than that of primary microglia . Thus, tWe subsequently found that CX3CR1 mRNA was downregulated by LPS. As shown in The disruption of FKN signaling in microglia results in various neurological symptoms elicited by various forms of stress. To extend the suppression of microglial activity by the FKN signal to other inflammatory responses, pro-inflammatory stimuli were administered, and the CX3CR1 gene expression was assessed. We observed that CX3CR1 gene expression was downregulated by ATP and ethanol. Given that mice with defective CX3CR1 in microglia show various neurological symptoms under various forms of stress , 46, theIn conclusion, we have demonstrated that delayed FKN administration reduces LPS-induced NO production without changing the iNOS protein concentration. In contrast, neither the migratory nor the proliferative activities were altered by FKN; thus, the effect of delayed FKN administration may be limited. We have also found that LPS and other pro-inflammatory stimuli decrease the expression of CX3CR1, the mechanism of which is still unknown, and CX3CR1 overexpression restores LPS-induced NO production. These findings suggest that the attenuation of FKN signaling is common under inflammatory conditions. Thus, compensating for the signal via the administration of FKN or the upregulation of CX3CR1  may improve anti-inflammatory responses and lead this to be considered as a therapeutic targeting method.S1 FigPrimary microglial cells were isolated by shaking from mixed glial cells, and plated see . Those c(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigBV-2 cells were incubated in DMEM with either 5% FBS or 0.5% bovine serum albumin and treated with 500 ng/ml LPS for 24 h. The media were obtained, and NO production was measured by assessing nitrite levels. NO production was not enhanced without the addition of serum.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.