β-catenin Search Results


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  • 95
    Thermo Fisher β catenin
    <t>β-catenin</t> is required for the formation of differentiated proximal tubule cells with strong LTL staining. Hnf4a marks both presumptive and differentiated proximal tubules. In the control kidney, presumptive proximal tubules show strong Cdh6 signal and weak LTL staining while differentiated proximal tubules show weak Cdh6 signal and strong LTL staining. In the β-catenin loss-of-function mutant kidney by Osr2Cre , all Hnf4a+ cells show strong Cdh6 signal, failing to form differentiated proximal tubules with strong LTL staining. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.
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    Millipore β catenin
    Knockdown of <t>β-catenin</t> or TCF-4 enhances Tat-mediated transactivation of the LTR independent of the nt −143 site. (a) U87MG cells were transfected with scrambled siRNA, β-catenin siRNA, or TCF-4 siRNA; 48 h postknockdown, the cells were transfected with WT-LTR or Δ-143-LTR luciferase constructs with either vector control (pcDNA3.1) or Tat expression plasmid for an additional 24 h. The cells were lysed, and a dual-luciferase assay was performed. (b) U87MG cells that stably express an integrated copy of the WT or a Δ-143-LTR-luciferase construct were transfected with the indicated siRNAs for 48 h, followed by transfection with vector control or Tat for an additional 24 h before the cells were lysed and a dual-luciferase assay was performed. Luciferase readings from panels a and b were normalized to the total protein content as measured by BCA assay. The data represent three independent experiments. *, P
    β Catenin, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cell signaling technology inc β catenin
    Relative expression of osteogenic markers (a) Runx2, (b) ALP, (c) β -catenin, and (d) Smurf2, which is associated with <t>β</t> -catenin degradation and reduced osteogenesis, were determined by qRT-PCR at day 7. All the samples were performed in triplicate. *** p
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    Cell Signaling Technology Inc phosphor β catenin
    Schematic diagram of the gene regulation of ESRRG and Wnt signaling. a ESRRG induces <t>β-catenin</t> degradation via an unknown mechanism and inhibits the binding of the Wnt component TCF/LEF to the CCND1 gene promoter region by direct interaction, thus potentially repressing the Wnt signaling pathway and blocking tumorigenesis. b The balance of ESRRG and Wnt signaling activity is crucial for tumor suppression or formation in GC
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    Cell Signaling Technology Inc α β catenin
    ER71 inhibits canonical Wnt signaling. (A) iEr71 ES cells were differentiated in the absence (Control) or presence (+CHIR99021, 1μM) of CHIR99021 from days 2-3 of differentiation, either with (+DOX) or without (−DOX) DOX from days 2-3 of differentiation. Flk-1 and PDGFRα expression were analyzed at day 3. (B) Er71 −/− ES cells were differentiated and treated with CHIR99021 or DKK1 from day 2. Flk-1 and PDGFRα expression was analyzed at day 3. (C-D) ST2 cells were cotransfected with Lef1-luciferase ± MSCV- Er71 and ± Wnt3a conditioned medium (C) or ± <t>β-catenin</t> expression vector (D). Luciferase activity was measured 48 hours later. Firefly luciferase activity was normalized by Renilla luciferase activity. (E) Flk-1 + mesoderm sorted from iEr71 EBs was cotransfected with Lef1-luciferase and β-catenin. DOX was added to culture and luciferase activity was measured 48 hours later. (F) Flk-1 + mesoderm sorted from Er71 +/+ and Er71 −/− EBs were cotransfected with Lef1-luciferase ± MSCV -Er71 . Luciferase activity was measured 48 hours later. Data are presented as the means ± SD of at least 4 independent experiments. * P
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    Abcam β catenin
    The effect of CD317 knockdown in polarized Caco-2 cells. (A) Left panels are maximum intensity projection XY images showing Alexa Fluor 594–phalloidin decoration of F-actin in polarized control and CD317 knockdown Caco-2 cells. Middle panels show XZ sections taken along the lines in left panels. Right panels show corresponding SEM images of the surface of control and CD317 knockdown cells. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (B) ZO-1 labeling of tight junctions in polarized CD317 knockdown and control Caco-2 cells and biotin labeling of proteins in the apical membrane of polarized CD317 knockdown Caco-2 cells as indicated. Left panels are maximum intensity projection XY images, and right panels are XZ sections taken along the lines in the left panels. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (C) Detection of <t>β-catenin</t> in the lateral membranes and sucrase-isomaltase in the apical membrane of polarized CD317 knockdown Caco-2 cells. XZ sections derived from maximum intensity projection XY images are presented. The right panel is a merge of the left and middle images. Bars, 10 µm.
    β Catenin, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 3959 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson β catenin
    GSK3β phosphorylates Dzip1. (A and B) Dzip1 interacts with GSK3β. Endogenous GSK3β was immunoprecipitated by Dzip1 but not IgG (A), and endogenous Dzip1 was immunoprecipitated with GFP-GSK3β in HEK 293T cells (B). (C and D) Dzip1 is co-localized with GSK3β at the basal body. G0-phase NIH 3T3 cells expressing GFP-GSK3β were immunostained for Dzip1 and AcTub (C), or cells expressing BFP-Centrin2 were immunostained with GSK3β and Dzip1 (D). Scale bar: 5 μm. (E) GSK3β binds Dzip1 in a kinase-substrate interaction manner. Wild-type (WT) GFP-GSK3β and the mutants S9A, K85R, and R96A were each co-expressed with Myc-Dzip1 in G0-phase HEK 293T cells, and treated with the CK1 inhibitor D4476 or the CK2 inhibitor CX4945. Note that treatment with CX4945 but not D4476 led to a significant decrease in the extent of the up-shifted Dzip1 bands, although the binding of Dzip1 to the GFP-GSK3β variants showed no difference. The extent of the up-shifting of the Dzip1 bands was decreased in K85R-expressing cells. (F) Phosphorylation of Dzip1 is coordinated with GSK3β activation. The kinase activity of GSK3β was negatively correlated with serum stimulation in NIH 3T3 cells. Note that the up-shifted bands (arrowheads) of Dzip1 became evident after serum depletion for 24–48 h, and disappeared after serum restimulation. γ-Tubulin was set as a loading control. (G) GSK3β phosphorylates Dzip1 in vivo. In resting mouse embryo fibroblast (MEFs) treated versus not treated with GSK3 and CK2 inhibitors, the Dzip1 bands were up-shifted less in GSK3- and CK2-inhibited cells. The protein levels of total <t>β-Catenin</t> and GSK3β were steady, but the phosphorylated (S33/37/T41) β-Catenin specifically disappeared from GSK3-inhibited cells. α-Tubulin was set as a loading control. (H) GSK3β phosphorylates Dzip1 in vitro. Auto-phosphorylation of GSK3β (55 kD), and the phosphorylated bands of the middle (28 kD), C-terminus (36 kD), and N- terminus (50 kD) of Dzip1 are shown (left panel). Coomassie blue staining of the gel shows the loaded amounts of Dzip1 fragments (right panel). Note that the S520A mutation resulted in decreased phosphorylation of Dzip1 by GSK3β. (I) Inhibition of GSK3 by BIO causes loss of phospho-S520 in Dzip1.
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    Novocastra β catenin
    Hematoxylin and eosin stain gastric sections and immunohistochemical staining for E-cadherin and <t>β-catenin.</t> (A) Hematoxylin and eosin staining in normal gastric tissue. (B) Hematoxylin and eosin staining in the tumor tissue of GC S10 with signet ring cell carcinoma: signet ring cells are highlighted by black arrows. (C) Hematoxylin and eosin staining evidence the diffuse histotype of GC S46. (D) E-cadherin staining in normal gastric tissue. (E) Reduction of E-cadherin staining in the signet ring cells of GC S10 respect to adjacent normal cells; signet ring cells are highlighted by black arrows. (F) Loss of E-cadherin expression in the diffuse tumor cells of GC S46 compared to normal tissue (on the right side of the photomicrograph). (G) β-catenin staining in normal gastric tissue. (H) Weakly β-catenin staining in signet ring cells (black arrows) of GC S10. (I) Loss of β-catenin stai ning in the tumor tissue of GC S46 compared to normal tissue (on the right side of the photomicrograph). All the photomicrographs were taken at 400× magnification.
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    SLIT2 LTD β catenin
    SLIT/ROBO1 signaling regulates the subcellular localization of <t>β-catenin</t>
    β Catenin, supplied by SLIT2 LTD, used in various techniques. Bioz Stars score: 92/100, based on 316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AnaSpec β catenin
    Exd4 treatment stabilizes β <t>-catenin,</t> siRNA-mediated knockdown of β -catenin abrogates Exd4-stimulated Wnt signaling, and Exd4 enhances phosphorylation of β -catenin on the stabilizing PKA site, Ser-675. A, Exd4 stabilizes cytosolic
    β Catenin, supplied by AnaSpec, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology β catenin
    FoxO1 knockdown promotes migration, invasion potential, and EMT in OSCC cells. (A) FoxO1 expression was detected by qRT-PCR and Western blot after transfected with FoxO1 shRNA in OSCC cells. (B) Images of the wound closure of monolayer Cal-27 and Tca8113 cells with FoxO1 knockdown at the time point of 0 and 24 h are presented on the left. Quantitative results are illustrated on the right. (C) The effect of FoxO1 knockdown on OSCC cells invasion were determined by Transwell assay with Matrigel, and the representative images are on the left. Quantitative results are illustrated on the right. (D) The effects of Cal-27-FoxO1 knockdown on expressions of EMT markers, E-cadherin, N-cadherin, <t>β-catenin,</t> and Vimentin, were measured using qRT-PCR and Western blot. And the effects of Cal-27-sh-2-STAT3/sh-1-FoxO1 on expressions of EMT markers, E-cadherin, N-cadherin, β-catenin, and Vimentin, were measured using qRT-PCR compared with those of sh-2-STAT3/scramble group. β-actin was used as a loading control. (E) Flow cytometry was used to examine the percentage of apoptotic cells in Cal-27 cells with FoxO1 knockdown and scramble control cells. All assays were carried out in triplicate. Results were shown as means ± SD. * P
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    Ventana Medical β catenin
    Representative examples of <t>β-catenin</t> expression in colorectal liver metastasis. (A) Only membranous staining was observed (score 0 for cytoplasm and nucleus). (B) Cytoplasmic expression of β-catenin with some positive nuclei (score 1 for cytoplasm and nuclei). (C) Diffuse and strong cytoplasmic and nuclear staining (score 1 for cytoplasm, and score 2 for nuclei). Magnification, ×200.
    β Catenin, supplied by Ventana Medical, used in various techniques. Bioz Stars score: 92/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Biolabs β catenin
    Acute exposure to lenalidomide induces <t>β-catenin</t> transcription. RT-PCR was performed to detect β-catenin or β-actin transcripts from cells propagated continuously in lenalidomide (R10R), or R10R cells that were removed from lenalidomide for 1 week (RR), to detect β-catenin, and comparisons were made to vehicle-treated, drug-naive cells ( WT ) ( A ). RT-PCR products were separated by acrylamide gel electrophoresis, and detected by ethidium bromide staining. The left panel shows representative agarose gels, and densitometry was performed to calculate the increase in β-catenin after correcting for β-actin, which was normalized to the WT cells, which were set at 1.0. Drug-naive ANBL-6 ( B ), KAS-6/1 ( C ), and MM1.S ( D ) cells were exposed to the indicated lenalidomide concentrations for 72 (ANBL-6) or 168 h (KAS-6/1, MM1.S). Each top panel provides a representative photomicrograph, and densitometry was performed to calculate the β-catenin/β-actin ratio. Values for each cell line are provided in the lower panels after normalization to the vehicle-treated control, which was arbitrarily set at 1.0.
    β Catenin, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech β catenin
    1C inhibited <t>Wnt/β-catenin</t> signaling pathway. The expression levels of Wnt/β-catenin signaling pathway involving proliferation were examined by Western blotting assay. Data are expressed as means ± SDs of triplicate experiments performed independently. ∗ p
    β Catenin, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Marque β catenin
    Effect of U94 on cell signaling pathways Western blot analysis of MDA-MB 231 lysates was performed using mAbs to Src pTyr418, total Src, <t>β-catenin</t> pTyr654, total β-catenin, FAK pTyr397, total FAK, STAT3 pTyr705, total STAT3, Cortactin pTyr421, total Cortactin, ARP2/3, GAPDH, Akt pSer473, total Akt, ERK pThr202 and total ERK as specific reagents. Quantification was carried out by densitometric analysis and plotting of the Src pTyr418/Src, β-catenin pTyr654/β-catenin, FAK pTyr397/FAK, STAT3 pTyr705/STAT3, Cortactin pTyr421/Cortactin, ARP2/3/GAPDH, Akt pSer473/Akt and ERK pThr202/ERK. Left panels, blots are representative of three independent experiments with similar results. Right panels, values reported are the means ± the SD of three independent experiments. Statistical analysis was performed by 1-way ANOVA, and the Bonferroni post-test was used to compare data (* P
    β Catenin, supplied by Cell Marque, used in various techniques. Bioz Stars score: 92/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company β catenin
    Involvement of <t>β-catenin</t> and survival signal of Akt and Src in the process of CLDN1 regulated anoikis (A) WB analysis, as compared to negative control cells, CLDN1-KD cells showed a distinct decrease of β-catenin, phosphorylated (Ser473) Akt and phosphorylated (Tyr416) Src expression. (B) WB analysis, as compared to control cells, CLDN1-overexpressed cells showed a strong increase of β-catenin, phosphorylated (Ser473) Akt and phosphorylated (Tyr416) Src expression.
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    VANGL2 LTD β catenin
    Decreased <t>β-catenin</t> and Vangl2 levels are observed in Atmin Gpg6/Gpg6 E13.5 embryonic kidneys compared with WT littermates. Kidney sections from WT ( A and C ) and Atmin Gpg6/Gpg6 ( B and D ) embryos were immunostained for β-catenin (A and B) and Vangl2 (C and D). β-Catenin immunostaining was significantly reduced in Atmin Gpg6/Gpg6 (B) compared with WT (A). Vangl2 immunostaining was also dramatically decreased and apical enrichment was altered in Atmin Gpg6/Gpg6 (D) compared with WT (C). Overlays of DAPI and Vangl2 localization emphasize the loss of apical enrichment of Vangl2 protein in Atmin Gpg6/Gpg6 (D inset) versus WT (C inset; n = 4). All images are maximum projections of confocal z stacks taken at 0.4 µm intervals. Images were taken in identical conditions and post-acquisition manipulations were identical. Western blotting revealed a 0.4-fold decrease ( I ) in β-catenin ( P
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    Bioworld Antibodies β catenin
    Representative micrographs of immunohistochemistry staining in the 5/6 Nx group rat renal cortex sections (400×). Notes: Immunohistochemistry staining of 5/6 Nx group for Wnt1 ( A ), <t>β-catenin</t> ( B ), GSK-3β ( C ), Dkk1 ( D ), TCF4 ( E ), Fn1 ( F ), and IgG negative control ( G ). Abbreviations: Nx, 5/6 nephrectomy; GSK-3β, glycogen synthase kinase-3β; Dkk1, dickkopf 1; TCF4, transcription factor 4; Fn1, fibronectin 1.
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    GeneTex β catenin
    Model of the RPIA mechanism for induction of <t>β-catenin</t> signaling in CRCs. Schematic representation shows the role of RPIA and β-catenin signaling in normal and CRC cells. In normal cells, RPIA does not interfere with GSK3β-mediated β-catenin degradation. In CRC, RPIA is overexpressed in both the cytoplasm and the nucleus; RPIA then binds to β-catenin in cytoplasm, and this prevents the degradation of β-catenin mediated by GSK3β. Within the nucleus, increased levels of β-catenin protein might also be caused by RPIA interrupting the APC-mediated transport of β-catenin from the nucleus to the cytoplasm. Overall, this would result in activation of downstream β-catenin target genes. β-cat, β-catenin; APC, adenomatous polyposis coli; CRC, colorectal cancer; GSK3β, glycogen synthase kinase-3′; RPIA, ribose-5-phosphate isomerase A, TCF, T-cell transcription factor.
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    Genechem β catenin
    FAT4 knockdown-induced gastric cancer cell growth, invasion, and migration are mediated by the Wnt/ <t>β</t> -catenin signalling pathway. BGC-823 and HGC-27 cells were transfected with or without shRNA (shRNA1) against FAT4 and siRNA targeting β -catenin, and analysed by western blotting ( A ), MTT assay ( B ), colony-formation assay ( C ), and migration and invasion assays ( D and E ). * P
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    Image Search Results


    β-catenin is required for the formation of differentiated proximal tubule cells with strong LTL staining. Hnf4a marks both presumptive and differentiated proximal tubules. In the control kidney, presumptive proximal tubules show strong Cdh6 signal and weak LTL staining while differentiated proximal tubules show weak Cdh6 signal and strong LTL staining. In the β-catenin loss-of-function mutant kidney by Osr2Cre , all Hnf4a+ cells show strong Cdh6 signal, failing to form differentiated proximal tubules with strong LTL staining. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Journal: Scientific Reports

    Article Title: β-catenin regulates the formation of multiple nephron segments in the mouse kidney

    doi: 10.1038/s41598-019-52255-w

    Figure Lengend Snippet: β-catenin is required for the formation of differentiated proximal tubule cells with strong LTL staining. Hnf4a marks both presumptive and differentiated proximal tubules. In the control kidney, presumptive proximal tubules show strong Cdh6 signal and weak LTL staining while differentiated proximal tubules show weak Cdh6 signal and strong LTL staining. In the β-catenin loss-of-function mutant kidney by Osr2Cre , all Hnf4a+ cells show strong Cdh6 signal, failing to form differentiated proximal tubules with strong LTL staining. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Article Snippet: Immunofluorescence Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 (TS1.15 H, rat, 1:20, Developmental Studies Hybridoma Bank), Wt1 (sc-7385, mouse IgG1, 1:100, Santa Cruz), GFP (GFP-1020, chick IgY, 1:500, Aves Labs), Slc12a3 (HPA028748, rabbit, 1:300, Sigma), LTL (FL-1321, FITC, 1:200, Vector Laboratories), Slc12a1 (18970–1-AP, rabbit, 1:200, Proteintech), β-catenin (71–2700, rabbit, 1:500, Invitrogen), β-catenin (sc-7963, mouse IgG1, 1:50, Santa Cruz), Pecam1 (sc-18916, rat, 1:300, Santa Cruz), Mafb (HPA005653, rabbit, 1:300, Sigma), Akap12 (25199–1-AP, rabbit, 1:300, Proteintech), Cdh6 (HPA007047, rabbit, 1:300, Sigma), Hnf4a (ab41898, mouse IgG2a, 1:500, Abcam), Pax2 (21385–1-AP, rabbit, 1:200, Proteintech), Slc5a2 (HPA041603, rabbit, 1:100, Sigma) Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution.

    Techniques: Staining, Mutagenesis

    ( A – C ) In the β-catenin loss-of-function mutant kidney by Osr2Cre , the podocytes, proximal tubules, and loops of Henle show little β-catenin staining, suggesting that Ctnnb1 , the gene encoding β-catenin, was deleted by Osr2Cre . Cells lacking β-catenin are able to form Wt1+ podocytes ( A ), LTL + proximal tubules ( B ), and Slc12a1+ loops of Henle ( C ). Note, however, that the β-catenin mutant kidney shows aberrant arrangement of Wt1+ podocytes, weaker LTL staining, and thinner Slc12a1+ tubules, suggesting developmental defects. ( D ) In the β-catenin mutant kidney, β-catenin is still present in Slc12a3+ distal tubules, consistent with the fact that Osr2Cre does not target distal tubules. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Journal: Scientific Reports

    Article Title: β-catenin regulates the formation of multiple nephron segments in the mouse kidney

    doi: 10.1038/s41598-019-52255-w

    Figure Lengend Snippet: ( A – C ) In the β-catenin loss-of-function mutant kidney by Osr2Cre , the podocytes, proximal tubules, and loops of Henle show little β-catenin staining, suggesting that Ctnnb1 , the gene encoding β-catenin, was deleted by Osr2Cre . Cells lacking β-catenin are able to form Wt1+ podocytes ( A ), LTL + proximal tubules ( B ), and Slc12a1+ loops of Henle ( C ). Note, however, that the β-catenin mutant kidney shows aberrant arrangement of Wt1+ podocytes, weaker LTL staining, and thinner Slc12a1+ tubules, suggesting developmental defects. ( D ) In the β-catenin mutant kidney, β-catenin is still present in Slc12a3+ distal tubules, consistent with the fact that Osr2Cre does not target distal tubules. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Article Snippet: Immunofluorescence Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 (TS1.15 H, rat, 1:20, Developmental Studies Hybridoma Bank), Wt1 (sc-7385, mouse IgG1, 1:100, Santa Cruz), GFP (GFP-1020, chick IgY, 1:500, Aves Labs), Slc12a3 (HPA028748, rabbit, 1:300, Sigma), LTL (FL-1321, FITC, 1:200, Vector Laboratories), Slc12a1 (18970–1-AP, rabbit, 1:200, Proteintech), β-catenin (71–2700, rabbit, 1:500, Invitrogen), β-catenin (sc-7963, mouse IgG1, 1:50, Santa Cruz), Pecam1 (sc-18916, rat, 1:300, Santa Cruz), Mafb (HPA005653, rabbit, 1:300, Sigma), Akap12 (25199–1-AP, rabbit, 1:300, Proteintech), Cdh6 (HPA007047, rabbit, 1:300, Sigma), Hnf4a (ab41898, mouse IgG2a, 1:500, Abcam), Pax2 (21385–1-AP, rabbit, 1:200, Proteintech), Slc5a2 (HPA041603, rabbit, 1:100, Sigma) Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution.

    Techniques: Mutagenesis, Staining

    Expression of a stable form of β-catenin prevents epithelial nephron progenitors from further differentiating. ( A ) Nascent (left) and mature (right) S-shaped bodies are shown. In the control kidney, Jag1 and Wt1 mark the medial and proximal segments of the S-shaped body, respectively. In the β-catenin gain-of-function mutant kidney by Wnt4GFPcre , the Jag1 expression domain expands into the proximal segment of the S-shaped body. Pax2 marks the collecting duct, the cap mesenchyme, and nascent developing nephrons. ( B ) In the control kidney, Pax2 expression in epithelial nephron progenitors is downregulated after S-shaped body stage. As a result, most of the Hnf4a+ cells are negative for Pax2. In the β-catenin gain-of-function mutant kidney, Pax2 is persistently expressed in the nephron lineage and all Hnf4a+ cells are also positive for Pax2, suggesting that β-catenin gain-of-function mutant cells fail to exit from their progenitor status. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Journal: Scientific Reports

    Article Title: β-catenin regulates the formation of multiple nephron segments in the mouse kidney

    doi: 10.1038/s41598-019-52255-w

    Figure Lengend Snippet: Expression of a stable form of β-catenin prevents epithelial nephron progenitors from further differentiating. ( A ) Nascent (left) and mature (right) S-shaped bodies are shown. In the control kidney, Jag1 and Wt1 mark the medial and proximal segments of the S-shaped body, respectively. In the β-catenin gain-of-function mutant kidney by Wnt4GFPcre , the Jag1 expression domain expands into the proximal segment of the S-shaped body. Pax2 marks the collecting duct, the cap mesenchyme, and nascent developing nephrons. ( B ) In the control kidney, Pax2 expression in epithelial nephron progenitors is downregulated after S-shaped body stage. As a result, most of the Hnf4a+ cells are negative for Pax2. In the β-catenin gain-of-function mutant kidney, Pax2 is persistently expressed in the nephron lineage and all Hnf4a+ cells are also positive for Pax2, suggesting that β-catenin gain-of-function mutant cells fail to exit from their progenitor status. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Article Snippet: Immunofluorescence Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 (TS1.15 H, rat, 1:20, Developmental Studies Hybridoma Bank), Wt1 (sc-7385, mouse IgG1, 1:100, Santa Cruz), GFP (GFP-1020, chick IgY, 1:500, Aves Labs), Slc12a3 (HPA028748, rabbit, 1:300, Sigma), LTL (FL-1321, FITC, 1:200, Vector Laboratories), Slc12a1 (18970–1-AP, rabbit, 1:200, Proteintech), β-catenin (71–2700, rabbit, 1:500, Invitrogen), β-catenin (sc-7963, mouse IgG1, 1:50, Santa Cruz), Pecam1 (sc-18916, rat, 1:300, Santa Cruz), Mafb (HPA005653, rabbit, 1:300, Sigma), Akap12 (25199–1-AP, rabbit, 1:300, Proteintech), Cdh6 (HPA007047, rabbit, 1:300, Sigma), Hnf4a (ab41898, mouse IgG2a, 1:500, Abcam), Pax2 (21385–1-AP, rabbit, 1:200, Proteintech), Slc5a2 (HPA041603, rabbit, 1:100, Sigma) Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution.

    Techniques: Expressing, Mutagenesis

    β-catenin is required for the proper formation of the renal corpuscle. ( A ) In the renal corpuscle of the control kidney, Wt1+ podocytes surround Pecam1+ endothelial cells in a crescent configuration and Akap12 marks parietal epithelial cells. By contrast, in the β-catenin mutant kidney, Pecam1 + endothelial cells fail to populate inside the renal corpuscle and Akap12 expression is considerably reduced. ( B ) Pecam1 + endothelial cells invade the vascular cleft in the S-shaped body in both control and mutant kidneys. In the proximal segment of the S-shaped body, Wt1 is detected in both visceral and parietal epithelial cells while Mafb is detected in the visceral epithelial cells only. The initial specification of visceral and parietal epithelial cells appears normal in the β-catenin mutant kidney. Images are representative of three independent experiments. HT, Hoechst. Stage E18.5. Scale bar: 50 μm.

    Journal: Scientific Reports

    Article Title: β-catenin regulates the formation of multiple nephron segments in the mouse kidney

    doi: 10.1038/s41598-019-52255-w

    Figure Lengend Snippet: β-catenin is required for the proper formation of the renal corpuscle. ( A ) In the renal corpuscle of the control kidney, Wt1+ podocytes surround Pecam1+ endothelial cells in a crescent configuration and Akap12 marks parietal epithelial cells. By contrast, in the β-catenin mutant kidney, Pecam1 + endothelial cells fail to populate inside the renal corpuscle and Akap12 expression is considerably reduced. ( B ) Pecam1 + endothelial cells invade the vascular cleft in the S-shaped body in both control and mutant kidneys. In the proximal segment of the S-shaped body, Wt1 is detected in both visceral and parietal epithelial cells while Mafb is detected in the visceral epithelial cells only. The initial specification of visceral and parietal epithelial cells appears normal in the β-catenin mutant kidney. Images are representative of three independent experiments. HT, Hoechst. Stage E18.5. Scale bar: 50 μm.

    Article Snippet: Immunofluorescence Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 (TS1.15 H, rat, 1:20, Developmental Studies Hybridoma Bank), Wt1 (sc-7385, mouse IgG1, 1:100, Santa Cruz), GFP (GFP-1020, chick IgY, 1:500, Aves Labs), Slc12a3 (HPA028748, rabbit, 1:300, Sigma), LTL (FL-1321, FITC, 1:200, Vector Laboratories), Slc12a1 (18970–1-AP, rabbit, 1:200, Proteintech), β-catenin (71–2700, rabbit, 1:500, Invitrogen), β-catenin (sc-7963, mouse IgG1, 1:50, Santa Cruz), Pecam1 (sc-18916, rat, 1:300, Santa Cruz), Mafb (HPA005653, rabbit, 1:300, Sigma), Akap12 (25199–1-AP, rabbit, 1:300, Proteintech), Cdh6 (HPA007047, rabbit, 1:300, Sigma), Hnf4a (ab41898, mouse IgG2a, 1:500, Abcam), Pax2 (21385–1-AP, rabbit, 1:200, Proteintech), Slc5a2 (HPA041603, rabbit, 1:100, Sigma) Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution.

    Techniques: Mutagenesis, Expressing

    Removal of β-catenin by Wnt4Cre inhibits loop of Henle elongation and distal tubule formation. ( A ) Slc12a1+ cells in the control kidney elongate, contributing to papilla formation. Slc12a1+ cells in the β-catenin loss-of-function mutant kidney by Wnt4Cre fail to elongate, causing a defective papilla formation. The ureter is marked by broken lines. ( B ) In the β-catenin loss-of-function mutant kidney by Wnt4Cre , there are less Slc12a3+ distal tubules and the few that have formed are positive for β-catenin, suggesting that these cells have escaped Wnt4Cre -mediated removal of β-catenin. The absence of β-catenin-negative distal tubule cells suggests that β-catenin is required for the formation of distal tubules. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Journal: Scientific Reports

    Article Title: β-catenin regulates the formation of multiple nephron segments in the mouse kidney

    doi: 10.1038/s41598-019-52255-w

    Figure Lengend Snippet: Removal of β-catenin by Wnt4Cre inhibits loop of Henle elongation and distal tubule formation. ( A ) Slc12a1+ cells in the control kidney elongate, contributing to papilla formation. Slc12a1+ cells in the β-catenin loss-of-function mutant kidney by Wnt4Cre fail to elongate, causing a defective papilla formation. The ureter is marked by broken lines. ( B ) In the β-catenin loss-of-function mutant kidney by Wnt4Cre , there are less Slc12a3+ distal tubules and the few that have formed are positive for β-catenin, suggesting that these cells have escaped Wnt4Cre -mediated removal of β-catenin. The absence of β-catenin-negative distal tubule cells suggests that β-catenin is required for the formation of distal tubules. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Article Snippet: Immunofluorescence Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 (TS1.15 H, rat, 1:20, Developmental Studies Hybridoma Bank), Wt1 (sc-7385, mouse IgG1, 1:100, Santa Cruz), GFP (GFP-1020, chick IgY, 1:500, Aves Labs), Slc12a3 (HPA028748, rabbit, 1:300, Sigma), LTL (FL-1321, FITC, 1:200, Vector Laboratories), Slc12a1 (18970–1-AP, rabbit, 1:200, Proteintech), β-catenin (71–2700, rabbit, 1:500, Invitrogen), β-catenin (sc-7963, mouse IgG1, 1:50, Santa Cruz), Pecam1 (sc-18916, rat, 1:300, Santa Cruz), Mafb (HPA005653, rabbit, 1:300, Sigma), Akap12 (25199–1-AP, rabbit, 1:300, Proteintech), Cdh6 (HPA007047, rabbit, 1:300, Sigma), Hnf4a (ab41898, mouse IgG2a, 1:500, Abcam), Pax2 (21385–1-AP, rabbit, 1:200, Proteintech), Slc5a2 (HPA041603, rabbit, 1:100, Sigma) Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution.

    Techniques: Mutagenesis

    ( A – E ) Lineage analysis with Osr2Cre in the developing mouse kidney. Cre-mediated recombination activates expression of EYFP reporter, which labels Osr2 -expressing cells and their descendants. ( A ) Osr2Cre targets the proximal (Wt1+) and medial (Jag1+) segments of the S-shaped body. In the nephron, Osr2Cre targets podocytes ( B ), proximal tubules ( C ), and loops of Henle ( D ), but not distal tubules ( B ). White arrowhead in ( B ) points to podocytes that escaped Osr2Cre . ( E ) From the absence of EYFP signal (green), we conclude that Osr2Cre targets neither the cap mesenchyme (white arrowhead, for example) nor the interstitial cells (asterisk). ( F ) β-catenin is ubiquitously expressed in the S-shaped body. ( G ) Osr2Cre removes β-catenin from the proximal (yellow arrowhead) and medial (white arrowhead) segments of the S-shaped body. Images are representative of three independent experiments. HT, Hoechst. Stage E18.5. Scale bar: 50 μm.

    Journal: Scientific Reports

    Article Title: β-catenin regulates the formation of multiple nephron segments in the mouse kidney

    doi: 10.1038/s41598-019-52255-w

    Figure Lengend Snippet: ( A – E ) Lineage analysis with Osr2Cre in the developing mouse kidney. Cre-mediated recombination activates expression of EYFP reporter, which labels Osr2 -expressing cells and their descendants. ( A ) Osr2Cre targets the proximal (Wt1+) and medial (Jag1+) segments of the S-shaped body. In the nephron, Osr2Cre targets podocytes ( B ), proximal tubules ( C ), and loops of Henle ( D ), but not distal tubules ( B ). White arrowhead in ( B ) points to podocytes that escaped Osr2Cre . ( E ) From the absence of EYFP signal (green), we conclude that Osr2Cre targets neither the cap mesenchyme (white arrowhead, for example) nor the interstitial cells (asterisk). ( F ) β-catenin is ubiquitously expressed in the S-shaped body. ( G ) Osr2Cre removes β-catenin from the proximal (yellow arrowhead) and medial (white arrowhead) segments of the S-shaped body. Images are representative of three independent experiments. HT, Hoechst. Stage E18.5. Scale bar: 50 μm.

    Article Snippet: Immunofluorescence Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 (TS1.15 H, rat, 1:20, Developmental Studies Hybridoma Bank), Wt1 (sc-7385, mouse IgG1, 1:100, Santa Cruz), GFP (GFP-1020, chick IgY, 1:500, Aves Labs), Slc12a3 (HPA028748, rabbit, 1:300, Sigma), LTL (FL-1321, FITC, 1:200, Vector Laboratories), Slc12a1 (18970–1-AP, rabbit, 1:200, Proteintech), β-catenin (71–2700, rabbit, 1:500, Invitrogen), β-catenin (sc-7963, mouse IgG1, 1:50, Santa Cruz), Pecam1 (sc-18916, rat, 1:300, Santa Cruz), Mafb (HPA005653, rabbit, 1:300, Sigma), Akap12 (25199–1-AP, rabbit, 1:300, Proteintech), Cdh6 (HPA007047, rabbit, 1:300, Sigma), Hnf4a (ab41898, mouse IgG2a, 1:500, Abcam), Pax2 (21385–1-AP, rabbit, 1:200, Proteintech), Slc5a2 (HPA041603, rabbit, 1:100, Sigma) Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution.

    Techniques: Expressing

    Expression of a stable form of β-catenin inhibits proper nephron patterning. The β-catenin gain-of-function mutant kidney by Wnt4GFPcre fails to form properly patterned nephrons. ( A ) No proximal tubules or Slc12a1+ loops of Henle are formed in the mutant kidney. ( B ) No glomeruli or Slc12a3+ distal tubules are formed in the mutant kidney. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Journal: Scientific Reports

    Article Title: β-catenin regulates the formation of multiple nephron segments in the mouse kidney

    doi: 10.1038/s41598-019-52255-w

    Figure Lengend Snippet: Expression of a stable form of β-catenin inhibits proper nephron patterning. The β-catenin gain-of-function mutant kidney by Wnt4GFPcre fails to form properly patterned nephrons. ( A ) No proximal tubules or Slc12a1+ loops of Henle are formed in the mutant kidney. ( B ) No glomeruli or Slc12a3+ distal tubules are formed in the mutant kidney. Images are representative of three independent experiments. Stage E18.5. Scale bar: 100 μm.

    Article Snippet: Immunofluorescence Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 (TS1.15 H, rat, 1:20, Developmental Studies Hybridoma Bank), Wt1 (sc-7385, mouse IgG1, 1:100, Santa Cruz), GFP (GFP-1020, chick IgY, 1:500, Aves Labs), Slc12a3 (HPA028748, rabbit, 1:300, Sigma), LTL (FL-1321, FITC, 1:200, Vector Laboratories), Slc12a1 (18970–1-AP, rabbit, 1:200, Proteintech), β-catenin (71–2700, rabbit, 1:500, Invitrogen), β-catenin (sc-7963, mouse IgG1, 1:50, Santa Cruz), Pecam1 (sc-18916, rat, 1:300, Santa Cruz), Mafb (HPA005653, rabbit, 1:300, Sigma), Akap12 (25199–1-AP, rabbit, 1:300, Proteintech), Cdh6 (HPA007047, rabbit, 1:300, Sigma), Hnf4a (ab41898, mouse IgG2a, 1:500, Abcam), Pax2 (21385–1-AP, rabbit, 1:200, Proteintech), Slc5a2 (HPA041603, rabbit, 1:100, Sigma) Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution.

    Techniques: Expressing, Mutagenesis

    Knockdown of β-catenin or TCF-4 enhances Tat-mediated transactivation of the LTR independent of the nt −143 site. (a) U87MG cells were transfected with scrambled siRNA, β-catenin siRNA, or TCF-4 siRNA; 48 h postknockdown, the cells were transfected with WT-LTR or Δ-143-LTR luciferase constructs with either vector control (pcDNA3.1) or Tat expression plasmid for an additional 24 h. The cells were lysed, and a dual-luciferase assay was performed. (b) U87MG cells that stably express an integrated copy of the WT or a Δ-143-LTR-luciferase construct were transfected with the indicated siRNAs for 48 h, followed by transfection with vector control or Tat for an additional 24 h before the cells were lysed and a dual-luciferase assay was performed. Luciferase readings from panels a and b were normalized to the total protein content as measured by BCA assay. The data represent three independent experiments. *, P

    Journal: Journal of Virology

    Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

    doi: 10.1128/JVI.00486-12

    Figure Lengend Snippet: Knockdown of β-catenin or TCF-4 enhances Tat-mediated transactivation of the LTR independent of the nt −143 site. (a) U87MG cells were transfected with scrambled siRNA, β-catenin siRNA, or TCF-4 siRNA; 48 h postknockdown, the cells were transfected with WT-LTR or Δ-143-LTR luciferase constructs with either vector control (pcDNA3.1) or Tat expression plasmid for an additional 24 h. The cells were lysed, and a dual-luciferase assay was performed. (b) U87MG cells that stably express an integrated copy of the WT or a Δ-143-LTR-luciferase construct were transfected with the indicated siRNAs for 48 h, followed by transfection with vector control or Tat for an additional 24 h before the cells were lysed and a dual-luciferase assay was performed. Luciferase readings from panels a and b were normalized to the total protein content as measured by BCA assay. The data represent three independent experiments. *, P

    Article Snippet: U87MG cells were transiently transfected with WT-LTR or Δ-143-LTR plasmid, and after 24 h, chromatin immunoprecipitation (ChIP) was performed on the cells using a Millipore kit with antibodies for TCF-4 (Cell Signaling), LEF1 (Cell Signaling), β-catenin (Sigma), or SMAR1/BANP (Abcam).

    Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Expressing, Stable Transfection, BIA-KA

    ), we propose that β-catenin/TCF-4/SMAR form a complex at nt −143 on the HIV LTR. This complex pulls HIV DNA spanning this region into the nuclear matrix and away from transcription machinery. Inhibition of β-catenin/TCF-4 by siRNA disrupts this chromatin repression complex and allows Pol II docking and recruitment of TCoA, such as NF-κB and C/EBP, to drive basal LTR activity.

    Journal: Journal of Virology

    Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

    doi: 10.1128/JVI.00486-12

    Figure Lengend Snippet: ), we propose that β-catenin/TCF-4/SMAR form a complex at nt −143 on the HIV LTR. This complex pulls HIV DNA spanning this region into the nuclear matrix and away from transcription machinery. Inhibition of β-catenin/TCF-4 by siRNA disrupts this chromatin repression complex and allows Pol II docking and recruitment of TCoA, such as NF-κB and C/EBP, to drive basal LTR activity.

    Article Snippet: U87MG cells were transiently transfected with WT-LTR or Δ-143-LTR plasmid, and after 24 h, chromatin immunoprecipitation (ChIP) was performed on the cells using a Millipore kit with antibodies for TCF-4 (Cell Signaling), LEF1 (Cell Signaling), β-catenin (Sigma), or SMAR1/BANP (Abcam).

    Techniques: Inhibition, Activity Assay

    Deletion of TCF-4 binding sites at nt −143 enhances LTR activity, in combination with β-catenin/TCF-4 knockdown. (a) Schematic representation of HIV Bal LTR constructs inserted into the pGL4.19 luciferase vector. The bold X denotes the deletion of the −143 site from the HIV LTR. (b) U87MG cells were transfected with the indicated siRNAs; 48 h later, the cells were transfected with WT LTR or Δ-143-LTR for an additional 24 h. The cells were lysed, and a dual-luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. (c) U87MG cells containing integrated WT LTR-luciferase or Δ-143-LTR-luciferase plasmid were transfected with the indicated siRNAs; 72 h postknockdown, the cells were lysed and a dual-luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. (d) U87MG cells containing integrated WT LTR-luciferase were transfected with control siRNA, β-catenin siRNA, TCF-4 siRNA, or both β-catenin and TCF-4 siRNA; 72 h postknockdown, the cells were lysed and a dual-luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. The data represent three independent experiments. *, P ). The error bars represent SD.

    Journal: Journal of Virology

    Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

    doi: 10.1128/JVI.00486-12

    Figure Lengend Snippet: Deletion of TCF-4 binding sites at nt −143 enhances LTR activity, in combination with β-catenin/TCF-4 knockdown. (a) Schematic representation of HIV Bal LTR constructs inserted into the pGL4.19 luciferase vector. The bold X denotes the deletion of the −143 site from the HIV LTR. (b) U87MG cells were transfected with the indicated siRNAs; 48 h later, the cells were transfected with WT LTR or Δ-143-LTR for an additional 24 h. The cells were lysed, and a dual-luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. (c) U87MG cells containing integrated WT LTR-luciferase or Δ-143-LTR-luciferase plasmid were transfected with the indicated siRNAs; 72 h postknockdown, the cells were lysed and a dual-luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. (d) U87MG cells containing integrated WT LTR-luciferase were transfected with control siRNA, β-catenin siRNA, TCF-4 siRNA, or both β-catenin and TCF-4 siRNA; 72 h postknockdown, the cells were lysed and a dual-luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. The data represent three independent experiments. *, P ). The error bars represent SD.

    Article Snippet: U87MG cells were transiently transfected with WT-LTR or Δ-143-LTR plasmid, and after 24 h, chromatin immunoprecipitation (ChIP) was performed on the cells using a Millipore kit with antibodies for TCF-4 (Cell Signaling), LEF1 (Cell Signaling), β-catenin (Sigma), or SMAR1/BANP (Abcam).

    Techniques: Binding Assay, Activity Assay, Construct, Luciferase, Plasmid Preparation, Transfection, BIA-KA

    The nuclear matrix protein SMAR1 binds to the HIV LTR in astrocytes and associates with TCF-4 in vivo . (a) U87MG-LTR-luc containing an integrated HIV LTR-luciferase construct (integrated) or U87MG astrocytoma cells (episomal) was transfected with SMAR1 siRNA. After 48 h, U87MG astrocytoma cells were transfected with the LTR-luciferase construct for an additional 24 h. After 72 h of knockdown, all cells were lysed, and a luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. (b) U87MG astrocytoma cells were transfected with the indicated LTR-luciferase plasmid for 24 h. Chromatin immunoprecipitation was performed using antibodies to SMAR1 or rabbit IgG, followed by real-time PCR using primers encompassing nt −143, as described in Materials and Methods. (c to h) TCF-4, SMAR1, or β-catenin was immunoprecipitated from nuclear extracts generated from U87MG cells, followed by Western blotting to detect coimmunoprecipitated proteins, as indicated. For blocking experiments (e and f), SMAR1 antibody was incubated with a 5-fold excess of SMAR1-blocking peptide for 30 min prior to Western blotting to show the specificity of binding. The data are representative of three independent experiments. *, P

    Journal: Journal of Virology

    Article Title: Identification of Novel T Cell Factor 4 (TCF-4) Binding Sites on the HIV Long Terminal Repeat Which Associate with TCF-4, ?-Catenin, and SMAR1 To Repress HIV Transcription

    doi: 10.1128/JVI.00486-12

    Figure Lengend Snippet: The nuclear matrix protein SMAR1 binds to the HIV LTR in astrocytes and associates with TCF-4 in vivo . (a) U87MG-LTR-luc containing an integrated HIV LTR-luciferase construct (integrated) or U87MG astrocytoma cells (episomal) was transfected with SMAR1 siRNA. After 48 h, U87MG astrocytoma cells were transfected with the LTR-luciferase construct for an additional 24 h. After 72 h of knockdown, all cells were lysed, and a luciferase assay was performed. Readings were normalized to the total protein content as measured by BCA assay. (b) U87MG astrocytoma cells were transfected with the indicated LTR-luciferase plasmid for 24 h. Chromatin immunoprecipitation was performed using antibodies to SMAR1 or rabbit IgG, followed by real-time PCR using primers encompassing nt −143, as described in Materials and Methods. (c to h) TCF-4, SMAR1, or β-catenin was immunoprecipitated from nuclear extracts generated from U87MG cells, followed by Western blotting to detect coimmunoprecipitated proteins, as indicated. For blocking experiments (e and f), SMAR1 antibody was incubated with a 5-fold excess of SMAR1-blocking peptide for 30 min prior to Western blotting to show the specificity of binding. The data are representative of three independent experiments. *, P

    Article Snippet: U87MG cells were transiently transfected with WT-LTR or Δ-143-LTR plasmid, and after 24 h, chromatin immunoprecipitation (ChIP) was performed on the cells using a Millipore kit with antibodies for TCF-4 (Cell Signaling), LEF1 (Cell Signaling), β-catenin (Sigma), or SMAR1/BANP (Abcam).

    Techniques: In Vivo, Luciferase, Construct, Transfection, BIA-KA, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Generated, Western Blot, Blocking Assay, Incubation, Binding Assay

    Relative expression of osteogenic markers (a) Runx2, (b) ALP, (c) β -catenin, and (d) Smurf2, which is associated with β -catenin degradation and reduced osteogenesis, were determined by qRT-PCR at day 7. All the samples were performed in triplicate. *** p

    Journal: ACS biomaterials science & engineering

    Article Title: Treating Titanium Particle-Induced Inflammation with Genetically Modified NF-κB Sensing IL-4 Secreting or Preconditioned Mesenchymal Stem Cells in Vitro

    doi: 10.1021/acsbiomaterials.9b00560

    Figure Lengend Snippet: Relative expression of osteogenic markers (a) Runx2, (b) ALP, (c) β -catenin, and (d) Smurf2, which is associated with β -catenin degradation and reduced osteogenesis, were determined by qRT-PCR at day 7. All the samples were performed in triplicate. *** p

    Article Snippet: The images were captured using a fluorescence microscope (Axio Observer.Z1; Carl Zeiss, Jena, Germany) in 3 randomly chosen fields of view, and the signal of β -catenin was quantified using ImageJ.

    Techniques: Expressing, Quantitative RT-PCR

    Immunofluorescent staining of β -catenin (green). Cell nuclei were counterstained with DAPI (blue). (a) The staining of the IL4MSC group at day 7. (b) The staining signal of β -catenin was quantified in 3 randomly selected fields of view using ImageJ.

    Journal: ACS biomaterials science & engineering

    Article Title: Treating Titanium Particle-Induced Inflammation with Genetically Modified NF-κB Sensing IL-4 Secreting or Preconditioned Mesenchymal Stem Cells in Vitro

    doi: 10.1021/acsbiomaterials.9b00560

    Figure Lengend Snippet: Immunofluorescent staining of β -catenin (green). Cell nuclei were counterstained with DAPI (blue). (a) The staining of the IL4MSC group at day 7. (b) The staining signal of β -catenin was quantified in 3 randomly selected fields of view using ImageJ.

    Article Snippet: The images were captured using a fluorescence microscope (Axio Observer.Z1; Carl Zeiss, Jena, Germany) in 3 randomly chosen fields of view, and the signal of β -catenin was quantified using ImageJ.

    Techniques: Staining

    CCN1 activates HSCs and affects cell function. LX-2 cells were infected with adenovirus AdCCN1 or AdRFP, respectively. (A) Following infection for 24 h, western blot analysis was performed to detect the expression levels of CCN1 in LX-2 cells, LX-2-RFP cells and LX-2-CCN1 cells. (B) Following infection for 72 h, α-SMA and collagen I, which are markers of HSC activation and fibrosis, were detected in the LX-2-CCN1 cells. (C) Expression levels of p-β-catenin, β-catenin, cyclin D1, VEGF, CD31 and CD34 were analyzed. CCN1 antibody (0.5 μ g/ml) was used to neutralize CCN1 in control groups. (D) Viability of LX-2-CCN1 cells was analyzed using MTT assays. (E) Following treatment with RCCN1 (0.6 μ g/ml), LX-2 cells were analyzed using MTT assays. CCN1 antibody (0.5 mg/ml) was used in assays. The final concentrations were 0.5 and 2.5 μ g/ml. (F) Monolayer scratch assay was used to analyze the migration of LX-2-CCN1 cells. * P

    Journal: International Journal of Molecular Medicine

    Article Title: CCN1/Cyr61 enhances the function of hepatic stellate cells in promoting the progression of hepatocellular carcinoma

    doi: 10.3892/ijmm.2017.3356

    Figure Lengend Snippet: CCN1 activates HSCs and affects cell function. LX-2 cells were infected with adenovirus AdCCN1 or AdRFP, respectively. (A) Following infection for 24 h, western blot analysis was performed to detect the expression levels of CCN1 in LX-2 cells, LX-2-RFP cells and LX-2-CCN1 cells. (B) Following infection for 72 h, α-SMA and collagen I, which are markers of HSC activation and fibrosis, were detected in the LX-2-CCN1 cells. (C) Expression levels of p-β-catenin, β-catenin, cyclin D1, VEGF, CD31 and CD34 were analyzed. CCN1 antibody (0.5 μ g/ml) was used to neutralize CCN1 in control groups. (D) Viability of LX-2-CCN1 cells was analyzed using MTT assays. (E) Following treatment with RCCN1 (0.6 μ g/ml), LX-2 cells were analyzed using MTT assays. CCN1 antibody (0.5 mg/ml) was used in assays. The final concentrations were 0.5 and 2.5 μ g/ml. (F) Monolayer scratch assay was used to analyze the migration of LX-2-CCN1 cells. * P

    Article Snippet: The membrane was then incubated with antibodies against phosphorylated-β-catenin (cat. no 9566, 1:1,000), total-β-catenin (cat. no. 8480, 1:1,000) (both from Cell Signaling Technology, Inc., Danvers, MA, USA), cyclin D1 (cat. no. 60186-1-Ig, 1:1,000), VEGF (cat. no. 19003-1-AP, 1:1,000), CD34 (cat. no. 14486-1-AP, 1:1,000), CD31 (cat. no. 66065-1-Ig; 1:1,000) (all from ProteinTech Group, Inc.), survivin (cat. no. ab76424; 1:1,000 dilution; Abcam, Cambridge, UK), and c-myc (cat. no. MA1-980, 1:1,000; Thermo Fisher Scientific, Inc.), respectively at 4°C overnight.

    Techniques: Cell Function Assay, Infection, Western Blot, Expressing, Activation Assay, MTT Assay, Wound Healing Assay, Migration

    Schematic diagram of the gene regulation of ESRRG and Wnt signaling. a ESRRG induces β-catenin degradation via an unknown mechanism and inhibits the binding of the Wnt component TCF/LEF to the CCND1 gene promoter region by direct interaction, thus potentially repressing the Wnt signaling pathway and blocking tumorigenesis. b The balance of ESRRG and Wnt signaling activity is crucial for tumor suppression or formation in GC

    Journal: Nature Communications

    Article Title: Estrogen-related receptor gamma functions as a tumor suppressor in gastric cancer

    doi: 10.1038/s41467-018-04244-2

    Figure Lengend Snippet: Schematic diagram of the gene regulation of ESRRG and Wnt signaling. a ESRRG induces β-catenin degradation via an unknown mechanism and inhibits the binding of the Wnt component TCF/LEF to the CCND1 gene promoter region by direct interaction, thus potentially repressing the Wnt signaling pathway and blocking tumorigenesis. b The balance of ESRRG and Wnt signaling activity is crucial for tumor suppression or formation in GC

    Article Snippet: Western blotting Western blot analysis was performed as described previously using antibodies against ESRRG (#H6812; R & D Systems), total and phosphor-β-catenin (#9561 and #9562; Cell Signaling Technology (CST), Danvers, MA), TCF4 (#2569;CST), Flag (#2368;CST), LEF1 (#A303-487A; Bethyl Laboratories), HDAC1 (#7872; Santa Cruz Biotechnology), β-actin (#A5316; Sigma-Aldrich), pGSK3α/β (#9331; CST), GSK3α/β (#ab15314; Abcam), and α-tubulin (#3873; CST).

    Techniques: Binding Assay, Blocking Assay, Activity Assay

    Defective turnover of β-catenin in cells genetically deficient in mouse presenilins and rescued by hPS1-bearing FAD-linked mutations. A: Confluent cultures of PS −/− cells and PS −/− cells reconstituted with either

    Journal:

    Article Title: Perturbed Neurogenesis in the Adult Hippocampus Associated with Presenilin-1 A246E Mutation

    doi:

    Figure Lengend Snippet: Defective turnover of β-catenin in cells genetically deficient in mouse presenilins and rescued by hPS1-bearing FAD-linked mutations. A: Confluent cultures of PS −/− cells and PS −/− cells reconstituted with either

    Article Snippet: Resistant cells were pooled and analyzed without further clonal selection. β-Catenin degradation was monitored by treating confluent cultures with 25 μg/ml cycloheximide for the indicated times as described, and CHAPS lysates were subjected to immunoblotting for phospho-45-specific β-catenin antibody (Cell Signaling, Beverly, MA).

    Techniques:

    CCL5 promotes prostate cancer invasion and PCSCs self-renewal via activating the CCR5/β-catenin/STAT3 pathway. a – c Heatmaps of 94 metastasis-related DEGs ( a ), 42 stemness-related DEGs ( b ), as well as 30 STAT3 pathway-related DEGs ( c ). RNA-Seq analysis was conducted to characterize the cellular responses of PC3 cells to 40 ng/ml CCL5 treatment. Differential gene expression analysis was conducted to identify the DEGs ( n = 3). d Venn diagram of the DEGs in the indicated groups. CTNNB1, also known as β-catenin, was the most significant one of them. e Western blotting assay indicated that the CD133 + PCSCs sorted from PC3 cells by MACS method exhibited increased expression of CCR5, β-catenin, and STAT3 when compared with the CD133 - subpopulation ( n = 3). f CCL5 treatment significantly elevated the promoter activity of STAT3 in PC3 cells while XAV-939, the specific inhibitor of β-catenin, partly abrogated that ( n = 6). g Immunofluorescence assay indicated that CCL5 treatment (40 ng/ml) could significantly induce the expression and nuclear translocation of β-catenin in prostate cancer cells. Scale bar, 10 μm. h CHIP assay suggested that β-catenin could bind to the promoter region of STAT3 , while the specific inhibitor of β-catenin (XAV-939) partly decreased their binding activity. i CCR5 specific siRNAs could significantly abrogate the activation effect of CCL5 on β-catenin/STAT3 pathway ( n = 3). All values are presented as the mean ± SD, * p

    Journal: Cell Death & Disease

    Article Title: CCL5 derived from tumor-associated macrophages promotes prostate cancer stem cells and metastasis via activating β-catenin/STAT3 signaling

    doi: 10.1038/s41419-020-2435-y

    Figure Lengend Snippet: CCL5 promotes prostate cancer invasion and PCSCs self-renewal via activating the CCR5/β-catenin/STAT3 pathway. a – c Heatmaps of 94 metastasis-related DEGs ( a ), 42 stemness-related DEGs ( b ), as well as 30 STAT3 pathway-related DEGs ( c ). RNA-Seq analysis was conducted to characterize the cellular responses of PC3 cells to 40 ng/ml CCL5 treatment. Differential gene expression analysis was conducted to identify the DEGs ( n = 3). d Venn diagram of the DEGs in the indicated groups. CTNNB1, also known as β-catenin, was the most significant one of them. e Western blotting assay indicated that the CD133 + PCSCs sorted from PC3 cells by MACS method exhibited increased expression of CCR5, β-catenin, and STAT3 when compared with the CD133 - subpopulation ( n = 3). f CCL5 treatment significantly elevated the promoter activity of STAT3 in PC3 cells while XAV-939, the specific inhibitor of β-catenin, partly abrogated that ( n = 6). g Immunofluorescence assay indicated that CCL5 treatment (40 ng/ml) could significantly induce the expression and nuclear translocation of β-catenin in prostate cancer cells. Scale bar, 10 μm. h CHIP assay suggested that β-catenin could bind to the promoter region of STAT3 , while the specific inhibitor of β-catenin (XAV-939) partly decreased their binding activity. i CCR5 specific siRNAs could significantly abrogate the activation effect of CCL5 on β-catenin/STAT3 pathway ( n = 3). All values are presented as the mean ± SD, * p

    Article Snippet: After 40 ng/ml CCL5 treatment for 24 h, cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS and then permeabilized with 0.25% Triton X-100 for 20 min. After blocking with 5% BSA for 1 h, cells were incubated with STAT3 antibody (A11216, Abclonal) or β-catenin antibody (8480S, CST) overnight at 4 °C.

    Techniques: RNA Sequencing Assay, Expressing, Western Blot, Magnetic Cell Separation, Activity Assay, Immunofluorescence, Translocation Assay, Chromatin Immunoprecipitation, Binding Assay, Activation Assay

    CCL5 knockdown in TAMs suppresses prostate cancer growth, bone metastasis and PCSCs activity in vivo. a , b THP1-derived TAMs co-injection not only significantly accelerated PC-3-Luc xenografts growth but also increased the bone metastasis of prostate cancer xenografts, whereas CCL5 knockdown in THP1-derived TAMs could partly abrogate that. Mice were imaged using the IVIS Lumina XR in vivo imaging system to monitor PC3-Luc xenografts growth and bone metastasis ( n = 12). Scale bar, 200 μm. c Immunohistochemistry indicated that CCL5 knockdown in THP1-derived TAMs could also significantly abrogate the up-regulation effect of TAMs on metastasis and stemness-related markers including β-catenin, STAT3, CD133, and Vimentin in PC-3-Luc xenografts ( n = 6). Scale bar, 50 μm. d CCL5 knockdown in THP1-derived TAMs could also significantly abrogate the promotion effect of THP1-derived TAMs on PCSCs self-renewal activity in vivo. Primary cells were isolated from fresh tumors by mechanical methods and subjected to PCSCs subpopulation analysis ( n = 3). e In vivo limiting dilution assay was conducted to compare the tumorigenicity changes of PCSCs after CCL5 knockdown in co-cultured TAMs. The tumorigenic cell frequency estimate and p values were calculated by the limiting dilution analysis tool ( http://bioinf.wehi.edu.au/software/elda/ ) ( n = 8). * p

    Journal: Cell Death & Disease

    Article Title: CCL5 derived from tumor-associated macrophages promotes prostate cancer stem cells and metastasis via activating β-catenin/STAT3 signaling

    doi: 10.1038/s41419-020-2435-y

    Figure Lengend Snippet: CCL5 knockdown in TAMs suppresses prostate cancer growth, bone metastasis and PCSCs activity in vivo. a , b THP1-derived TAMs co-injection not only significantly accelerated PC-3-Luc xenografts growth but also increased the bone metastasis of prostate cancer xenografts, whereas CCL5 knockdown in THP1-derived TAMs could partly abrogate that. Mice were imaged using the IVIS Lumina XR in vivo imaging system to monitor PC3-Luc xenografts growth and bone metastasis ( n = 12). Scale bar, 200 μm. c Immunohistochemistry indicated that CCL5 knockdown in THP1-derived TAMs could also significantly abrogate the up-regulation effect of TAMs on metastasis and stemness-related markers including β-catenin, STAT3, CD133, and Vimentin in PC-3-Luc xenografts ( n = 6). Scale bar, 50 μm. d CCL5 knockdown in THP1-derived TAMs could also significantly abrogate the promotion effect of THP1-derived TAMs on PCSCs self-renewal activity in vivo. Primary cells were isolated from fresh tumors by mechanical methods and subjected to PCSCs subpopulation analysis ( n = 3). e In vivo limiting dilution assay was conducted to compare the tumorigenicity changes of PCSCs after CCL5 knockdown in co-cultured TAMs. The tumorigenic cell frequency estimate and p values were calculated by the limiting dilution analysis tool ( http://bioinf.wehi.edu.au/software/elda/ ) ( n = 8). * p

    Article Snippet: After 40 ng/ml CCL5 treatment for 24 h, cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS and then permeabilized with 0.25% Triton X-100 for 20 min. After blocking with 5% BSA for 1 h, cells were incubated with STAT3 antibody (A11216, Abclonal) or β-catenin antibody (8480S, CST) overnight at 4 °C.

    Techniques: Activity Assay, In Vivo, Derivative Assay, Injection, Mouse Assay, In Vivo Imaging, Immunohistochemistry, Isolation, Limiting Dilution Assay, Cell Culture, Software

    ER71 inhibits canonical Wnt signaling. (A) iEr71 ES cells were differentiated in the absence (Control) or presence (+CHIR99021, 1μM) of CHIR99021 from days 2-3 of differentiation, either with (+DOX) or without (−DOX) DOX from days 2-3 of differentiation. Flk-1 and PDGFRα expression were analyzed at day 3. (B) Er71 −/− ES cells were differentiated and treated with CHIR99021 or DKK1 from day 2. Flk-1 and PDGFRα expression was analyzed at day 3. (C-D) ST2 cells were cotransfected with Lef1-luciferase ± MSCV- Er71 and ± Wnt3a conditioned medium (C) or ± β-catenin expression vector (D). Luciferase activity was measured 48 hours later. Firefly luciferase activity was normalized by Renilla luciferase activity. (E) Flk-1 + mesoderm sorted from iEr71 EBs was cotransfected with Lef1-luciferase and β-catenin. DOX was added to culture and luciferase activity was measured 48 hours later. (F) Flk-1 + mesoderm sorted from Er71 +/+ and Er71 −/− EBs were cotransfected with Lef1-luciferase ± MSCV -Er71 . Luciferase activity was measured 48 hours later. Data are presented as the means ± SD of at least 4 independent experiments. * P

    Journal: Blood

    Article Title: ER71 specifies Flk-1+ hemangiogenic mesoderm by inhibiting cardiac mesoderm and Wnt signaling

    doi: 10.1182/blood-2012-01-403766

    Figure Lengend Snippet: ER71 inhibits canonical Wnt signaling. (A) iEr71 ES cells were differentiated in the absence (Control) or presence (+CHIR99021, 1μM) of CHIR99021 from days 2-3 of differentiation, either with (+DOX) or without (−DOX) DOX from days 2-3 of differentiation. Flk-1 and PDGFRα expression were analyzed at day 3. (B) Er71 −/− ES cells were differentiated and treated with CHIR99021 or DKK1 from day 2. Flk-1 and PDGFRα expression was analyzed at day 3. (C-D) ST2 cells were cotransfected with Lef1-luciferase ± MSCV- Er71 and ± Wnt3a conditioned medium (C) or ± β-catenin expression vector (D). Luciferase activity was measured 48 hours later. Firefly luciferase activity was normalized by Renilla luciferase activity. (E) Flk-1 + mesoderm sorted from iEr71 EBs was cotransfected with Lef1-luciferase and β-catenin. DOX was added to culture and luciferase activity was measured 48 hours later. (F) Flk-1 + mesoderm sorted from Er71 +/+ and Er71 −/− EBs were cotransfected with Lef1-luciferase ± MSCV -Er71 . Luciferase activity was measured 48 hours later. Data are presented as the means ± SD of at least 4 independent experiments. * P

    Article Snippet: The following Abs were used: α- β-tubulin (Abcam), α-lamin (Abcam), α-GAPDH (Santa Cruz Biotechnology), α-VE-cadherin (eBioBV13; eBioscience), α-β-catenin (Cell Signaling Technology), and α-Flk-1 (55B11; Cell Signaling Technology).

    Techniques: Expressing, Luciferase, Plasmid Preparation, Activity Assay

    ER71 up-regulates VE-cadherin expression, which forms a VE-cadherin/ β-catenin/Flk-1 complex. (A) Cdh5 gene expression is shown in Flk-1 + PDGFRα + (DP) and Flk-1 + PDGFRα − (SP) cell populations. (B-C) qRT-PCR analyses of Cdh5 gene expression in Er71 +/+ , Er71 −/− , and iEr71 EBs (± DOX, with DOX during days 2-4) on day 4 (B; n = 4) and embryonic day 9.5 heart trunk region of Er71 +/+ and Er71 −/− embryos (n = 12; C). (D) Western blot analyses of VE-cadherin expression in day 4 Er71 +/+ , Er71 −/− , or iEr71 EBs (± DOX, with DOX during days 2-4). (E) ER71 ChIP-Seq peaks in the Cdh5 gene region. The number of ChIP-Seq–aligned tags near the mouse Cdh5 gene locus is displayed in the UCSC genome browser. The location of the enriched ER71 ChIP-Seq peak relative to the transcription start site of the Cdh5 gene (−5.48 kb) is indicated with an arrow. (F) ChIP–qRT-PCR analysis for ER71 binding on the Cdh5 promoter region in iEr71 ES cells. Primers flanking an open reading frame–free intergenic region in mouse chromosome 6 were used as a negative control. Primers flanking the Flk-1 promoter region were used as a positive control. The relative enrichment of ER71 binding is represented as the -fold change. (G) Day 4 iEr71 EBs (± DOX) were subjected to immunoprecipitation using VE-cadherin (left), β-catenin (middle), or Flk1 Ab (right). Western blot analyses were performed using the Abs indicated on the left. DOX was added from day 2. (H-I) Day 4 iEr71 (H) and i β- catenin-2A-Er71 (I) EBs (± DOX) were separated for cytosolic or nuclear fractions, and β-catenin levels were analyzed by Western blot. Cytosolic and nuclear signals were normalized to β-tubulin and lamin B, respectively. Data are presented as means ± SD of at least 4 independent experiments.

    Journal: Blood

    Article Title: ER71 specifies Flk-1+ hemangiogenic mesoderm by inhibiting cardiac mesoderm and Wnt signaling

    doi: 10.1182/blood-2012-01-403766

    Figure Lengend Snippet: ER71 up-regulates VE-cadherin expression, which forms a VE-cadherin/ β-catenin/Flk-1 complex. (A) Cdh5 gene expression is shown in Flk-1 + PDGFRα + (DP) and Flk-1 + PDGFRα − (SP) cell populations. (B-C) qRT-PCR analyses of Cdh5 gene expression in Er71 +/+ , Er71 −/− , and iEr71 EBs (± DOX, with DOX during days 2-4) on day 4 (B; n = 4) and embryonic day 9.5 heart trunk region of Er71 +/+ and Er71 −/− embryos (n = 12; C). (D) Western blot analyses of VE-cadherin expression in day 4 Er71 +/+ , Er71 −/− , or iEr71 EBs (± DOX, with DOX during days 2-4). (E) ER71 ChIP-Seq peaks in the Cdh5 gene region. The number of ChIP-Seq–aligned tags near the mouse Cdh5 gene locus is displayed in the UCSC genome browser. The location of the enriched ER71 ChIP-Seq peak relative to the transcription start site of the Cdh5 gene (−5.48 kb) is indicated with an arrow. (F) ChIP–qRT-PCR analysis for ER71 binding on the Cdh5 promoter region in iEr71 ES cells. Primers flanking an open reading frame–free intergenic region in mouse chromosome 6 were used as a negative control. Primers flanking the Flk-1 promoter region were used as a positive control. The relative enrichment of ER71 binding is represented as the -fold change. (G) Day 4 iEr71 EBs (± DOX) were subjected to immunoprecipitation using VE-cadherin (left), β-catenin (middle), or Flk1 Ab (right). Western blot analyses were performed using the Abs indicated on the left. DOX was added from day 2. (H-I) Day 4 iEr71 (H) and i β- catenin-2A-Er71 (I) EBs (± DOX) were separated for cytosolic or nuclear fractions, and β-catenin levels were analyzed by Western blot. Cytosolic and nuclear signals were normalized to β-tubulin and lamin B, respectively. Data are presented as means ± SD of at least 4 independent experiments.

    Article Snippet: The following Abs were used: α- β-tubulin (Abcam), α-lamin (Abcam), α-GAPDH (Santa Cruz Biotechnology), α-VE-cadherin (eBioBV13; eBioscience), α-β-catenin (Cell Signaling Technology), and α-Flk-1 (55B11; Cell Signaling Technology).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Positive Control, Immunoprecipitation

    Growth arrest of established murine tumours upon stromal Hh activation. ( a ) Protocol schematic. ( b ) Example of a 3D μUS reconstruction; tumour volume (arrow) is highlighted in red, other hypoechoic (black) structure is the urinary bladder (ub); lu: colonic lumen, filled with US gel; vaw: ventral abdominal wall. ( c ) Tomato + cells in an AOM/DSS-induced tumour from a Col1a2CreER;R26-LSL-tdTomato mouse 3 days after Tam administration; β-catenin (green) marks tumour cells; nuclear stain: 4,6-diamidino-2-phenylindole (DAPI); scale bar, 100 μm. ( d ) Tumour size as measured by μUS after Tam instillation (week 0). A total of n =5 Ptch1 ΔCol1 mice with n =11 tumours and n =7 controls (littermates lacking Cre recombinase, floxed Ptch1 alleles or both) with n =12 tumours are included; 1 to 5 tumours per mouse were analysed. The adjusted P -values (fdr) from t -tests are indicated: P =0.406 (week 0), P =0.022 (week 1), P =0.029 (week 2), P =0.026 (week 3), P =0.026 (week 4) and P =0.078 (week 5). Box-and-whisker plots: whiskers represent minima and maxima. In the control group, mice had to be killed because of bleeding per anus, leaving 2 tumours for US analysis in week 5 and none in week 6. ( e ) Quantification of cleaved caspase-3 positivity per high-power field (original magnification × 400): controls ( n =9 tumours), Ptch1 ΔCol1 mice ( n =5 tumours), P =0.0034 ( t -test). Middle and right panels show representative IHC images for similar-sized tumours 3 weeks after Tam treatment for the indicated genotypes. Scale bars, 20 μm. ( f ) Proliferative index as a percentage of Ki67 + nuclei for n =7 controls and n =4 tumours from Ptch1 ΔCol1 mice, P =0.0467 ( t -test) and representative images (middle and right panels). Scale bars, 50/20 μm. ( g ) ISH semi-quantification of Grem1 in control mice ( n =5 tumours) and Ptch1 ΔCol1 mice ( n =7 tumours). Stacked bars represent the frequency of intensity grading; P =0.0076 (Mann–Whitney test), middle and right panels show representative images for the two groups; scale bars, 200/100 μm. ( h ) Quantification of p-Smad1/5 IHC in control mice ( n =8 tumours) and Ptch1 ΔCol1 mice ( n =9 tumours); P =0.0028 ( t -test), middle and right panels show representative images for the two groups; scale bars, 50 μm; ( e – h ) boxed areas show magnified regions. * P

    Journal: Nature Communications

    Article Title: Stromal Hedgehog signalling is downregulated in colon cancer and its restoration restrains tumour growth

    doi: 10.1038/ncomms12321

    Figure Lengend Snippet: Growth arrest of established murine tumours upon stromal Hh activation. ( a ) Protocol schematic. ( b ) Example of a 3D μUS reconstruction; tumour volume (arrow) is highlighted in red, other hypoechoic (black) structure is the urinary bladder (ub); lu: colonic lumen, filled with US gel; vaw: ventral abdominal wall. ( c ) Tomato + cells in an AOM/DSS-induced tumour from a Col1a2CreER;R26-LSL-tdTomato mouse 3 days after Tam administration; β-catenin (green) marks tumour cells; nuclear stain: 4,6-diamidino-2-phenylindole (DAPI); scale bar, 100 μm. ( d ) Tumour size as measured by μUS after Tam instillation (week 0). A total of n =5 Ptch1 ΔCol1 mice with n =11 tumours and n =7 controls (littermates lacking Cre recombinase, floxed Ptch1 alleles or both) with n =12 tumours are included; 1 to 5 tumours per mouse were analysed. The adjusted P -values (fdr) from t -tests are indicated: P =0.406 (week 0), P =0.022 (week 1), P =0.029 (week 2), P =0.026 (week 3), P =0.026 (week 4) and P =0.078 (week 5). Box-and-whisker plots: whiskers represent minima and maxima. In the control group, mice had to be killed because of bleeding per anus, leaving 2 tumours for US analysis in week 5 and none in week 6. ( e ) Quantification of cleaved caspase-3 positivity per high-power field (original magnification × 400): controls ( n =9 tumours), Ptch1 ΔCol1 mice ( n =5 tumours), P =0.0034 ( t -test). Middle and right panels show representative IHC images for similar-sized tumours 3 weeks after Tam treatment for the indicated genotypes. Scale bars, 20 μm. ( f ) Proliferative index as a percentage of Ki67 + nuclei for n =7 controls and n =4 tumours from Ptch1 ΔCol1 mice, P =0.0467 ( t -test) and representative images (middle and right panels). Scale bars, 50/20 μm. ( g ) ISH semi-quantification of Grem1 in control mice ( n =5 tumours) and Ptch1 ΔCol1 mice ( n =7 tumours). Stacked bars represent the frequency of intensity grading; P =0.0076 (Mann–Whitney test), middle and right panels show representative images for the two groups; scale bars, 200/100 μm. ( h ) Quantification of p-Smad1/5 IHC in control mice ( n =8 tumours) and Ptch1 ΔCol1 mice ( n =9 tumours); P =0.0028 ( t -test), middle and right panels show representative images for the two groups; scale bars, 50 μm; ( e – h ) boxed areas show magnified regions. * P

    Article Snippet: The antibodies and dilutions were as follows: anti-vimentin (Santa Cruz, sc7557, 1:500); anti-desmin (Abcam, ab8592, 1:500); anti-α-sma (Abcam, ab5694, 1:500); anti-collagen1 (Abcam, ab34710, 1:200); anti-β-catenin (Cell Signalling, #9582, 1:100); anti-Ita6 (CD49f, BD Pharmigen, # 555734, 1:100).

    Techniques: Activation Assay, Staining, Mouse Assay, Whisker Assay, Immunohistochemistry, In Situ Hybridization, MANN-WHITNEY

    The effect of CD317 knockdown in polarized Caco-2 cells. (A) Left panels are maximum intensity projection XY images showing Alexa Fluor 594–phalloidin decoration of F-actin in polarized control and CD317 knockdown Caco-2 cells. Middle panels show XZ sections taken along the lines in left panels. Right panels show corresponding SEM images of the surface of control and CD317 knockdown cells. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (B) ZO-1 labeling of tight junctions in polarized CD317 knockdown and control Caco-2 cells and biotin labeling of proteins in the apical membrane of polarized CD317 knockdown Caco-2 cells as indicated. Left panels are maximum intensity projection XY images, and right panels are XZ sections taken along the lines in the left panels. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (C) Detection of β-catenin in the lateral membranes and sucrase-isomaltase in the apical membrane of polarized CD317 knockdown Caco-2 cells. XZ sections derived from maximum intensity projection XY images are presented. The right panel is a merge of the left and middle images. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

    doi: 10.1083/jcb.200804154

    Figure Lengend Snippet: The effect of CD317 knockdown in polarized Caco-2 cells. (A) Left panels are maximum intensity projection XY images showing Alexa Fluor 594–phalloidin decoration of F-actin in polarized control and CD317 knockdown Caco-2 cells. Middle panels show XZ sections taken along the lines in left panels. Right panels show corresponding SEM images of the surface of control and CD317 knockdown cells. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (B) ZO-1 labeling of tight junctions in polarized CD317 knockdown and control Caco-2 cells and biotin labeling of proteins in the apical membrane of polarized CD317 knockdown Caco-2 cells as indicated. Left panels are maximum intensity projection XY images, and right panels are XZ sections taken along the lines in the left panels. Arrows indicate the positions of the top (A) and bottom (B) of the cell monolayer. (C) Detection of β-catenin in the lateral membranes and sucrase-isomaltase in the apical membrane of polarized CD317 knockdown Caco-2 cells. XZ sections derived from maximum intensity projection XY images are presented. The right panel is a merge of the left and middle images. Bars, 10 µm.

    Article Snippet: Other antibodies used were mouse monoclonal antibodies to ezrin (Cell Signaling Technology), myc-9E10 (Sigma-Aldrich), α-tubulin (Sigma-Aldrich), Rac (BD), ZO-1 (Invitrogen), β-actin (Sigma-Aldrich), CD99 , GFP (Clontech Laboratories, Inc.), and sucrase-isomaltase (a gift from H.-P. Hauri, University of Basel, Basel, Switzerland) and rabbit polyclonal antibodies to β-catenin (Abcam), EBP50 (Abcam), phospho–MLC 2 (Ser19; Cell Signaling Technologies), phospho-ERM (Cell Signaling Technologies), Na+ /K+ exchanger (Millipore), and RICH2RhoGap (in house).

    Techniques: Labeling, Derivative Assay

    Localization of CD317 in polarized Caco-2 cells, its knockdown in Caco-2 cells, and the effect of knockdown on F-actin. (A) Localization of CD317 in polarized Caco-2 cells. The top row shows XY images of CD317 (detected with an anti-CD317 antibody) and F-actin (Alexa Fluor 594–phalloidin) localization as indicated. The second row shows an XZ section from the first row. The third row shows XY images of CD317 and β-catenin localization as indicated (β-catenin is a marker of lateral membranes). The bottom row shows an XZ section from the third row. Panels on the right show merged images of the left and middle panels, with DAPI-stained nuclei on the right in the second row. (B) Immunoblot analysis (using an anti-CD317 antibody) of lysates from Caco-2 cells stably expressing CD317 siRNA or control GFP siRNA as indicated. The bands representing higher molecular weight proteins in the control siRNA lane correspond to glycosylated CD317. An immunoblot of α-tubulin was used as a loading control. Molecular mass is indicated in kilodaltons. (C) Alexa Fluor 594–phalloidin decoration of F-actin in nonpolarized CD317 knockdown cells (top) and control cells (bottom). Each image shows a colony of cells with DAPI-stained nuclei. The top panel is an XZ section from a field of CD317 knockdown cells. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

    doi: 10.1083/jcb.200804154

    Figure Lengend Snippet: Localization of CD317 in polarized Caco-2 cells, its knockdown in Caco-2 cells, and the effect of knockdown on F-actin. (A) Localization of CD317 in polarized Caco-2 cells. The top row shows XY images of CD317 (detected with an anti-CD317 antibody) and F-actin (Alexa Fluor 594–phalloidin) localization as indicated. The second row shows an XZ section from the first row. The third row shows XY images of CD317 and β-catenin localization as indicated (β-catenin is a marker of lateral membranes). The bottom row shows an XZ section from the third row. Panels on the right show merged images of the left and middle panels, with DAPI-stained nuclei on the right in the second row. (B) Immunoblot analysis (using an anti-CD317 antibody) of lysates from Caco-2 cells stably expressing CD317 siRNA or control GFP siRNA as indicated. The bands representing higher molecular weight proteins in the control siRNA lane correspond to glycosylated CD317. An immunoblot of α-tubulin was used as a loading control. Molecular mass is indicated in kilodaltons. (C) Alexa Fluor 594–phalloidin decoration of F-actin in nonpolarized CD317 knockdown cells (top) and control cells (bottom). Each image shows a colony of cells with DAPI-stained nuclei. The top panel is an XZ section from a field of CD317 knockdown cells. Bars, 10 µm.

    Article Snippet: Other antibodies used were mouse monoclonal antibodies to ezrin (Cell Signaling Technology), myc-9E10 (Sigma-Aldrich), α-tubulin (Sigma-Aldrich), Rac (BD), ZO-1 (Invitrogen), β-actin (Sigma-Aldrich), CD99 , GFP (Clontech Laboratories, Inc.), and sucrase-isomaltase (a gift from H.-P. Hauri, University of Basel, Basel, Switzerland) and rabbit polyclonal antibodies to β-catenin (Abcam), EBP50 (Abcam), phospho–MLC 2 (Ser19; Cell Signaling Technologies), phospho-ERM (Cell Signaling Technologies), Na+ /K+ exchanger (Millipore), and RICH2RhoGap (in house).

    Techniques: Marker, Staining, Stable Transfection, Expressing, Molecular Weight

    RICH2 knockdown leads to a change in the localization of CD317. Localization of CD317 in polarized RICH2 knockdown Caco-2 cells. The top row shows maximum intensity projection XY images of CD317 and β-catenin, as indicated, in methanol-fixed cells. Arrows indicate potential intracellular populations of CD317 molecules. The second row shows detection of CD317 in PFA-fixed (nonpermeabilized) cells (XZ sections); the right panel shows DAPI-stained nuclei in addition to the apical CD317. The third and fourth rows present XZ sections from methanol-fixed (permeabilized) cells showing CD317 and β-catenin localization in RICH2 knockdown and control polarized Caco-2 cells as indicated. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

    doi: 10.1083/jcb.200804154

    Figure Lengend Snippet: RICH2 knockdown leads to a change in the localization of CD317. Localization of CD317 in polarized RICH2 knockdown Caco-2 cells. The top row shows maximum intensity projection XY images of CD317 and β-catenin, as indicated, in methanol-fixed cells. Arrows indicate potential intracellular populations of CD317 molecules. The second row shows detection of CD317 in PFA-fixed (nonpermeabilized) cells (XZ sections); the right panel shows DAPI-stained nuclei in addition to the apical CD317. The third and fourth rows present XZ sections from methanol-fixed (permeabilized) cells showing CD317 and β-catenin localization in RICH2 knockdown and control polarized Caco-2 cells as indicated. Bars, 10 µm.

    Article Snippet: Other antibodies used were mouse monoclonal antibodies to ezrin (Cell Signaling Technology), myc-9E10 (Sigma-Aldrich), α-tubulin (Sigma-Aldrich), Rac (BD), ZO-1 (Invitrogen), β-actin (Sigma-Aldrich), CD99 , GFP (Clontech Laboratories, Inc.), and sucrase-isomaltase (a gift from H.-P. Hauri, University of Basel, Basel, Switzerland) and rabbit polyclonal antibodies to β-catenin (Abcam), EBP50 (Abcam), phospho–MLC 2 (Ser19; Cell Signaling Technologies), phospho-ERM (Cell Signaling Technologies), Na+ /K+ exchanger (Millipore), and RICH2RhoGap (in house).

    Techniques: Staining

    GSK3β phosphorylates Dzip1. (A and B) Dzip1 interacts with GSK3β. Endogenous GSK3β was immunoprecipitated by Dzip1 but not IgG (A), and endogenous Dzip1 was immunoprecipitated with GFP-GSK3β in HEK 293T cells (B). (C and D) Dzip1 is co-localized with GSK3β at the basal body. G0-phase NIH 3T3 cells expressing GFP-GSK3β were immunostained for Dzip1 and AcTub (C), or cells expressing BFP-Centrin2 were immunostained with GSK3β and Dzip1 (D). Scale bar: 5 μm. (E) GSK3β binds Dzip1 in a kinase-substrate interaction manner. Wild-type (WT) GFP-GSK3β and the mutants S9A, K85R, and R96A were each co-expressed with Myc-Dzip1 in G0-phase HEK 293T cells, and treated with the CK1 inhibitor D4476 or the CK2 inhibitor CX4945. Note that treatment with CX4945 but not D4476 led to a significant decrease in the extent of the up-shifted Dzip1 bands, although the binding of Dzip1 to the GFP-GSK3β variants showed no difference. The extent of the up-shifting of the Dzip1 bands was decreased in K85R-expressing cells. (F) Phosphorylation of Dzip1 is coordinated with GSK3β activation. The kinase activity of GSK3β was negatively correlated with serum stimulation in NIH 3T3 cells. Note that the up-shifted bands (arrowheads) of Dzip1 became evident after serum depletion for 24–48 h, and disappeared after serum restimulation. γ-Tubulin was set as a loading control. (G) GSK3β phosphorylates Dzip1 in vivo. In resting mouse embryo fibroblast (MEFs) treated versus not treated with GSK3 and CK2 inhibitors, the Dzip1 bands were up-shifted less in GSK3- and CK2-inhibited cells. The protein levels of total β-Catenin and GSK3β were steady, but the phosphorylated (S33/37/T41) β-Catenin specifically disappeared from GSK3-inhibited cells. α-Tubulin was set as a loading control. (H) GSK3β phosphorylates Dzip1 in vitro. Auto-phosphorylation of GSK3β (55 kD), and the phosphorylated bands of the middle (28 kD), C-terminus (36 kD), and N- terminus (50 kD) of Dzip1 are shown (left panel). Coomassie blue staining of the gel shows the loaded amounts of Dzip1 fragments (right panel). Note that the S520A mutation resulted in decreased phosphorylation of Dzip1 by GSK3β. (I) Inhibition of GSK3 by BIO causes loss of phospho-S520 in Dzip1.

    Journal: PLoS Biology

    Article Title: GSK3β-Dzip1-Rab8 Cascade Regulates Ciliogenesis after Mitosis

    doi: 10.1371/journal.pbio.1002129

    Figure Lengend Snippet: GSK3β phosphorylates Dzip1. (A and B) Dzip1 interacts with GSK3β. Endogenous GSK3β was immunoprecipitated by Dzip1 but not IgG (A), and endogenous Dzip1 was immunoprecipitated with GFP-GSK3β in HEK 293T cells (B). (C and D) Dzip1 is co-localized with GSK3β at the basal body. G0-phase NIH 3T3 cells expressing GFP-GSK3β were immunostained for Dzip1 and AcTub (C), or cells expressing BFP-Centrin2 were immunostained with GSK3β and Dzip1 (D). Scale bar: 5 μm. (E) GSK3β binds Dzip1 in a kinase-substrate interaction manner. Wild-type (WT) GFP-GSK3β and the mutants S9A, K85R, and R96A were each co-expressed with Myc-Dzip1 in G0-phase HEK 293T cells, and treated with the CK1 inhibitor D4476 or the CK2 inhibitor CX4945. Note that treatment with CX4945 but not D4476 led to a significant decrease in the extent of the up-shifted Dzip1 bands, although the binding of Dzip1 to the GFP-GSK3β variants showed no difference. The extent of the up-shifting of the Dzip1 bands was decreased in K85R-expressing cells. (F) Phosphorylation of Dzip1 is coordinated with GSK3β activation. The kinase activity of GSK3β was negatively correlated with serum stimulation in NIH 3T3 cells. Note that the up-shifted bands (arrowheads) of Dzip1 became evident after serum depletion for 24–48 h, and disappeared after serum restimulation. γ-Tubulin was set as a loading control. (G) GSK3β phosphorylates Dzip1 in vivo. In resting mouse embryo fibroblast (MEFs) treated versus not treated with GSK3 and CK2 inhibitors, the Dzip1 bands were up-shifted less in GSK3- and CK2-inhibited cells. The protein levels of total β-Catenin and GSK3β were steady, but the phosphorylated (S33/37/T41) β-Catenin specifically disappeared from GSK3-inhibited cells. α-Tubulin was set as a loading control. (H) GSK3β phosphorylates Dzip1 in vitro. Auto-phosphorylation of GSK3β (55 kD), and the phosphorylated bands of the middle (28 kD), C-terminus (36 kD), and N- terminus (50 kD) of Dzip1 are shown (left panel). Coomassie blue staining of the gel shows the loaded amounts of Dzip1 fragments (right panel). Note that the S520A mutation resulted in decreased phosphorylation of Dzip1 by GSK3β. (I) Inhibition of GSK3 by BIO causes loss of phospho-S520 in Dzip1.

    Article Snippet: Antibodies and Reagents The CK1 inhibitor D4476 (Tocris Bioscience, 2902), rabbit anti-GSK3β (Santa Cruz Biotechnology, sc-9166), β-Catenin (BD, 610154), phosphorylated GSK3β (S9, Cell Signaling Technology, #93365), and phosphorylated β-Catenin (N3, Cell Signaling Technology, #9561) were kind gifts from Dr. Wei Wu (Tsinghua University).

    Techniques: Immunoprecipitation, Expressing, Binding Assay, Activation Assay, Activity Assay, Serum Depletion, In Vivo, In Vitro, Staining, Mutagenesis, Inhibition

    Hematoxylin and eosin stain gastric sections and immunohistochemical staining for E-cadherin and β-catenin. (A) Hematoxylin and eosin staining in normal gastric tissue. (B) Hematoxylin and eosin staining in the tumor tissue of GC S10 with signet ring cell carcinoma: signet ring cells are highlighted by black arrows. (C) Hematoxylin and eosin staining evidence the diffuse histotype of GC S46. (D) E-cadherin staining in normal gastric tissue. (E) Reduction of E-cadherin staining in the signet ring cells of GC S10 respect to adjacent normal cells; signet ring cells are highlighted by black arrows. (F) Loss of E-cadherin expression in the diffuse tumor cells of GC S46 compared to normal tissue (on the right side of the photomicrograph). (G) β-catenin staining in normal gastric tissue. (H) Weakly β-catenin staining in signet ring cells (black arrows) of GC S10. (I) Loss of β-catenin stai ning in the tumor tissue of GC S46 compared to normal tissue (on the right side of the photomicrograph). All the photomicrographs were taken at 400× magnification.

    Journal: PLoS ONE

    Article Title: Identification and Characterization of CDH1 Germline Variants in Sporadic Gastric Cancer Patients and in Individuals at Risk of Gastric Cancer

    doi: 10.1371/journal.pone.0077035

    Figure Lengend Snippet: Hematoxylin and eosin stain gastric sections and immunohistochemical staining for E-cadherin and β-catenin. (A) Hematoxylin and eosin staining in normal gastric tissue. (B) Hematoxylin and eosin staining in the tumor tissue of GC S10 with signet ring cell carcinoma: signet ring cells are highlighted by black arrows. (C) Hematoxylin and eosin staining evidence the diffuse histotype of GC S46. (D) E-cadherin staining in normal gastric tissue. (E) Reduction of E-cadherin staining in the signet ring cells of GC S10 respect to adjacent normal cells; signet ring cells are highlighted by black arrows. (F) Loss of E-cadherin expression in the diffuse tumor cells of GC S46 compared to normal tissue (on the right side of the photomicrograph). (G) β-catenin staining in normal gastric tissue. (H) Weakly β-catenin staining in signet ring cells (black arrows) of GC S10. (I) Loss of β-catenin stai ning in the tumor tissue of GC S46 compared to normal tissue (on the right side of the photomicrograph). All the photomicrographs were taken at 400× magnification.

    Article Snippet: Immunohistochemistry (IHC) was performed using the mouse monoclonal antibody against human E-cadherin (clone 36, Ventana Medical System, Tucson, AZ, USA), and β-catenin (clone 17C2 Novocastra, Newcastle upon Tyne, UK).

    Techniques: H&E Stain, Immunohistochemistry, Staining, Expressing

    SLIT/ROBO1 signaling regulates the subcellular localization of β-catenin

    Journal: Developmental cell

    Article Title: SLIT/ROBO1 signaling suppresses mammary branching morphogenesis by limiting basal cell number

    doi: 10.1016/j.devcel.2011.05.012

    Figure Lengend Snippet: SLIT/ROBO1 signaling regulates the subcellular localization of β-catenin

    Article Snippet: To investigate whether β-catenin is downstream of SLIT/ROBO1 in basal cells, we treated HME50 cells with SLIT2 and, using biochemical fractionation, detected a shift in β-catenin from the nuclear to the cytosolic/membrane fractions ( ).

    Techniques:

    Exd4 treatment stabilizes β -catenin, siRNA-mediated knockdown of β -catenin abrogates Exd4-stimulated Wnt signaling, and Exd4 enhances phosphorylation of β -catenin on the stabilizing PKA site, Ser-675. A, Exd4 stabilizes cytosolic

    Journal:

    Article Title: Glucagon-like Peptide-1 Activation of TCF7L2-dependent Wnt Signaling Enhances Pancreatic Beta Cell Proliferation *Glucagon-like Peptide-1 Activation of TCF7L2-dependent Wnt Signaling Enhances Pancreatic Beta Cell Proliferation * S⃞

    doi: 10.1074/jbc.M706105200

    Figure Lengend Snippet: Exd4 treatment stabilizes β -catenin, siRNA-mediated knockdown of β -catenin abrogates Exd4-stimulated Wnt signaling, and Exd4 enhances phosphorylation of β -catenin on the stabilizing PKA site, Ser-675. A, Exd4 stabilizes cytosolic

    Article Snippet: Western Immunoblots —Membrane immunoblots were prepared from extracts of INS-1 cells and were interrogated with antisera to β-catenin as follows: total protein, the destabilizing GSK-3 phosphorylation sites (UpState catalog number 46-626), and the stabilizing PKA-mediated phosphorylation site, Ser-675, that stabilizes β-catenin (AnaSpec catalog number 29619). siRNA-mediated Knockdown of β -Catenin Expression —siRNAs against rat β-catenin (GenBank™ accession number ) were from Dharmacon (siRNA1 catalog number J100628-05, siRNA2 catalog number J-100628-06).

    Techniques:

    Exd4 treatment enhances cyclin D1 and c-Myc transcription through a β -catenin/TCF4-binding site. A, immunoblot shows endogenous expression of TCF7L2 in INS-1 cells. Left lane shows transient ectopic expression of Myc-tagged TCF7L2 in INS-1

    Journal:

    Article Title: Glucagon-like Peptide-1 Activation of TCF7L2-dependent Wnt Signaling Enhances Pancreatic Beta Cell Proliferation *Glucagon-like Peptide-1 Activation of TCF7L2-dependent Wnt Signaling Enhances Pancreatic Beta Cell Proliferation * S⃞

    doi: 10.1074/jbc.M706105200

    Figure Lengend Snippet: Exd4 treatment enhances cyclin D1 and c-Myc transcription through a β -catenin/TCF4-binding site. A, immunoblot shows endogenous expression of TCF7L2 in INS-1 cells. Left lane shows transient ectopic expression of Myc-tagged TCF7L2 in INS-1

    Article Snippet: Western Immunoblots —Membrane immunoblots were prepared from extracts of INS-1 cells and were interrogated with antisera to β-catenin as follows: total protein, the destabilizing GSK-3 phosphorylation sites (UpState catalog number 46-626), and the stabilizing PKA-mediated phosphorylation site, Ser-675, that stabilizes β-catenin (AnaSpec catalog number 29619). siRNA-mediated Knockdown of β -Catenin Expression —siRNAs against rat β-catenin (GenBank™ accession number ) were from Dharmacon (siRNA1 catalog number J100628-05, siRNA2 catalog number J-100628-06).

    Techniques: Binding Assay, Expressing

    FoxO1 knockdown promotes migration, invasion potential, and EMT in OSCC cells. (A) FoxO1 expression was detected by qRT-PCR and Western blot after transfected with FoxO1 shRNA in OSCC cells. (B) Images of the wound closure of monolayer Cal-27 and Tca8113 cells with FoxO1 knockdown at the time point of 0 and 24 h are presented on the left. Quantitative results are illustrated on the right. (C) The effect of FoxO1 knockdown on OSCC cells invasion were determined by Transwell assay with Matrigel, and the representative images are on the left. Quantitative results are illustrated on the right. (D) The effects of Cal-27-FoxO1 knockdown on expressions of EMT markers, E-cadherin, N-cadherin, β-catenin, and Vimentin, were measured using qRT-PCR and Western blot. And the effects of Cal-27-sh-2-STAT3/sh-1-FoxO1 on expressions of EMT markers, E-cadherin, N-cadherin, β-catenin, and Vimentin, were measured using qRT-PCR compared with those of sh-2-STAT3/scramble group. β-actin was used as a loading control. (E) Flow cytometry was used to examine the percentage of apoptotic cells in Cal-27 cells with FoxO1 knockdown and scramble control cells. All assays were carried out in triplicate. Results were shown as means ± SD. * P

    Journal: Frontiers in Oncology

    Article Title: STAT3 Promotes Invasion and Aerobic Glycolysis of Human Oral Squamous Cell Carcinoma via Inhibiting FoxO1

    doi: 10.3389/fonc.2019.01175

    Figure Lengend Snippet: FoxO1 knockdown promotes migration, invasion potential, and EMT in OSCC cells. (A) FoxO1 expression was detected by qRT-PCR and Western blot after transfected with FoxO1 shRNA in OSCC cells. (B) Images of the wound closure of monolayer Cal-27 and Tca8113 cells with FoxO1 knockdown at the time point of 0 and 24 h are presented on the left. Quantitative results are illustrated on the right. (C) The effect of FoxO1 knockdown on OSCC cells invasion were determined by Transwell assay with Matrigel, and the representative images are on the left. Quantitative results are illustrated on the right. (D) The effects of Cal-27-FoxO1 knockdown on expressions of EMT markers, E-cadherin, N-cadherin, β-catenin, and Vimentin, were measured using qRT-PCR and Western blot. And the effects of Cal-27-sh-2-STAT3/sh-1-FoxO1 on expressions of EMT markers, E-cadherin, N-cadherin, β-catenin, and Vimentin, were measured using qRT-PCR compared with those of sh-2-STAT3/scramble group. β-actin was used as a loading control. (E) Flow cytometry was used to examine the percentage of apoptotic cells in Cal-27 cells with FoxO1 knockdown and scramble control cells. All assays were carried out in triplicate. Results were shown as means ± SD. * P

    Article Snippet: Reagents Monoclonal mouse anti-human antibodies to STAT3 (sc-293151), FoxO1 (H-128), β-catenin (sc-1496) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, Transfection, shRNA, Transwell Assay, Flow Cytometry, Cytometry

    STAT3 knockdown inhibits migration, invasion potential, and EMT in OSCC cells. (A) STAT3 mRNA levels were evaluated using qRT-PCR in SCC25, Cal-27, HSC3, Tca8113, and UM1 cells. (B) STAT3 expression was detected by qRT-PCR and Western blot after transfected with STAT3 shRNA in OSCC cells. (C) Images of the wound closure of monolayer Cal-27 and Tca8113 cells with STAT3 knockdown at the time point of 0 and 24 h are presented on the left. Quantitative results are illustrated on the right. (D) The effect of STAT3 knockdown on OSCC cells invasion were determined by Transwell assay with Matrigel, and the representative images are on the left. Quantitative results are illustrated on the right. (E) The effects of Cal-27-STAT3 knockdown on expressions of EMT markers, E-cadherin, N-cadherin, β-catenin, and Vimentin, were measured using qRT-PCR and Western blot. β-actin was used as a loading control. (F) Flow cytometry was used to examine the percentage of apoptotic cells in Cal-27 cells with STAT3 knockdown and scramble control cells. All assays were carried out in triplicate. Results were shown as means ± SD. * P

    Journal: Frontiers in Oncology

    Article Title: STAT3 Promotes Invasion and Aerobic Glycolysis of Human Oral Squamous Cell Carcinoma via Inhibiting FoxO1

    doi: 10.3389/fonc.2019.01175

    Figure Lengend Snippet: STAT3 knockdown inhibits migration, invasion potential, and EMT in OSCC cells. (A) STAT3 mRNA levels were evaluated using qRT-PCR in SCC25, Cal-27, HSC3, Tca8113, and UM1 cells. (B) STAT3 expression was detected by qRT-PCR and Western blot after transfected with STAT3 shRNA in OSCC cells. (C) Images of the wound closure of monolayer Cal-27 and Tca8113 cells with STAT3 knockdown at the time point of 0 and 24 h are presented on the left. Quantitative results are illustrated on the right. (D) The effect of STAT3 knockdown on OSCC cells invasion were determined by Transwell assay with Matrigel, and the representative images are on the left. Quantitative results are illustrated on the right. (E) The effects of Cal-27-STAT3 knockdown on expressions of EMT markers, E-cadherin, N-cadherin, β-catenin, and Vimentin, were measured using qRT-PCR and Western blot. β-actin was used as a loading control. (F) Flow cytometry was used to examine the percentage of apoptotic cells in Cal-27 cells with STAT3 knockdown and scramble control cells. All assays were carried out in triplicate. Results were shown as means ± SD. * P

    Article Snippet: Reagents Monoclonal mouse anti-human antibodies to STAT3 (sc-293151), FoxO1 (H-128), β-catenin (sc-1496) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Migration, Quantitative RT-PCR, Expressing, Western Blot, Transfection, shRNA, Transwell Assay, Flow Cytometry, Cytometry

    Representative examples of β-catenin expression in colorectal liver metastasis. (A) Only membranous staining was observed (score 0 for cytoplasm and nucleus). (B) Cytoplasmic expression of β-catenin with some positive nuclei (score 1 for cytoplasm and nuclei). (C) Diffuse and strong cytoplasmic and nuclear staining (score 1 for cytoplasm, and score 2 for nuclei). Magnification, ×200.

    Journal: Oncology Letters

    Article Title: Loss of CDX2 gene expression is associated with DNA repair proteins and is a crucial member of the Wnt signaling pathway in liver metastasis of colorectal cancer

    doi: 10.3892/ol.2018.7756

    Figure Lengend Snippet: Representative examples of β-catenin expression in colorectal liver metastasis. (A) Only membranous staining was observed (score 0 for cytoplasm and nucleus). (B) Cytoplasmic expression of β-catenin with some positive nuclei (score 1 for cytoplasm and nuclei). (C) Diffuse and strong cytoplasmic and nuclear staining (score 1 for cytoplasm, and score 2 for nuclei). Magnification, ×200.

    Article Snippet: For ERCC1 (8F1, Neomarkers; dilution: 1:100) and β-catenin (14, RTU; Ventana Medical Systems, Inc.; Roche Diagnostics) slides were stained by a computer-controlled autostainer (Ventana BenchMark Ultra; Ventana Medical Systems, Inc.; Roche Diagnostics).

    Techniques: Expressing, Staining

    Acute exposure to lenalidomide induces β-catenin transcription. RT-PCR was performed to detect β-catenin or β-actin transcripts from cells propagated continuously in lenalidomide (R10R), or R10R cells that were removed from lenalidomide for 1 week (RR), to detect β-catenin, and comparisons were made to vehicle-treated, drug-naive cells ( WT ) ( A ). RT-PCR products were separated by acrylamide gel electrophoresis, and detected by ethidium bromide staining. The left panel shows representative agarose gels, and densitometry was performed to calculate the increase in β-catenin after correcting for β-actin, which was normalized to the WT cells, which were set at 1.0. Drug-naive ANBL-6 ( B ), KAS-6/1 ( C ), and MM1.S ( D ) cells were exposed to the indicated lenalidomide concentrations for 72 (ANBL-6) or 168 h (KAS-6/1, MM1.S). Each top panel provides a representative photomicrograph, and densitometry was performed to calculate the β-catenin/β-actin ratio. Values for each cell line are provided in the lower panels after normalization to the vehicle-treated control, which was arbitrarily set at 1.0.

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M110.180208

    Figure Lengend Snippet: Acute exposure to lenalidomide induces β-catenin transcription. RT-PCR was performed to detect β-catenin or β-actin transcripts from cells propagated continuously in lenalidomide (R10R), or R10R cells that were removed from lenalidomide for 1 week (RR), to detect β-catenin, and comparisons were made to vehicle-treated, drug-naive cells ( WT ) ( A ). RT-PCR products were separated by acrylamide gel electrophoresis, and detected by ethidium bromide staining. The left panel shows representative agarose gels, and densitometry was performed to calculate the increase in β-catenin after correcting for β-actin, which was normalized to the WT cells, which were set at 1.0. Drug-naive ANBL-6 ( B ), KAS-6/1 ( C ), and MM1.S ( D ) cells were exposed to the indicated lenalidomide concentrations for 72 (ANBL-6) or 168 h (KAS-6/1, MM1.S). Each top panel provides a representative photomicrograph, and densitometry was performed to calculate the β-catenin/β-actin ratio. Values for each cell line are provided in the lower panels after normalization to the vehicle-treated control, which was arbitrarily set at 1.0.

    Article Snippet: Adenoviruses directing the expression of GFP as a negative control, or β-catenin (Vector Biolabs, Philadelphia, PA), were infected into myeloma cell lines in triplicate experiments prior to exposure to lenalidomide.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Acrylamide Gel Assay, Electrophoresis, Staining

    Activation of β-catenin induces cyclin D1 and c-Myc levels. Wild-type drug-naive U266 ( upper profiles ) and MM1.S ( middle profiles ) cell populations ( dashed lines ), and their comparable R10R variants ( solid lines ) were evaluated by FACS for expression of β-catenin ( A ), cyclin D1 ( B ), and c-Myc ( C ). The lower panels display semiquantitative analyses of the mean fluorescent intensity for each R10R cell line compared with the WT controls, which were arbitrarily set at 1.0. Each dotted line represents 1.0, and values above that level represent increased expression of the proteins of interest. The Student's paired t test was used to determine statistical significance, and * denotes p

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M110.180208

    Figure Lengend Snippet: Activation of β-catenin induces cyclin D1 and c-Myc levels. Wild-type drug-naive U266 ( upper profiles ) and MM1.S ( middle profiles ) cell populations ( dashed lines ), and their comparable R10R variants ( solid lines ) were evaluated by FACS for expression of β-catenin ( A ), cyclin D1 ( B ), and c-Myc ( C ). The lower panels display semiquantitative analyses of the mean fluorescent intensity for each R10R cell line compared with the WT controls, which were arbitrarily set at 1.0. Each dotted line represents 1.0, and values above that level represent increased expression of the proteins of interest. The Student's paired t test was used to determine statistical significance, and * denotes p

    Article Snippet: Adenoviruses directing the expression of GFP as a negative control, or β-catenin (Vector Biolabs, Philadelphia, PA), were infected into myeloma cell lines in triplicate experiments prior to exposure to lenalidomide.

    Techniques: Activation Assay, FACS, Expressing

    Lenalidomide induces β-catenin protein accumulation. Protein extracts from myeloma cells propagated continuously in lenalidomide (R10R), or R10R cells that were removed from lenalidomide for 1 week (RR), were probed to detect β-catenin, and compared with vehicle-treated drug-naive cells ( WT ) ( A ). The left panel shows a representative Western blot, and densitometry was performed to calculate the increase in β-catenin in each cell line after correcting for β-actin, which was normalized to the WT cells, which were set at 1.0. Acute exposure was studied in drug-naive ANBL-6 ( B ), KAS-6/1 ( C ), and MM1.S ( D ) cells, which were treated with the indicated lenalidomide concentrations for 72 (ANBL-6) or 168 h (KAS-6/1, MM1.S).

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M110.180208

    Figure Lengend Snippet: Lenalidomide induces β-catenin protein accumulation. Protein extracts from myeloma cells propagated continuously in lenalidomide (R10R), or R10R cells that were removed from lenalidomide for 1 week (RR), were probed to detect β-catenin, and compared with vehicle-treated drug-naive cells ( WT ) ( A ). The left panel shows a representative Western blot, and densitometry was performed to calculate the increase in β-catenin in each cell line after correcting for β-actin, which was normalized to the WT cells, which were set at 1.0. Acute exposure was studied in drug-naive ANBL-6 ( B ), KAS-6/1 ( C ), and MM1.S ( D ) cells, which were treated with the indicated lenalidomide concentrations for 72 (ANBL-6) or 168 h (KAS-6/1, MM1.S).

    Article Snippet: Adenoviruses directing the expression of GFP as a negative control, or β-catenin (Vector Biolabs, Philadelphia, PA), were infected into myeloma cell lines in triplicate experiments prior to exposure to lenalidomide.

    Techniques: Western Blot

    1C inhibited Wnt/β-catenin signaling pathway. The expression levels of Wnt/β-catenin signaling pathway involving proliferation were examined by Western blotting assay. Data are expressed as means ± SDs of triplicate experiments performed independently. ∗ p

    Journal: Journal of Ginseng Research

    Article Title: Anticancer activity and potential mechanisms of 1C, a ginseng saponin derivative, on prostate cancer cells

    doi: 10.1016/j.jgr.2016.12.014

    Figure Lengend Snippet: 1C inhibited Wnt/β-catenin signaling pathway. The expression levels of Wnt/β-catenin signaling pathway involving proliferation were examined by Western blotting assay. Data are expressed as means ± SDs of triplicate experiments performed independently. ∗ p

    Article Snippet: After electrophoresis, the resolved protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane, and the membrane was blocked with 5% bovine serum albumin (BSA) in TBST buffer for 1 h. After blocking, the membrane was incubated with a 1:1000 dilution of primary antibody against mouse double minute 2 (MDM2; Bioworld Technology, Inc., Minnesota, USA), P53 (Proteintech Group, Inc., Chicago, USA), Cl-caspase-3 (Cell Singaling Technology, Inc., Danvers, MA, USA), Cl-caspase-9 (Cell Singaling Technology Inc), PARP (Cell Singaling Technology, Inc), Bcl-2 (Santa Cruz Biotechnology, Inc), Cytochrome C (Proteintech Group, Inc), Bax (Proteintech Group, Inc), CCND1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), β-catenin (Proteintech Group, Inc), TCF-4 (Proteintech Group, Inc), C-Myc (Proteintech Group, Inc) or β-actin (Ding-Guo Biotech Ltd., Beijing) in 5% BSA and at 4○ C for overnight.

    Techniques: Expressing, Western Blot

    Effect of U94 on cell signaling pathways Western blot analysis of MDA-MB 231 lysates was performed using mAbs to Src pTyr418, total Src, β-catenin pTyr654, total β-catenin, FAK pTyr397, total FAK, STAT3 pTyr705, total STAT3, Cortactin pTyr421, total Cortactin, ARP2/3, GAPDH, Akt pSer473, total Akt, ERK pThr202 and total ERK as specific reagents. Quantification was carried out by densitometric analysis and plotting of the Src pTyr418/Src, β-catenin pTyr654/β-catenin, FAK pTyr397/FAK, STAT3 pTyr705/STAT3, Cortactin pTyr421/Cortactin, ARP2/3/GAPDH, Akt pSer473/Akt and ERK pThr202/ERK. Left panels, blots are representative of three independent experiments with similar results. Right panels, values reported are the means ± the SD of three independent experiments. Statistical analysis was performed by 1-way ANOVA, and the Bonferroni post-test was used to compare data (* P

    Journal: Oncotarget

    Article Title: U94 of human herpesvirus 6 down-modulates Src, promotes a partial mesenchymal-to-epithelial transition and inhibits tumor cell growth, invasion and metastasis

    doi: 10.18632/oncotarget.17817

    Figure Lengend Snippet: Effect of U94 on cell signaling pathways Western blot analysis of MDA-MB 231 lysates was performed using mAbs to Src pTyr418, total Src, β-catenin pTyr654, total β-catenin, FAK pTyr397, total FAK, STAT3 pTyr705, total STAT3, Cortactin pTyr421, total Cortactin, ARP2/3, GAPDH, Akt pSer473, total Akt, ERK pThr202 and total ERK as specific reagents. Quantification was carried out by densitometric analysis and plotting of the Src pTyr418/Src, β-catenin pTyr654/β-catenin, FAK pTyr397/FAK, STAT3 pTyr705/STAT3, Cortactin pTyr421/Cortactin, ARP2/3/GAPDH, Akt pSer473/Akt and ERK pThr202/ERK. Left panels, blots are representative of three independent experiments with similar results. Right panels, values reported are the means ± the SD of three independent experiments. Statistical analysis was performed by 1-way ANOVA, and the Bonferroni post-test was used to compare data (* P

    Article Snippet: Each section was then incubated for 30 min with monoclonal antibody to β-catenin (diluition: 1:120; Cell Marque), vimentin (diluition 1:150; Leica Biosystems), E-cadherin (diluition 1:50; Invitrogen) or with rabbit polyclonal antibody to mouse CD31 (dilution 1:50; Abcam).

    Techniques: Western Blot, Multiple Displacement Amplification

    U94 expression induces a partial MET in MDA-MB 231 cells After 3D culture, cells were harvested and processed for staining. Five μm thick sections were prepared and stained with (A) H E for morphological analysis, or IHC for (B) β-catenin, (C) Vimentin, and (D) E-cadherin expression. (Original magnification: 20x upper panels; 40x lower panels). The arrows in panel A show cytonuclear atypias, such as nuclear pleomorphism, multinuclearity (black arrow) and “cell in cell” aspects (red arrow). (E) Real-time-PCR analysis of mesenchymal markers Snail1, Twist, MMP-2 and N-Cadherin was performed on NT cells and on EGFP + and U94 + cells at day 4 p.i. Gene expression is represented as relative mRNA levels. Data represent the mean (± SD) of three independent experiments performed in triplicate.

    Journal: Oncotarget

    Article Title: U94 of human herpesvirus 6 down-modulates Src, promotes a partial mesenchymal-to-epithelial transition and inhibits tumor cell growth, invasion and metastasis

    doi: 10.18632/oncotarget.17817

    Figure Lengend Snippet: U94 expression induces a partial MET in MDA-MB 231 cells After 3D culture, cells were harvested and processed for staining. Five μm thick sections were prepared and stained with (A) H E for morphological analysis, or IHC for (B) β-catenin, (C) Vimentin, and (D) E-cadherin expression. (Original magnification: 20x upper panels; 40x lower panels). The arrows in panel A show cytonuclear atypias, such as nuclear pleomorphism, multinuclearity (black arrow) and “cell in cell” aspects (red arrow). (E) Real-time-PCR analysis of mesenchymal markers Snail1, Twist, MMP-2 and N-Cadherin was performed on NT cells and on EGFP + and U94 + cells at day 4 p.i. Gene expression is represented as relative mRNA levels. Data represent the mean (± SD) of three independent experiments performed in triplicate.

    Article Snippet: Each section was then incubated for 30 min with monoclonal antibody to β-catenin (diluition: 1:120; Cell Marque), vimentin (diluition 1:150; Leica Biosystems), E-cadherin (diluition 1:50; Invitrogen) or with rabbit polyclonal antibody to mouse CD31 (dilution 1:50; Abcam).

    Techniques: Expressing, Multiple Displacement Amplification, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

    Involvement of β-catenin and survival signal of Akt and Src in the process of CLDN1 regulated anoikis (A) WB analysis, as compared to negative control cells, CLDN1-KD cells showed a distinct decrease of β-catenin, phosphorylated (Ser473) Akt and phosphorylated (Tyr416) Src expression. (B) WB analysis, as compared to control cells, CLDN1-overexpressed cells showed a strong increase of β-catenin, phosphorylated (Ser473) Akt and phosphorylated (Tyr416) Src expression.

    Journal: Oncotarget

    Article Title: Claudin-1 enhances tumor proliferation and metastasis by regulating cell anoikis in gastric cancer

    doi:

    Figure Lengend Snippet: Involvement of β-catenin and survival signal of Akt and Src in the process of CLDN1 regulated anoikis (A) WB analysis, as compared to negative control cells, CLDN1-KD cells showed a distinct decrease of β-catenin, phosphorylated (Ser473) Akt and phosphorylated (Tyr416) Src expression. (B) WB analysis, as compared to control cells, CLDN1-overexpressed cells showed a strong increase of β-catenin, phosphorylated (Ser473) Akt and phosphorylated (Tyr416) Src expression.

    Article Snippet: Another construct of LV5-EF1a-GFP/β-catenin lentiviral transduction particles for ectopic overexpression of β-catenin was also purchased from Genepharma (Shanghai, CHINA) and transfected into HS-746T/CLDN1-KD cells.

    Techniques: Western Blot, Negative Control, Expressing

    Overexpression of β-catenin in CLDN1-KD cell of HS-746T restored cell aggregation, anoikis resistance and survival signals of Akt and Src activation (A) WB analysis, the expression of β-catenin decreased in CLDN1-KD cells and re-increased in HS-746T/CLDN1-KD/β-catenin cells. (B) Representative photographs of cell aggregation after 6 hrs of suspension (original magnifications: ×40). (C) FCM analysis of cell apoptosis after 3h of suspension. Relative apoptotic rate of HS-746T/CLDN1-KD/β-catenin significantly decreased after 3h of suspension as compared to that of HS-746T/CLDN1-KD (*, P

    Journal: Oncotarget

    Article Title: Claudin-1 enhances tumor proliferation and metastasis by regulating cell anoikis in gastric cancer

    doi:

    Figure Lengend Snippet: Overexpression of β-catenin in CLDN1-KD cell of HS-746T restored cell aggregation, anoikis resistance and survival signals of Akt and Src activation (A) WB analysis, the expression of β-catenin decreased in CLDN1-KD cells and re-increased in HS-746T/CLDN1-KD/β-catenin cells. (B) Representative photographs of cell aggregation after 6 hrs of suspension (original magnifications: ×40). (C) FCM analysis of cell apoptosis after 3h of suspension. Relative apoptotic rate of HS-746T/CLDN1-KD/β-catenin significantly decreased after 3h of suspension as compared to that of HS-746T/CLDN1-KD (*, P

    Article Snippet: Another construct of LV5-EF1a-GFP/β-catenin lentiviral transduction particles for ectopic overexpression of β-catenin was also purchased from Genepharma (Shanghai, CHINA) and transfected into HS-746T/CLDN1-KD cells.

    Techniques: Over Expression, Activation Assay, Western Blot, Expressing

    Decreased β-catenin and Vangl2 levels are observed in Atmin Gpg6/Gpg6 E13.5 embryonic kidneys compared with WT littermates. Kidney sections from WT ( A and C ) and Atmin Gpg6/Gpg6 ( B and D ) embryos were immunostained for β-catenin (A and B) and Vangl2 (C and D). β-Catenin immunostaining was significantly reduced in Atmin Gpg6/Gpg6 (B) compared with WT (A). Vangl2 immunostaining was also dramatically decreased and apical enrichment was altered in Atmin Gpg6/Gpg6 (D) compared with WT (C). Overlays of DAPI and Vangl2 localization emphasize the loss of apical enrichment of Vangl2 protein in Atmin Gpg6/Gpg6 (D inset) versus WT (C inset; n = 4). All images are maximum projections of confocal z stacks taken at 0.4 µm intervals. Images were taken in identical conditions and post-acquisition manipulations were identical. Western blotting revealed a 0.4-fold decrease ( I ) in β-catenin ( P

    Journal: Human Molecular Genetics

    Article Title: Atmin mediates kidney morphogenesis by modulating Wnt signaling

    doi: 10.1093/hmg/ddu246

    Figure Lengend Snippet: Decreased β-catenin and Vangl2 levels are observed in Atmin Gpg6/Gpg6 E13.5 embryonic kidneys compared with WT littermates. Kidney sections from WT ( A and C ) and Atmin Gpg6/Gpg6 ( B and D ) embryos were immunostained for β-catenin (A and B) and Vangl2 (C and D). β-Catenin immunostaining was significantly reduced in Atmin Gpg6/Gpg6 (B) compared with WT (A). Vangl2 immunostaining was also dramatically decreased and apical enrichment was altered in Atmin Gpg6/Gpg6 (D) compared with WT (C). Overlays of DAPI and Vangl2 localization emphasize the loss of apical enrichment of Vangl2 protein in Atmin Gpg6/Gpg6 (D inset) versus WT (C inset; n = 4). All images are maximum projections of confocal z stacks taken at 0.4 µm intervals. Images were taken in identical conditions and post-acquisition manipulations were identical. Western blotting revealed a 0.4-fold decrease ( I ) in β-catenin ( P

    Article Snippet: β-Catenin and Vangl2 proteins are altered in AtminGpg6/Gpg6 kidneys Since transcriptional differences relevant to the Wnt signaling pathway were observed between the AtminGpg6/Gpg6 and WT embryonic kidneys, the levels of β-catenin and Vangl2, two key proteins for the canonical and non-canonical/PCP Wnt signaling pathways, respectively, were also examined.

    Techniques: Immunostaining, Western Blot

    Representative micrographs of immunohistochemistry staining in the 5/6 Nx group rat renal cortex sections (400×). Notes: Immunohistochemistry staining of 5/6 Nx group for Wnt1 ( A ), β-catenin ( B ), GSK-3β ( C ), Dkk1 ( D ), TCF4 ( E ), Fn1 ( F ), and IgG negative control ( G ). Abbreviations: Nx, 5/6 nephrectomy; GSK-3β, glycogen synthase kinase-3β; Dkk1, dickkopf 1; TCF4, transcription factor 4; Fn1, fibronectin 1.

    Journal: Drug Design, Development and Therapy

    Article Title: Protective effect of Huang Gan formula in 5/6 nephrectomized rats by depressing the Wnt/β-catenin signaling pathway

    doi: 10.2147/DDDT.S81157

    Figure Lengend Snippet: Representative micrographs of immunohistochemistry staining in the 5/6 Nx group rat renal cortex sections (400×). Notes: Immunohistochemistry staining of 5/6 Nx group for Wnt1 ( A ), β-catenin ( B ), GSK-3β ( C ), Dkk1 ( D ), TCF4 ( E ), Fn1 ( F ), and IgG negative control ( G ). Abbreviations: Nx, 5/6 nephrectomy; GSK-3β, glycogen synthase kinase-3β; Dkk1, dickkopf 1; TCF4, transcription factor 4; Fn1, fibronectin 1.

    Article Snippet: Rabbit polyclonal antibody Wnt1 (ab85060; Abcam, Inc., Cambridge, MA, USA), β-catenin (BS1982; Bioworld Technology, Inc., St Louis Park, MN, USA), GSK-3β (sc9166; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-GSK-3β (sc-135653; Santa Cruz Biotechnology Inc., Dallas, TX, USA), transcription factor 4 (TCF4) (BS6172; Bioworld Technology, Inc.), dickkopf 1 (Dkk1) (BS7731; Bioworld Technology, Inc.), fibronectin 1 (Fn1) (ab2413; Abcam, Inc.), and glyceraldehyde 3-phosphate dehydro-genase (GAPDH) (ab9485; Abcam, Inc.) were purchased.

    Techniques: Immunohistochemistry, Staining, Negative Control

    Wnt1, β-catenin, TCF4, GSK-3β, Dkk1, and Fn1 mRNA expression in renal tissues (n=6). Notes: Compared with the 5/6 Nx group, * P

    Journal: Drug Design, Development and Therapy

    Article Title: Protective effect of Huang Gan formula in 5/6 nephrectomized rats by depressing the Wnt/β-catenin signaling pathway

    doi: 10.2147/DDDT.S81157

    Figure Lengend Snippet: Wnt1, β-catenin, TCF4, GSK-3β, Dkk1, and Fn1 mRNA expression in renal tissues (n=6). Notes: Compared with the 5/6 Nx group, * P

    Article Snippet: Rabbit polyclonal antibody Wnt1 (ab85060; Abcam, Inc., Cambridge, MA, USA), β-catenin (BS1982; Bioworld Technology, Inc., St Louis Park, MN, USA), GSK-3β (sc9166; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-GSK-3β (sc-135653; Santa Cruz Biotechnology Inc., Dallas, TX, USA), transcription factor 4 (TCF4) (BS6172; Bioworld Technology, Inc.), dickkopf 1 (Dkk1) (BS7731; Bioworld Technology, Inc.), fibronectin 1 (Fn1) (ab2413; Abcam, Inc.), and glyceraldehyde 3-phosphate dehydro-genase (GAPDH) (ab9485; Abcam, Inc.) were purchased.

    Techniques: Expressing

    Western blot analysis for detection of Wnt1, β-catenin, TCF4, GSK-3β, Dkk1, and Fn1 expression in the kidney. Notes: Western blot bands of different proteins ( A ); semi-quantitative analysis of the bands (from ( B ) to ( G ) are Wnt1, β-catenin, GSK-3β, Dkk1, TCF4, and Fn1 in sequence). Compared with 5/6 Nx group, * P

    Journal: Drug Design, Development and Therapy

    Article Title: Protective effect of Huang Gan formula in 5/6 nephrectomized rats by depressing the Wnt/β-catenin signaling pathway

    doi: 10.2147/DDDT.S81157

    Figure Lengend Snippet: Western blot analysis for detection of Wnt1, β-catenin, TCF4, GSK-3β, Dkk1, and Fn1 expression in the kidney. Notes: Western blot bands of different proteins ( A ); semi-quantitative analysis of the bands (from ( B ) to ( G ) are Wnt1, β-catenin, GSK-3β, Dkk1, TCF4, and Fn1 in sequence). Compared with 5/6 Nx group, * P

    Article Snippet: Rabbit polyclonal antibody Wnt1 (ab85060; Abcam, Inc., Cambridge, MA, USA), β-catenin (BS1982; Bioworld Technology, Inc., St Louis Park, MN, USA), GSK-3β (sc9166; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-GSK-3β (sc-135653; Santa Cruz Biotechnology Inc., Dallas, TX, USA), transcription factor 4 (TCF4) (BS6172; Bioworld Technology, Inc.), dickkopf 1 (Dkk1) (BS7731; Bioworld Technology, Inc.), fibronectin 1 (Fn1) (ab2413; Abcam, Inc.), and glyceraldehyde 3-phosphate dehydro-genase (GAPDH) (ab9485; Abcam, Inc.) were purchased.

    Techniques: Western Blot, Expressing, Sequencing

    Model of the RPIA mechanism for induction of β-catenin signaling in CRCs. Schematic representation shows the role of RPIA and β-catenin signaling in normal and CRC cells. In normal cells, RPIA does not interfere with GSK3β-mediated β-catenin degradation. In CRC, RPIA is overexpressed in both the cytoplasm and the nucleus; RPIA then binds to β-catenin in cytoplasm, and this prevents the degradation of β-catenin mediated by GSK3β. Within the nucleus, increased levels of β-catenin protein might also be caused by RPIA interrupting the APC-mediated transport of β-catenin from the nucleus to the cytoplasm. Overall, this would result in activation of downstream β-catenin target genes. β-cat, β-catenin; APC, adenomatous polyposis coli; CRC, colorectal cancer; GSK3β, glycogen synthase kinase-3′; RPIA, ribose-5-phosphate isomerase A, TCF, T-cell transcription factor.

    Journal: PLoS Biology

    Article Title: Identification of a noncanonical function for ribose-5-phosphate isomerase A promotes colorectal cancer formation by stabilizing and activating β-catenin via a novel C-terminal domain

    doi: 10.1371/journal.pbio.2003714

    Figure Lengend Snippet: Model of the RPIA mechanism for induction of β-catenin signaling in CRCs. Schematic representation shows the role of RPIA and β-catenin signaling in normal and CRC cells. In normal cells, RPIA does not interfere with GSK3β-mediated β-catenin degradation. In CRC, RPIA is overexpressed in both the cytoplasm and the nucleus; RPIA then binds to β-catenin in cytoplasm, and this prevents the degradation of β-catenin mediated by GSK3β. Within the nucleus, increased levels of β-catenin protein might also be caused by RPIA interrupting the APC-mediated transport of β-catenin from the nucleus to the cytoplasm. Overall, this would result in activation of downstream β-catenin target genes. β-cat, β-catenin; APC, adenomatous polyposis coli; CRC, colorectal cancer; GSK3β, glycogen synthase kinase-3′; RPIA, ribose-5-phosphate isomerase A, TCF, T-cell transcription factor.

    Article Snippet: Primary antibodies include RPIA (Cat# ab67080; Abcam, Cambridge, United Kingdom), β-actin (Cat# GTX109639; GeneTex, Inc, Irvine, CA), β-catenin (Cat# GTX61089, GeneTex; Cat# ab22656, Abcam), β-catenin (phospho Ser33/Ser37) (Cat# GTX11350 GeneTex), APC (Cat# GTX61328, GeneTex), Ubiquitin (Cat# 3936; Cell Signaling Technology, Danvers, MA), K48-linkage specific polyubiquitin (Cat# 4289, Cell Signaling Tecnology), β-Tubulin (Cat# ab52866, Abcam), and Lamin A/C (Cat# ab108922, Abcam).

    Techniques: Activation Assay

    RPIA promotes β-catenin expression and tumorigenesis in vivo. (A and B) In the upper panels, the HE stain was examined in the AB line (WT) and in (A) 3M and (B) 5M Tg ( ifabp : RPIA ) zebrafish. As shown in the bottom panels, via IHC, β-catenin expression levels were increased in (A) 3M and (B) 5M Tg ( ifabp : RPIA ) fish as well as in different regions of the intestine: IB, MI, and PI. Magnification: 400X for HE and 200X for IHC. Scale bar: 20 μm for HE and 50 μm for IHC. (C-E) β-catenin target genes were elevated in 3M Tg ( ifabp : RPIA ) fish, especially in (E) the PI. The expression level of β-catenin target genes was analyzed in 3M control fish ( n = 6) and RPIA Tg fish ( n = 18) from three portions of guts. The gene expression levels were analyzed with qPCR. There are extreme data in each group, so they are removed for the statistical analysis. (C) For IB, the number of WT is 6, and the number for RPIA is 7. (D) For the MI, the number of WT is 6, and the number for RPIA is 13. (E) For PI, the number of WT is 3, and the number for RPIA is 9. The statistical significance was calculated with Student t test (* 0.01

    Journal: PLoS Biology

    Article Title: Identification of a noncanonical function for ribose-5-phosphate isomerase A promotes colorectal cancer formation by stabilizing and activating β-catenin via a novel C-terminal domain

    doi: 10.1371/journal.pbio.2003714

    Figure Lengend Snippet: RPIA promotes β-catenin expression and tumorigenesis in vivo. (A and B) In the upper panels, the HE stain was examined in the AB line (WT) and in (A) 3M and (B) 5M Tg ( ifabp : RPIA ) zebrafish. As shown in the bottom panels, via IHC, β-catenin expression levels were increased in (A) 3M and (B) 5M Tg ( ifabp : RPIA ) fish as well as in different regions of the intestine: IB, MI, and PI. Magnification: 400X for HE and 200X for IHC. Scale bar: 20 μm for HE and 50 μm for IHC. (C-E) β-catenin target genes were elevated in 3M Tg ( ifabp : RPIA ) fish, especially in (E) the PI. The expression level of β-catenin target genes was analyzed in 3M control fish ( n = 6) and RPIA Tg fish ( n = 18) from three portions of guts. The gene expression levels were analyzed with qPCR. There are extreme data in each group, so they are removed for the statistical analysis. (C) For IB, the number of WT is 6, and the number for RPIA is 7. (D) For the MI, the number of WT is 6, and the number for RPIA is 13. (E) For PI, the number of WT is 3, and the number for RPIA is 9. The statistical significance was calculated with Student t test (* 0.01

    Article Snippet: Primary antibodies include RPIA (Cat# ab67080; Abcam, Cambridge, United Kingdom), β-actin (Cat# GTX109639; GeneTex, Inc, Irvine, CA), β-catenin (Cat# GTX61089, GeneTex; Cat# ab22656, Abcam), β-catenin (phospho Ser33/Ser37) (Cat# GTX11350 GeneTex), APC (Cat# GTX61328, GeneTex), Ubiquitin (Cat# 3936; Cell Signaling Technology, Danvers, MA), K48-linkage specific polyubiquitin (Cat# 4289, Cell Signaling Tecnology), β-Tubulin (Cat# ab52866, Abcam), and Lamin A/C (Cat# ab108922, Abcam).

    Techniques: Expressing, In Vivo, H&E Stain, Immunohistochemistry, Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction

    RPIA localizes in the nucleus and interacts with APC and β-catenin in HCT116 cells. (A) Nuclear localization of RPIA was detected by immunostaining with an anti-RPIA antibody (green) in HCT116 cells with and without overexpression of RPIA. DAPI was used to counterstain nuclei (blue). Scale bar: 50 μm. (B) Left panels: The cell lysates were precipitated by anti-APC, anti-β-catenin, and anti-RPIA antibodies in HCT116 cells. The APC, β-catenin, and RPIA interaction can be increased by RPIA-WT but not by RPIA-ΔD. Right panels: Protein loading input for IP assay of HCT116 cells. The orange boxes indicate the signals were enhanced by RPIA-WT but not in RPIA-ΔD. (C) Model of RPIA-β-catenin-APC interaction in HCT116 cell line. APC, adenomatous polyposis coli; Cyt, cytoplasm; IgA, immunoglobulin A; IP, immunoprecipitation; pcDNA, pcDNA3 vector control; Nu, nucleus; RPIA-ΔD, RPIA deletion domain D mutant; RPIA, ribose-5-phosphate isomerase A; RPIA-WT, RPIA wild type.

    Journal: PLoS Biology

    Article Title: Identification of a noncanonical function for ribose-5-phosphate isomerase A promotes colorectal cancer formation by stabilizing and activating β-catenin via a novel C-terminal domain

    doi: 10.1371/journal.pbio.2003714

    Figure Lengend Snippet: RPIA localizes in the nucleus and interacts with APC and β-catenin in HCT116 cells. (A) Nuclear localization of RPIA was detected by immunostaining with an anti-RPIA antibody (green) in HCT116 cells with and without overexpression of RPIA. DAPI was used to counterstain nuclei (blue). Scale bar: 50 μm. (B) Left panels: The cell lysates were precipitated by anti-APC, anti-β-catenin, and anti-RPIA antibodies in HCT116 cells. The APC, β-catenin, and RPIA interaction can be increased by RPIA-WT but not by RPIA-ΔD. Right panels: Protein loading input for IP assay of HCT116 cells. The orange boxes indicate the signals were enhanced by RPIA-WT but not in RPIA-ΔD. (C) Model of RPIA-β-catenin-APC interaction in HCT116 cell line. APC, adenomatous polyposis coli; Cyt, cytoplasm; IgA, immunoglobulin A; IP, immunoprecipitation; pcDNA, pcDNA3 vector control; Nu, nucleus; RPIA-ΔD, RPIA deletion domain D mutant; RPIA, ribose-5-phosphate isomerase A; RPIA-WT, RPIA wild type.

    Article Snippet: Primary antibodies include RPIA (Cat# ab67080; Abcam, Cambridge, United Kingdom), β-actin (Cat# GTX109639; GeneTex, Inc, Irvine, CA), β-catenin (Cat# GTX61089, GeneTex; Cat# ab22656, Abcam), β-catenin (phospho Ser33/Ser37) (Cat# GTX11350 GeneTex), APC (Cat# GTX61328, GeneTex), Ubiquitin (Cat# 3936; Cell Signaling Technology, Danvers, MA), K48-linkage specific polyubiquitin (Cat# 4289, Cell Signaling Tecnology), β-Tubulin (Cat# ab52866, Abcam), and Lamin A/C (Cat# ab108922, Abcam).

    Techniques: Immunostaining, Over Expression, Immunoprecipitation, Plasmid Preparation, Mutagenesis

    The C-terminal domain of RPIA containing amino acids 290 to 311 is required for cell proliferation and β-catenin stabilization in HCT116 cells. (A) The RPIA amino acid sequence in several species. Identical sequences are represented by red text. Blue text indicates > 50% but

    Journal: PLoS Biology

    Article Title: Identification of a noncanonical function for ribose-5-phosphate isomerase A promotes colorectal cancer formation by stabilizing and activating β-catenin via a novel C-terminal domain

    doi: 10.1371/journal.pbio.2003714

    Figure Lengend Snippet: The C-terminal domain of RPIA containing amino acids 290 to 311 is required for cell proliferation and β-catenin stabilization in HCT116 cells. (A) The RPIA amino acid sequence in several species. Identical sequences are represented by red text. Blue text indicates > 50% but

    Article Snippet: Primary antibodies include RPIA (Cat# ab67080; Abcam, Cambridge, United Kingdom), β-actin (Cat# GTX109639; GeneTex, Inc, Irvine, CA), β-catenin (Cat# GTX61089, GeneTex; Cat# ab22656, Abcam), β-catenin (phospho Ser33/Ser37) (Cat# GTX11350 GeneTex), APC (Cat# GTX61328, GeneTex), Ubiquitin (Cat# 3936; Cell Signaling Technology, Danvers, MA), K48-linkage specific polyubiquitin (Cat# 4289, Cell Signaling Tecnology), β-Tubulin (Cat# ab52866, Abcam), and Lamin A/C (Cat# ab108922, Abcam).

    Techniques: Sequencing

    RPIA expression is positively correlated with β-catenin protein levels and stability in HCT116 cells. (A) Knockdown of RPIA reduced β-catenin protein levels and overexpression of RPIA increased β-catenin protein levels in both the cytoplasmic and nuclear fractions of HCT116 cells. (B) Knockdown of RPIA did not decrease ERK and pERK protein levels, which were measured by western blotting in total protein analysis (up panel) in HCT116. Conversely, overexpression of RPIA did not increase ERK and pERK protein levels (up panel). In the lower panel, both cytoplasmic and nuclear fraction showed that ERK and pERK protein levels did not up-regulate in HCT116. (C) Scatter plots show a positive correlation between RPIA and β-catenin expression in the colon tissue or nucleus. (D) To determine the half-life of β-catenin protein, western blots were used to measure the abundance of β-catenin at different time points following the addition of 10 μg/ml of the protein synthesis inhibitor CHX to HCT116 cells transfected with either control siRNA or RPIA-siRNA. The lower panels show plots of the relative β-catenin protein level, expressed as a percentage as a function of time after CHX treatment. (E) RPIA-ΔD lost the ability to stabilize β-catenin. Relative β-catenin protein levels as measured by quantification of western blot are shown in HCT116 cells. (F) The reduced β-catenin levels by RPIA knockdown were rescued by 5 μM of MG132 treatment (left panel). Inhibition of RPIA stimulated ubiquitination of β-catenin (right panel). β-Catenin was precipitated by specific antibody. Coprecipitated ubiquitin levels were examined via western blot with antiubiquitin antibody. (G) Phosphorylated β-catenin (at Ser33/Ser37) versus total β-catenin was elevated upon RPIA knockdown. Gel images are shown in the up panel. (H) Overexpression of nondegradable β-catenin can overcome the growth inhibition induced by RPIA knockdown in HCT116 cells. The proliferation fold is compared to pMCV6 transfected control cell at first day. (I) The elevated viability induced by expression of RPIA was decreased upon ICRT14 (β-catenin inhibitor) treatment. Dose-dependent effects were revealed in HCT116 cells. (J) pGSK3β Ser9 protein expression levels were up-regulated in the cytoplasmic extract upon overexpression of RPIA-WT but not upon RPIA-ΔD in HCT116 cells. (K) Cell proliferation was measured in RPIA knockdown HCT116 cells combined with 2.5 mM LiCl or 5 μM CHIR99021, respectively. The statistical significance was calculated with the Student t test (*** P

    Journal: PLoS Biology

    Article Title: Identification of a noncanonical function for ribose-5-phosphate isomerase A promotes colorectal cancer formation by stabilizing and activating β-catenin via a novel C-terminal domain

    doi: 10.1371/journal.pbio.2003714

    Figure Lengend Snippet: RPIA expression is positively correlated with β-catenin protein levels and stability in HCT116 cells. (A) Knockdown of RPIA reduced β-catenin protein levels and overexpression of RPIA increased β-catenin protein levels in both the cytoplasmic and nuclear fractions of HCT116 cells. (B) Knockdown of RPIA did not decrease ERK and pERK protein levels, which were measured by western blotting in total protein analysis (up panel) in HCT116. Conversely, overexpression of RPIA did not increase ERK and pERK protein levels (up panel). In the lower panel, both cytoplasmic and nuclear fraction showed that ERK and pERK protein levels did not up-regulate in HCT116. (C) Scatter plots show a positive correlation between RPIA and β-catenin expression in the colon tissue or nucleus. (D) To determine the half-life of β-catenin protein, western blots were used to measure the abundance of β-catenin at different time points following the addition of 10 μg/ml of the protein synthesis inhibitor CHX to HCT116 cells transfected with either control siRNA or RPIA-siRNA. The lower panels show plots of the relative β-catenin protein level, expressed as a percentage as a function of time after CHX treatment. (E) RPIA-ΔD lost the ability to stabilize β-catenin. Relative β-catenin protein levels as measured by quantification of western blot are shown in HCT116 cells. (F) The reduced β-catenin levels by RPIA knockdown were rescued by 5 μM of MG132 treatment (left panel). Inhibition of RPIA stimulated ubiquitination of β-catenin (right panel). β-Catenin was precipitated by specific antibody. Coprecipitated ubiquitin levels were examined via western blot with antiubiquitin antibody. (G) Phosphorylated β-catenin (at Ser33/Ser37) versus total β-catenin was elevated upon RPIA knockdown. Gel images are shown in the up panel. (H) Overexpression of nondegradable β-catenin can overcome the growth inhibition induced by RPIA knockdown in HCT116 cells. The proliferation fold is compared to pMCV6 transfected control cell at first day. (I) The elevated viability induced by expression of RPIA was decreased upon ICRT14 (β-catenin inhibitor) treatment. Dose-dependent effects were revealed in HCT116 cells. (J) pGSK3β Ser9 protein expression levels were up-regulated in the cytoplasmic extract upon overexpression of RPIA-WT but not upon RPIA-ΔD in HCT116 cells. (K) Cell proliferation was measured in RPIA knockdown HCT116 cells combined with 2.5 mM LiCl or 5 μM CHIR99021, respectively. The statistical significance was calculated with the Student t test (*** P

    Article Snippet: Primary antibodies include RPIA (Cat# ab67080; Abcam, Cambridge, United Kingdom), β-actin (Cat# GTX109639; GeneTex, Inc, Irvine, CA), β-catenin (Cat# GTX61089, GeneTex; Cat# ab22656, Abcam), β-catenin (phospho Ser33/Ser37) (Cat# GTX11350 GeneTex), APC (Cat# GTX61328, GeneTex), Ubiquitin (Cat# 3936; Cell Signaling Technology, Danvers, MA), K48-linkage specific polyubiquitin (Cat# 4289, Cell Signaling Tecnology), β-Tubulin (Cat# ab52866, Abcam), and Lamin A/C (Cat# ab108922, Abcam).

    Techniques: Expressing, Over Expression, Western Blot, Transfection, Inhibition

    RPIA regulates colon cell proliferation through β-catenin expression in HCT116 cells. (A) Knockdown of RPIA significantly reduced cell proliferation and RPIA overexpression enhanced cell proliferation in HCT116 cells. Co-treatment of si-RPIA and pcDNA-RPIA rescued the reduction of cellular proliferation upon knockdown of RPIA in HCT116. Cell viability assays were performed by measuring the cells at the second, third, fourth, and fifth days and the proliferation fold is compared to control cell at the first day. Control: Co-transfect with scramble RNA and pcDNA empty vector as negative control. (B) RPIA knockdown significantly reduced colony formation ability, and RPIA overexpression enhanced colony formation ability in HCT116 cells. si-NC: Transfect with scramble siRNA as negative control. Representative images of colonies were shown on top of the quantification result. (C) Knockdown of RPIA reduced β-catenin protein levels as measured by western blotting (left panel) and quantified using Image J (middle panel) but did not significantly alter mRNA levels of β-catenin as measured by qPCR (right panel) in HCT116 cells. (D) RPIA overexpression increased β-catenin protein levels (left and middle panels) but did not affect β-catenin mRNA levels (right panel) in HCT116 cells. (E) Knock down of RPIA reduced the β-catenin/TCF luciferase reporter activity in HCT116 cells. (F) Overexpression of RPIA induced the β-catenin/TCF luciferase reporter activity in HCT116 cells. (G) Knockdown of RPIA decreased the mRNA levels of the β-catenin target genes CCND1 , CCNE2 , and AXIN2 in HCT116 cells. (H) Overexpression of RPIA increased the mRNA levels of the β-catenin target genes CCND1 , CCNE2 , and AXIN2 in HCT116 cells. The statistical significance was calculated with Student t test (* 0.01

    Journal: PLoS Biology

    Article Title: Identification of a noncanonical function for ribose-5-phosphate isomerase A promotes colorectal cancer formation by stabilizing and activating β-catenin via a novel C-terminal domain

    doi: 10.1371/journal.pbio.2003714

    Figure Lengend Snippet: RPIA regulates colon cell proliferation through β-catenin expression in HCT116 cells. (A) Knockdown of RPIA significantly reduced cell proliferation and RPIA overexpression enhanced cell proliferation in HCT116 cells. Co-treatment of si-RPIA and pcDNA-RPIA rescued the reduction of cellular proliferation upon knockdown of RPIA in HCT116. Cell viability assays were performed by measuring the cells at the second, third, fourth, and fifth days and the proliferation fold is compared to control cell at the first day. Control: Co-transfect with scramble RNA and pcDNA empty vector as negative control. (B) RPIA knockdown significantly reduced colony formation ability, and RPIA overexpression enhanced colony formation ability in HCT116 cells. si-NC: Transfect with scramble siRNA as negative control. Representative images of colonies were shown on top of the quantification result. (C) Knockdown of RPIA reduced β-catenin protein levels as measured by western blotting (left panel) and quantified using Image J (middle panel) but did not significantly alter mRNA levels of β-catenin as measured by qPCR (right panel) in HCT116 cells. (D) RPIA overexpression increased β-catenin protein levels (left and middle panels) but did not affect β-catenin mRNA levels (right panel) in HCT116 cells. (E) Knock down of RPIA reduced the β-catenin/TCF luciferase reporter activity in HCT116 cells. (F) Overexpression of RPIA induced the β-catenin/TCF luciferase reporter activity in HCT116 cells. (G) Knockdown of RPIA decreased the mRNA levels of the β-catenin target genes CCND1 , CCNE2 , and AXIN2 in HCT116 cells. (H) Overexpression of RPIA increased the mRNA levels of the β-catenin target genes CCND1 , CCNE2 , and AXIN2 in HCT116 cells. The statistical significance was calculated with Student t test (* 0.01

    Article Snippet: Primary antibodies include RPIA (Cat# ab67080; Abcam, Cambridge, United Kingdom), β-actin (Cat# GTX109639; GeneTex, Inc, Irvine, CA), β-catenin (Cat# GTX61089, GeneTex; Cat# ab22656, Abcam), β-catenin (phospho Ser33/Ser37) (Cat# GTX11350 GeneTex), APC (Cat# GTX61328, GeneTex), Ubiquitin (Cat# 3936; Cell Signaling Technology, Danvers, MA), K48-linkage specific polyubiquitin (Cat# 4289, Cell Signaling Tecnology), β-Tubulin (Cat# ab52866, Abcam), and Lamin A/C (Cat# ab108922, Abcam).

    Techniques: Expressing, Over Expression, Plasmid Preparation, Negative Control, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

    FAT4 knockdown-induced gastric cancer cell growth, invasion, and migration are mediated by the Wnt/ β -catenin signalling pathway. BGC-823 and HGC-27 cells were transfected with or without shRNA (shRNA1) against FAT4 and siRNA targeting β -catenin, and analysed by western blotting ( A ), MTT assay ( B ), colony-formation assay ( C ), and migration and invasion assays ( D and E ). * P

    Journal: British Journal of Cancer

    Article Title: FAT4 functions as a tumour suppressor in gastric cancer by modulating Wnt/β-catenin signalling

    doi: 10.1038/bjc.2015.367

    Figure Lengend Snippet: FAT4 knockdown-induced gastric cancer cell growth, invasion, and migration are mediated by the Wnt/ β -catenin signalling pathway. BGC-823 and HGC-27 cells were transfected with or without shRNA (shRNA1) against FAT4 and siRNA targeting β -catenin, and analysed by western blotting ( A ), MTT assay ( B ), colony-formation assay ( C ), and migration and invasion assays ( D and E ). * P

    Article Snippet: Small interfering RNA (siRNA) against β -catenin was purchased from GeneChem.

    Techniques: Migration, Transfection, shRNA, Western Blot, MTT Assay, Colony Assay

    Effect of FAT4 knockdown on Wnt/ β -catenin signalling. ( A ) Altered nuclear translocation of β -catenin in response to FAT4 knockdown. Nuclear and cytosolic extracts of BGC-823 and HGC-27 cells were analysed by western blotting with Lamin A and β -actin as a loading control. ( B ) Cells were transfected with TOPflash or FOPflash and Renilla pRL-TK plasmids and subjected to dual-luciferase assays 48 h after transfection. Reporter activity was normalised to Renilla luciferase activity. ( C ) Western blot analysis of Wnt target gene expression in response to FAT4 knockdown in BGC-823 and HGC-27 cells. β -Actin was used as the loading control. ** P

    Journal: British Journal of Cancer

    Article Title: FAT4 functions as a tumour suppressor in gastric cancer by modulating Wnt/β-catenin signalling

    doi: 10.1038/bjc.2015.367

    Figure Lengend Snippet: Effect of FAT4 knockdown on Wnt/ β -catenin signalling. ( A ) Altered nuclear translocation of β -catenin in response to FAT4 knockdown. Nuclear and cytosolic extracts of BGC-823 and HGC-27 cells were analysed by western blotting with Lamin A and β -actin as a loading control. ( B ) Cells were transfected with TOPflash or FOPflash and Renilla pRL-TK plasmids and subjected to dual-luciferase assays 48 h after transfection. Reporter activity was normalised to Renilla luciferase activity. ( C ) Western blot analysis of Wnt target gene expression in response to FAT4 knockdown in BGC-823 and HGC-27 cells. β -Actin was used as the loading control. ** P

    Article Snippet: Small interfering RNA (siRNA) against β -catenin was purchased from GeneChem.

    Techniques: Translocation Assay, Western Blot, Transfection, Luciferase, Activity Assay, Expressing