l wnt3a cells  (ATCC)


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    ATCC l wnt3a cells
    PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or <t>Wnt3a</t> for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.
    L Wnt3a Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/β-catenin Signaling"

    Article Title: Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/β-catenin Signaling

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.67842

    PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or Wnt3a for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.
    Figure Legend Snippet: PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or Wnt3a for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.

    Techniques Used: Activity Assay, Transfection, Over Expression, Western Blot, Expressing, RNA Sequencing Assay

    PANK1 phosphorylates the N-terminal serine and threonine residues of β-catenin. (A) The effect of knockdown or overexpression of PANK1 in Huh7 and QGY-7701 cells on the phosphorylation level of β-catenin was measured. After the cells were stimulated by Wnt3a for different periods of time, the phosphorylation level of β-catenin was measured by western blot. (B) Ubiquitin assay was performed to measure the effect of PANK1 expression on the ubiquitination level of β-catenin. After being treated with MG132 for 10h, the PVTT cells with overexpressed PANK1, Huh7 cells, with down-regulated expression of PANK1, and their respective control cells were lysed for supernatant collection. The antibody against β-catenin was used for CO-IP. Then, a western blot assay was performed, and the antibody against ubiquitin was used for measurement. (C) The interaction between exogenously expressed CK1α (Myc-CK1α) and PANK1 (Flag-PANK1) was measured in 293T cells. The Myc-CK1α and Flag-PANK1 plasmids were transiently transfected into 293T cells. 48h later, CO-IP was performed with an antibody against Myc or the Flag tag. (D) The interaction between endogenous CK1α and PANK1 in Huh7 and QGY-7701 cells was measured. (E) The Wnt3a stimulation-dependent interaction between CK1α and PANK1 in QGY-7701 cells was measured. After treatment with Wnt3a for different periods of time, the QGY-7701 cells with the overexpression PANK1 were lysed and centrifuged for supernatant collection. The antibody against Flag was used for CO-IP. (F) The phosphorylation of β-catenin by PANK was measured through the in vitro kinase reaction. See “Materials and Methods” for the details on the reaction.
    Figure Legend Snippet: PANK1 phosphorylates the N-terminal serine and threonine residues of β-catenin. (A) The effect of knockdown or overexpression of PANK1 in Huh7 and QGY-7701 cells on the phosphorylation level of β-catenin was measured. After the cells were stimulated by Wnt3a for different periods of time, the phosphorylation level of β-catenin was measured by western blot. (B) Ubiquitin assay was performed to measure the effect of PANK1 expression on the ubiquitination level of β-catenin. After being treated with MG132 for 10h, the PVTT cells with overexpressed PANK1, Huh7 cells, with down-regulated expression of PANK1, and their respective control cells were lysed for supernatant collection. The antibody against β-catenin was used for CO-IP. Then, a western blot assay was performed, and the antibody against ubiquitin was used for measurement. (C) The interaction between exogenously expressed CK1α (Myc-CK1α) and PANK1 (Flag-PANK1) was measured in 293T cells. The Myc-CK1α and Flag-PANK1 plasmids were transiently transfected into 293T cells. 48h later, CO-IP was performed with an antibody against Myc or the Flag tag. (D) The interaction between endogenous CK1α and PANK1 in Huh7 and QGY-7701 cells was measured. (E) The Wnt3a stimulation-dependent interaction between CK1α and PANK1 in QGY-7701 cells was measured. After treatment with Wnt3a for different periods of time, the QGY-7701 cells with the overexpression PANK1 were lysed and centrifuged for supernatant collection. The antibody against Flag was used for CO-IP. (F) The phosphorylation of β-catenin by PANK was measured through the in vitro kinase reaction. See “Materials and Methods” for the details on the reaction.

    Techniques Used: Over Expression, Western Blot, Ubiquitin Assay, Expressing, Co-Immunoprecipitation Assay, Transfection, FLAG-tag, In Vitro

    l wnt3a crl 2647 cells  (ATCC)


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    ATCC l wnt3a crl 2647 cells
    L Wnt3a Crl 2647 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    l wnt3a crl 2647 cells  (ATCC)


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    Structured Review

    ATCC l wnt3a crl 2647 cells
    (A) Schematic diagrams showing the domain structures of TcdB and the 2 short fragments derived from TcdB (TcdB FBD and TcdB mu ) used in this study. GTD, glucosyltransferase domain; CPD, cysteine protease domain; Delivery/RBD, membrane translocation and receptor-binding domain; CROPs, combined repetitive oligopeptides domain. The structural model of TcdB FBD -CRD7 complex shown is modeled based on the crystal structure of TcdB FBD -CRD2 (PDB code: 6C0B) and CRD7 (PDB code:5T44). TcdB FBD , CRD2, and CRD7 are colored pink, green, and blue, respectively. CRD, cysteine-rich domain. (B) TcdB FBD blocked <t>WNT3A-mediated</t> signaling in MDA-MB-231 cells in a dose-dependent manner, whereas TcdB mu showed no inhibition at nanomolar concentrations. Wnt signaling activity was analyzed using the TOPFLASH/TK-Renilla (TK/RL) dual luciferase reporter assay (error bars indicate mean ± SEM, 3 independent experiments). (C) Wnt signaling activity in MDA-MB-231 cells was monitored using TK/RL assays over 5 days after induction by WNT3A conditioned medium with the indicated concentrations of TcdB FBD or TcdB mu . Error bars indicate mean ± SEM, 3 independent experiments. (D) Nude mice were subcutaneously transplanted with TK/RL-transduced MDA-MB-231 cells and then treated at the indicated time point with TcdB FBD or TcdB mu (20 mg/kg of body weight) by intraperitoneal (i.p.) injection. D-Luciferin was injected 5 min before tumor tissues were isolated and the luciferase activity in tumor tissues was then measured ex vivo and quantified (error bars indicate mean ± SEM, n = 4–5 tumors). Numerical values are in .
    L Wnt3a Crl 2647 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth"

    Article Title: Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth

    Journal: PLOS Biology

    doi: 10.1371/journal.pbio.3002353

    (A) Schematic diagrams showing the domain structures of TcdB and the 2 short fragments derived from TcdB (TcdB FBD and TcdB mu ) used in this study. GTD, glucosyltransferase domain; CPD, cysteine protease domain; Delivery/RBD, membrane translocation and receptor-binding domain; CROPs, combined repetitive oligopeptides domain. The structural model of TcdB FBD -CRD7 complex shown is modeled based on the crystal structure of TcdB FBD -CRD2 (PDB code: 6C0B) and CRD7 (PDB code:5T44). TcdB FBD , CRD2, and CRD7 are colored pink, green, and blue, respectively. CRD, cysteine-rich domain. (B) TcdB FBD blocked WNT3A-mediated signaling in MDA-MB-231 cells in a dose-dependent manner, whereas TcdB mu showed no inhibition at nanomolar concentrations. Wnt signaling activity was analyzed using the TOPFLASH/TK-Renilla (TK/RL) dual luciferase reporter assay (error bars indicate mean ± SEM, 3 independent experiments). (C) Wnt signaling activity in MDA-MB-231 cells was monitored using TK/RL assays over 5 days after induction by WNT3A conditioned medium with the indicated concentrations of TcdB FBD or TcdB mu . Error bars indicate mean ± SEM, 3 independent experiments. (D) Nude mice were subcutaneously transplanted with TK/RL-transduced MDA-MB-231 cells and then treated at the indicated time point with TcdB FBD or TcdB mu (20 mg/kg of body weight) by intraperitoneal (i.p.) injection. D-Luciferin was injected 5 min before tumor tissues were isolated and the luciferase activity in tumor tissues was then measured ex vivo and quantified (error bars indicate mean ± SEM, n = 4–5 tumors). Numerical values are in .
    Figure Legend Snippet: (A) Schematic diagrams showing the domain structures of TcdB and the 2 short fragments derived from TcdB (TcdB FBD and TcdB mu ) used in this study. GTD, glucosyltransferase domain; CPD, cysteine protease domain; Delivery/RBD, membrane translocation and receptor-binding domain; CROPs, combined repetitive oligopeptides domain. The structural model of TcdB FBD -CRD7 complex shown is modeled based on the crystal structure of TcdB FBD -CRD2 (PDB code: 6C0B) and CRD7 (PDB code:5T44). TcdB FBD , CRD2, and CRD7 are colored pink, green, and blue, respectively. CRD, cysteine-rich domain. (B) TcdB FBD blocked WNT3A-mediated signaling in MDA-MB-231 cells in a dose-dependent manner, whereas TcdB mu showed no inhibition at nanomolar concentrations. Wnt signaling activity was analyzed using the TOPFLASH/TK-Renilla (TK/RL) dual luciferase reporter assay (error bars indicate mean ± SEM, 3 independent experiments). (C) Wnt signaling activity in MDA-MB-231 cells was monitored using TK/RL assays over 5 days after induction by WNT3A conditioned medium with the indicated concentrations of TcdB FBD or TcdB mu . Error bars indicate mean ± SEM, 3 independent experiments. (D) Nude mice were subcutaneously transplanted with TK/RL-transduced MDA-MB-231 cells and then treated at the indicated time point with TcdB FBD or TcdB mu (20 mg/kg of body weight) by intraperitoneal (i.p.) injection. D-Luciferin was injected 5 min before tumor tissues were isolated and the luciferase activity in tumor tissues was then measured ex vivo and quantified (error bars indicate mean ± SEM, n = 4–5 tumors). Numerical values are in .

    Techniques Used: Derivative Assay, Membrane, Translocation Assay, Binding Assay, Inhibition, Activity Assay, Luciferase, Reporter Assay, Injection, Isolation, Ex Vivo

    l wnt3a cells  (ATCC)


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    ATCC l wnt3a cells
    PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or <t>Wnt3a</t> for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.
    L Wnt3a Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/β-catenin Signaling"

    Article Title: Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/β-catenin Signaling

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.67842

    PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or Wnt3a for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.
    Figure Legend Snippet: PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or Wnt3a for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.

    Techniques Used: Activity Assay, Transfection, Over Expression, Western Blot, Expressing, RNA Sequencing Assay

    PANK1 phosphorylates the N-terminal serine and threonine residues of β-catenin. (A) The effect of knockdown or overexpression of PANK1 in Huh7 and QGY-7701 cells on the phosphorylation level of β-catenin was measured. After the cells were stimulated by Wnt3a for different periods of time, the phosphorylation level of β-catenin was measured by western blot. (B) Ubiquitin assay was performed to measure the effect of PANK1 expression on the ubiquitination level of β-catenin. After being treated with MG132 for 10h, the PVTT cells with overexpressed PANK1, Huh7 cells, with down-regulated expression of PANK1, and their respective control cells were lysed for supernatant collection. The antibody against β-catenin was used for CO-IP. Then, a western blot assay was performed, and the antibody against ubiquitin was used for measurement. (C) The interaction between exogenously expressed CK1α (Myc-CK1α) and PANK1 (Flag-PANK1) was measured in 293T cells. The Myc-CK1α and Flag-PANK1 plasmids were transiently transfected into 293T cells. 48h later, CO-IP was performed with an antibody against Myc or the Flag tag. (D) The interaction between endogenous CK1α and PANK1 in Huh7 and QGY-7701 cells was measured. (E) The Wnt3a stimulation-dependent interaction between CK1α and PANK1 in QGY-7701 cells was measured. After treatment with Wnt3a for different periods of time, the QGY-7701 cells with the overexpression PANK1 were lysed and centrifuged for supernatant collection. The antibody against Flag was used for CO-IP. (F) The phosphorylation of β-catenin by PANK was measured through the in vitro kinase reaction. See “Materials and Methods” for the details on the reaction.
    Figure Legend Snippet: PANK1 phosphorylates the N-terminal serine and threonine residues of β-catenin. (A) The effect of knockdown or overexpression of PANK1 in Huh7 and QGY-7701 cells on the phosphorylation level of β-catenin was measured. After the cells were stimulated by Wnt3a for different periods of time, the phosphorylation level of β-catenin was measured by western blot. (B) Ubiquitin assay was performed to measure the effect of PANK1 expression on the ubiquitination level of β-catenin. After being treated with MG132 for 10h, the PVTT cells with overexpressed PANK1, Huh7 cells, with down-regulated expression of PANK1, and their respective control cells were lysed for supernatant collection. The antibody against β-catenin was used for CO-IP. Then, a western blot assay was performed, and the antibody against ubiquitin was used for measurement. (C) The interaction between exogenously expressed CK1α (Myc-CK1α) and PANK1 (Flag-PANK1) was measured in 293T cells. The Myc-CK1α and Flag-PANK1 plasmids were transiently transfected into 293T cells. 48h later, CO-IP was performed with an antibody against Myc or the Flag tag. (D) The interaction between endogenous CK1α and PANK1 in Huh7 and QGY-7701 cells was measured. (E) The Wnt3a stimulation-dependent interaction between CK1α and PANK1 in QGY-7701 cells was measured. After treatment with Wnt3a for different periods of time, the QGY-7701 cells with the overexpression PANK1 were lysed and centrifuged for supernatant collection. The antibody against Flag was used for CO-IP. (F) The phosphorylation of β-catenin by PANK was measured through the in vitro kinase reaction. See “Materials and Methods” for the details on the reaction.

    Techniques Used: Over Expression, Western Blot, Ubiquitin Assay, Expressing, Co-Immunoprecipitation Assay, Transfection, FLAG-tag, In Vitro

    mouse l1 cells expressing wnt3a  (ATCC)


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    ATCC mouse l1 cells expressing wnt3a
    Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of <t>Wnt3a-CM</t> (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p < 0.05, ** p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.
    Mouse L1 Cells Expressing Wnt3a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity"

    Article Title: Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-13-26

    Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of Wnt3a-CM (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p < 0.05, ** p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.
    Figure Legend Snippet: Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of Wnt3a-CM (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p < 0.05, ** p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.

    Techniques Used: Luciferase, Activity Assay, Transfection, Mutagenesis

    A) Tph1 gene expression and B) 5- Htr1b gene expression in CHO cells transfected with wild type LRP5 (WT-LRP5) or mutant LRP5 and treated with Wnt3a-CM or L1-CM . Expression of the Tph1 and 5- Htr1b genes is shown as a fold change relative to the untreated control (pcDNA3.1+) and normalized to β-actin . The effects of the LRP5 mutants on the expression of these genes were examined by comparing the results for the mutants with those for WT-LRP5 using Student's t test. The statistical significance of each pair is denoted below the mutant columns; ** p < 0.01 as compared to WT-LRP5. Results are expressed as mean ± SD.
    Figure Legend Snippet: A) Tph1 gene expression and B) 5- Htr1b gene expression in CHO cells transfected with wild type LRP5 (WT-LRP5) or mutant LRP5 and treated with Wnt3a-CM or L1-CM . Expression of the Tph1 and 5- Htr1b genes is shown as a fold change relative to the untreated control (pcDNA3.1+) and normalized to β-actin . The effects of the LRP5 mutants on the expression of these genes were examined by comparing the results for the mutants with those for WT-LRP5 using Student's t test. The statistical significance of each pair is denoted below the mutant columns; ** p < 0.01 as compared to WT-LRP5. Results are expressed as mean ± SD.

    Techniques Used: Expressing, Transfection, Mutagenesis

    l wnt3a cells  (ATCC)


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    ATCC l wnt3a cells
    Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of <t>Wnt3a-CM</t> (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p < 0.05, ** p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.
    L Wnt3a Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity"

    Article Title: Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-13-26

    Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of Wnt3a-CM (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p < 0.05, ** p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.
    Figure Legend Snippet: Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of Wnt3a-CM (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p < 0.05, ** p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.

    Techniques Used: Luciferase, Activity Assay, Transfection, Mutagenesis

    A) Tph1 gene expression and B) 5- Htr1b gene expression in CHO cells transfected with wild type LRP5 (WT-LRP5) or mutant LRP5 and treated with Wnt3a-CM or L1-CM . Expression of the Tph1 and 5- Htr1b genes is shown as a fold change relative to the untreated control (pcDNA3.1+) and normalized to β-actin . The effects of the LRP5 mutants on the expression of these genes were examined by comparing the results for the mutants with those for WT-LRP5 using Student's t test. The statistical significance of each pair is denoted below the mutant columns; ** p < 0.01 as compared to WT-LRP5. Results are expressed as mean ± SD.
    Figure Legend Snippet: A) Tph1 gene expression and B) 5- Htr1b gene expression in CHO cells transfected with wild type LRP5 (WT-LRP5) or mutant LRP5 and treated with Wnt3a-CM or L1-CM . Expression of the Tph1 and 5- Htr1b genes is shown as a fold change relative to the untreated control (pcDNA3.1+) and normalized to β-actin . The effects of the LRP5 mutants on the expression of these genes were examined by comparing the results for the mutants with those for WT-LRP5 using Student's t test. The statistical significance of each pair is denoted below the mutant columns; ** p < 0.01 as compared to WT-LRP5. Results are expressed as mean ± SD.

    Techniques Used: Expressing, Transfection, Mutagenesis

    l wnt3a  (ATCC)


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    ATCC l wnt3a
    ( A ) Immuno-fluorescence detection of beta-catenin in 2D monolayers of EMT6 or 4T1 cells treated with L-CM or <t>Wnt3a-CM</t> in the presence or absence of C4S or CS-E treatment. Wnt3a-CM led to a drastic increase in nuclear beta-catenin levels; concomitant treatment with CS-E, but not C4S, severely reduced nuclear beta-catenin expression levels (red arrowheads: cell membrane staining; red arrows: nuclear staining) (beta-catenin: green; DAPI: blue; scale bar = 50 micrometer). ( B, C ) Effect of CS-E on TOPFLASH reporter assays. Treatment with Wnt3a-CM for 24 hours caused a significant increase in TOPFLASH luciferase activity when compared to treatment with L-CM in both EMT6 ( B ) and 4T1 cells ( C ). Concomitant treatment with CS-E, but not C4S, significantly reduced Wnt3a-stimulated TOPFLASH reporter activity. ( D ) Western blot analysis of Wnt/beta-catenin signaling receptor activation. Treatment of EMT6 cells with Wnt3a-CM, but not L-CM for 1 hour led to phosphorylation of LRP6 (pLRP6). Concomitant treatment with CS-E (100 microgram/ml) interfered with phosphorylation of the LRP6 protein. Note that only the upper bands of the LRP6 are phosphorylated in response to Wnt3a (black arrows). Quantitation of these Western blots demonstrated that CS-E treatment significantly reduced LRP6 receptor phosphorylation by 60% in both EMT6 and 4T1 cells (*p<0.01; n≥3).
    L Wnt3a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Chondroitin Sulfate-E Is a Negative Regulator of a Pro-Tumorigenic Wnt/Beta-Catenin-Collagen 1 Axis in Breast Cancer Cells"

    Article Title: Chondroitin Sulfate-E Is a Negative Regulator of a Pro-Tumorigenic Wnt/Beta-Catenin-Collagen 1 Axis in Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103966

    ( A ) Immuno-fluorescence detection of beta-catenin in 2D monolayers of EMT6 or 4T1 cells treated with L-CM or Wnt3a-CM in the presence or absence of C4S or CS-E treatment. Wnt3a-CM led to a drastic increase in nuclear beta-catenin levels; concomitant treatment with CS-E, but not C4S, severely reduced nuclear beta-catenin expression levels (red arrowheads: cell membrane staining; red arrows: nuclear staining) (beta-catenin: green; DAPI: blue; scale bar = 50 micrometer). ( B, C ) Effect of CS-E on TOPFLASH reporter assays. Treatment with Wnt3a-CM for 24 hours caused a significant increase in TOPFLASH luciferase activity when compared to treatment with L-CM in both EMT6 ( B ) and 4T1 cells ( C ). Concomitant treatment with CS-E, but not C4S, significantly reduced Wnt3a-stimulated TOPFLASH reporter activity. ( D ) Western blot analysis of Wnt/beta-catenin signaling receptor activation. Treatment of EMT6 cells with Wnt3a-CM, but not L-CM for 1 hour led to phosphorylation of LRP6 (pLRP6). Concomitant treatment with CS-E (100 microgram/ml) interfered with phosphorylation of the LRP6 protein. Note that only the upper bands of the LRP6 are phosphorylated in response to Wnt3a (black arrows). Quantitation of these Western blots demonstrated that CS-E treatment significantly reduced LRP6 receptor phosphorylation by 60% in both EMT6 and 4T1 cells (*p<0.01; n≥3).
    Figure Legend Snippet: ( A ) Immuno-fluorescence detection of beta-catenin in 2D monolayers of EMT6 or 4T1 cells treated with L-CM or Wnt3a-CM in the presence or absence of C4S or CS-E treatment. Wnt3a-CM led to a drastic increase in nuclear beta-catenin levels; concomitant treatment with CS-E, but not C4S, severely reduced nuclear beta-catenin expression levels (red arrowheads: cell membrane staining; red arrows: nuclear staining) (beta-catenin: green; DAPI: blue; scale bar = 50 micrometer). ( B, C ) Effect of CS-E on TOPFLASH reporter assays. Treatment with Wnt3a-CM for 24 hours caused a significant increase in TOPFLASH luciferase activity when compared to treatment with L-CM in both EMT6 ( B ) and 4T1 cells ( C ). Concomitant treatment with CS-E, but not C4S, significantly reduced Wnt3a-stimulated TOPFLASH reporter activity. ( D ) Western blot analysis of Wnt/beta-catenin signaling receptor activation. Treatment of EMT6 cells with Wnt3a-CM, but not L-CM for 1 hour led to phosphorylation of LRP6 (pLRP6). Concomitant treatment with CS-E (100 microgram/ml) interfered with phosphorylation of the LRP6 protein. Note that only the upper bands of the LRP6 are phosphorylated in response to Wnt3a (black arrows). Quantitation of these Western blots demonstrated that CS-E treatment significantly reduced LRP6 receptor phosphorylation by 60% in both EMT6 and 4T1 cells (*p<0.01; n≥3).

    Techniques Used: Fluorescence, Expressing, Staining, Luciferase, Activity Assay, Western Blot, Activation Assay, Quantitation Assay

    ( A ) RTqPCR to quantify Col1a1 mRNA expression in response to Wnt3a stimulation, in the presence or absence of CS-E. Treatment with Wnt3a-CM led to a 2-fold (EMT6) and 4-fold (4T1) increase in Col1a1 mRNA expression. Concomitant treatment with CS-E significantly interfered with this stimulatory effect of Wnt3a and reduced Col1a1 expression levels (*p<0.05). ( B ) Wnt/beta-catenin pathway inhibition by IWR-1. IWR-1 at both 5 microMolar and 15 microMolar completely inhibited Wnt3a-stimulated TOPFLASH activity to the level of control treated cells in EMT6 cells. In 4T1 cells, an IWR-1 concentration of 40 microM led to an almost complete loss of TOPFLASH activity. ( C ) RTqPCR analysis: inhibition of Wnt/beta-catenin signaling by IWR-1 (EMT6 cells: 5 microMolar; 4T1: 40 microMolar) led to a loss of Wnt3a-mediated induction of Col1a1 mRNA expression in EMT6 and 4T1 cells.
    Figure Legend Snippet: ( A ) RTqPCR to quantify Col1a1 mRNA expression in response to Wnt3a stimulation, in the presence or absence of CS-E. Treatment with Wnt3a-CM led to a 2-fold (EMT6) and 4-fold (4T1) increase in Col1a1 mRNA expression. Concomitant treatment with CS-E significantly interfered with this stimulatory effect of Wnt3a and reduced Col1a1 expression levels (*p<0.05). ( B ) Wnt/beta-catenin pathway inhibition by IWR-1. IWR-1 at both 5 microMolar and 15 microMolar completely inhibited Wnt3a-stimulated TOPFLASH activity to the level of control treated cells in EMT6 cells. In 4T1 cells, an IWR-1 concentration of 40 microM led to an almost complete loss of TOPFLASH activity. ( C ) RTqPCR analysis: inhibition of Wnt/beta-catenin signaling by IWR-1 (EMT6 cells: 5 microMolar; 4T1: 40 microMolar) led to a loss of Wnt3a-mediated induction of Col1a1 mRNA expression in EMT6 and 4T1 cells.

    Techniques Used: Expressing, Inhibition, Activity Assay, Concentration Assay

    l wnt3a cells  (ATCC)


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    ATCC l wnt3a cells
    Primer sequences used in qRT-PCR.
    L Wnt3a Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Xanthan Gum Ameliorates Osteoarthritis and Mitigates Cartilage Degradation via Regulation of the Wnt3a/β-Catenin Signaling Pathway"

    Article Title: Xanthan Gum Ameliorates Osteoarthritis and Mitigates Cartilage Degradation via Regulation of the Wnt3a/β-Catenin Signaling Pathway

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.916092

    Primer sequences used in qRT-PCR.
    Figure Legend Snippet: Primer sequences used in qRT-PCR.

    Techniques Used:

    Assessment of the protective effect of XG on SNP-stimulated chondrocytes and the suppressive effect of XG on Wnt/β-catenin signaling activity. ( A ) SD rat chondrocytes were treated with various concentrations of XG (0, 0.1, 1, 2, and 4 mg/mL) with or without SNP (1 mM, 24 h) followed by CCK8 assay analysis. n =6, *** p <0.001 compared with control group ; # p <0.05, ### p < 0.001 compared with the SNP-induced only group. ( B ) XG suppressed activation of the Wnt3a/β-catenin signaling pathway by 25% Wnt3a-CM in a dose-dependent manner. To examine the ability of XG to inhibit Wnt3a/β-catenin signaling, ATDC5 cells were treated with Wnt3a-CM (25%) and different concentrations of XG (0, 0.1, 1, 2 and 4 mg/mL) 24 h after transfection with the TOPFlash plasmid. FOPFlash was used as a negative control. Data represent the mean ±SD, n =3, ** p <0.01, compared with untreated group, # p <0.05, ## p <0.01, compared with Wnt3a-CM group.
    Figure Legend Snippet: Assessment of the protective effect of XG on SNP-stimulated chondrocytes and the suppressive effect of XG on Wnt/β-catenin signaling activity. ( A ) SD rat chondrocytes were treated with various concentrations of XG (0, 0.1, 1, 2, and 4 mg/mL) with or without SNP (1 mM, 24 h) followed by CCK8 assay analysis. n =6, *** p <0.001 compared with control group ; # p <0.05, ### p < 0.001 compared with the SNP-induced only group. ( B ) XG suppressed activation of the Wnt3a/β-catenin signaling pathway by 25% Wnt3a-CM in a dose-dependent manner. To examine the ability of XG to inhibit Wnt3a/β-catenin signaling, ATDC5 cells were treated with Wnt3a-CM (25%) and different concentrations of XG (0, 0.1, 1, 2 and 4 mg/mL) 24 h after transfection with the TOPFlash plasmid. FOPFlash was used as a negative control. Data represent the mean ±SD, n =3, ** p <0.01, compared with untreated group, # p <0.05, ## p <0.01, compared with Wnt3a-CM group.

    Techniques Used: Activity Assay, CCK-8 Assay, Activation Assay, Transfection, Plasmid Preparation, Negative Control

    XG regulates cartilage degradation by downregulating the Wnt/β catenin signaling pathway in vitro . ( A ) Following treatment with or without SNP (1 mM, 24 h) in the absence or presence of XG (1 mg/mL, 24 h). Representative blots of Wnt3a and β-catenin. ( B ) Quantification of Wnt3a and β-catenin protein expression levels. n =6, ** p <0.01, *** p <0.001 vs . control group, # p <0.05 vs . SNP-induced only group. ( C ) Quantification results obtained by qRT-PCR for Wnt3a and β-catenin mRNA expression levels in each group. n =6, *** p <0.001 vs . control group, # p <0.05 vs . SNP-induced only group.
    Figure Legend Snippet: XG regulates cartilage degradation by downregulating the Wnt/β catenin signaling pathway in vitro . ( A ) Following treatment with or without SNP (1 mM, 24 h) in the absence or presence of XG (1 mg/mL, 24 h). Representative blots of Wnt3a and β-catenin. ( B ) Quantification of Wnt3a and β-catenin protein expression levels. n =6, ** p <0.01, *** p <0.001 vs . control group, # p <0.05 vs . SNP-induced only group. ( C ) Quantification results obtained by qRT-PCR for Wnt3a and β-catenin mRNA expression levels in each group. n =6, *** p <0.001 vs . control group, # p <0.05 vs . SNP-induced only group.

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR

    XG affects the levels of the downstream proteins MMP13 and ADAMTS5 of the Wnt/β-catenin pathway. ( A ) Representative blots of MMP13 and ADAMTS5. ( B ) Quantified proteins expression levels of Wnt3a and β-catenin in SD rat chondrocytes incubated with or without SNP (1 mM, 24 h) with or without XG treatment. n =6, *** p <0.001 vs . control group, # p <0.05 vs . SNP-induced only group. ( C ) Quantification results obtained by qRT-PCR for MMP13 and ADAMTS5 mRNA expression levels in each group. n =6, *** p <0.001 vs . control group, # p <0.05 vs . SNP-induced only group.
    Figure Legend Snippet: XG affects the levels of the downstream proteins MMP13 and ADAMTS5 of the Wnt/β-catenin pathway. ( A ) Representative blots of MMP13 and ADAMTS5. ( B ) Quantified proteins expression levels of Wnt3a and β-catenin in SD rat chondrocytes incubated with or without SNP (1 mM, 24 h) with or without XG treatment. n =6, *** p <0.001 vs . control group, # p <0.05 vs . SNP-induced only group. ( C ) Quantification results obtained by qRT-PCR for MMP13 and ADAMTS5 mRNA expression levels in each group. n =6, *** p <0.001 vs . control group, # p <0.05 vs . SNP-induced only group.

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR

    wnt3a l  (ATCC)


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    ATCC wnt3a l
    NCI8642 is able to revert DKK1 inhibition of the Wnt pathway. (a) Luciferase activity detected in PC12-TCF-Luc cells stimulated with <t>Wnt3a</t> alone and in presence of increasing volumes of DKK1 CM. (b) Effect of NCI8642 on Wnt3a and DKK1 treated cells. Open square: basal signal; open triangle: cells treated with Wnt3a CM; open circle: cells treated with Wnt3a, DKK1 CM, and DMSO 1%; close symbols: cells treated with serial dilutions of NCI8642 (up to 50 μ M), either in the presence (close circles) or in the absence (close triangles) of DKK1 CM.
    Wnt3a L, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Functional Characterization of a Small-Molecule Inhibitor of the DKK1-LRP6 Interaction"

    Article Title: Functional Characterization of a Small-Molecule Inhibitor of the DKK1-LRP6 Interaction

    Journal: ISRN Molecular Biology

    doi: 10.5402/2012/823875

    NCI8642 is able to revert DKK1 inhibition of the Wnt pathway. (a) Luciferase activity detected in PC12-TCF-Luc cells stimulated with Wnt3a alone and in presence of increasing volumes of DKK1 CM. (b) Effect of NCI8642 on Wnt3a and DKK1 treated cells. Open square: basal signal; open triangle: cells treated with Wnt3a CM; open circle: cells treated with Wnt3a, DKK1 CM, and DMSO 1%; close symbols: cells treated with serial dilutions of NCI8642 (up to 50 μ M), either in the presence (close circles) or in the absence (close triangles) of DKK1 CM.
    Figure Legend Snippet: NCI8642 is able to revert DKK1 inhibition of the Wnt pathway. (a) Luciferase activity detected in PC12-TCF-Luc cells stimulated with Wnt3a alone and in presence of increasing volumes of DKK1 CM. (b) Effect of NCI8642 on Wnt3a and DKK1 treated cells. Open square: basal signal; open triangle: cells treated with Wnt3a CM; open circle: cells treated with Wnt3a, DKK1 CM, and DMSO 1%; close symbols: cells treated with serial dilutions of NCI8642 (up to 50 μ M), either in the presence (close circles) or in the absence (close triangles) of DKK1 CM.

    Techniques Used: Inhibition, Luciferase, Activity Assay

    DKK1 inhibits Wnt3a-mediated β -catenin translocation. (A) Immunostaining of β -catenin in L-cells following stimulation with Wnt3a CM, either without DKK1 or in the presence of increasing volumes of DKK1 CM. Panel (a), unstimulated cells; panel (b), Wnt3a-stimulated cells; panels (c–f), Wnt3a-stimulated cells in presence of DKK1 CM dilutions. Cell nuclei counterstaining with Hoechst 33342 dye are shown for each panel. (B) Histogram representation of the nuclear/cytoplasmic intensity ratios corresponding to the cells shown in (A).
    Figure Legend Snippet: DKK1 inhibits Wnt3a-mediated β -catenin translocation. (A) Immunostaining of β -catenin in L-cells following stimulation with Wnt3a CM, either without DKK1 or in the presence of increasing volumes of DKK1 CM. Panel (a), unstimulated cells; panel (b), Wnt3a-stimulated cells; panels (c–f), Wnt3a-stimulated cells in presence of DKK1 CM dilutions. Cell nuclei counterstaining with Hoechst 33342 dye are shown for each panel. (B) Histogram representation of the nuclear/cytoplasmic intensity ratios corresponding to the cells shown in (A).

    Techniques Used: Translocation Assay, Immunostaining

    NCI8642 is able to compete with DKK1 in β -catenin translocation assay. (A) Panel a: Wnt3a-stimulated cells; panel b: cells treated with Wnt3a and DKK1; panel c: cells treated with Wnt3a, DKK1, and 50 μ M NCI8642; panel d: cells treated with 50 μ M NCI8642 only. (B) Concentration-response curve of NCI8642 (from 1 to 50 μ M) expressed as % of activity. The activity was defined as the ratio of nuclear/cytoplasmic intensity normalized between Wnt3a + DMSO treatment (100% activity) and Wnt3a + DKK1 + DMSO treatment (0% activity).
    Figure Legend Snippet: NCI8642 is able to compete with DKK1 in β -catenin translocation assay. (A) Panel a: Wnt3a-stimulated cells; panel b: cells treated with Wnt3a and DKK1; panel c: cells treated with Wnt3a, DKK1, and 50 μ M NCI8642; panel d: cells treated with 50 μ M NCI8642 only. (B) Concentration-response curve of NCI8642 (from 1 to 50 μ M) expressed as % of activity. The activity was defined as the ratio of nuclear/cytoplasmic intensity normalized between Wnt3a + DMSO treatment (100% activity) and Wnt3a + DKK1 + DMSO treatment (0% activity).

    Techniques Used: Translocation Assay, Concentration Assay, Activity Assay

    l wnt3a cells  (ATCC)


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    ATCC l wnt3a cells
    mRNA expression of Wnt ligands 1–19, Fzd receptors 1–10, Wnt antagonists sFRP1-4, WIF1, Dkk1 and Dkk4
    L Wnt3a Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis"

    Article Title: Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis

    Journal: BMC Immunology

    doi: 10.1186/1471-2172-7-13

    mRNA expression of Wnt ligands 1–19, Fzd receptors 1–10, Wnt antagonists sFRP1-4, WIF1, Dkk1 and Dkk4
    Figure Legend Snippet: mRNA expression of Wnt ligands 1–19, Fzd receptors 1–10, Wnt antagonists sFRP1-4, WIF1, Dkk1 and Dkk4

    Techniques Used: Expressing

    Wnt3A induces β-catenin stabilization in BM B progenitor cells . Western blot analysis of β-catenin levels in BM CD10 + B lineage progenitor cells stimulated with Wnt3A (100 ng/ml) or vehicle (PBS with 0.1% detoxified BSA) for 3 hours. The blots were incubated with an Ab against β-catenin, followed by an Ab against β-actin to ascertain equal loading in the wells. The same results were found in cells from 4 out of 5 different donors, indicating some degree of donor variation in the response to Wnt3A.
    Figure Legend Snippet: Wnt3A induces β-catenin stabilization in BM B progenitor cells . Western blot analysis of β-catenin levels in BM CD10 + B lineage progenitor cells stimulated with Wnt3A (100 ng/ml) or vehicle (PBS with 0.1% detoxified BSA) for 3 hours. The blots were incubated with an Ab against β-catenin, followed by an Ab against β-actin to ascertain equal loading in the wells. The same results were found in cells from 4 out of 5 different donors, indicating some degree of donor variation in the response to Wnt3A.

    Techniques Used: Western Blot, Incubation

    Wnt3A inhibits in vitro B lymphopoiesis . BM CD133 + CD10 - HPC (A: assay 1) or CD10 + B progenitor cells (B: assay 2) were cocultured with a confluent layer of the murine stromal cell line MS-5 for 3 or 2 weeks, respectively, while treated with Wnt3A (100 ng/ml), Wnt3A + sFRP1 (2 μg/ml), Wnt3A + Dkk1 (500 ng/ml) or medium only. The number of resulting CD19 + B lineage cells in each sample was determined by quantitative flow cytometry. The percentage of CD34 + cells among the CD19 + cells were measured before and after culturing, with and without treatment with Wnt3A (C). The bars represent the mean of N experiments performed in duplicate, ± SEM. A) N = 6. B) Cells treated with control medium or Wnt3A: N = 11, Wnt3A + sFRP1: N = 3, Wnt3A + Dkk1: N = 2. C) day 0: N = 7, day 7: N = 3, Day 14: N = 8. *p ≤ 0.01, Wilcoxon Signed Ranks Test.
    Figure Legend Snippet: Wnt3A inhibits in vitro B lymphopoiesis . BM CD133 + CD10 - HPC (A: assay 1) or CD10 + B progenitor cells (B: assay 2) were cocultured with a confluent layer of the murine stromal cell line MS-5 for 3 or 2 weeks, respectively, while treated with Wnt3A (100 ng/ml), Wnt3A + sFRP1 (2 μg/ml), Wnt3A + Dkk1 (500 ng/ml) or medium only. The number of resulting CD19 + B lineage cells in each sample was determined by quantitative flow cytometry. The percentage of CD34 + cells among the CD19 + cells were measured before and after culturing, with and without treatment with Wnt3A (C). The bars represent the mean of N experiments performed in duplicate, ± SEM. A) N = 6. B) Cells treated with control medium or Wnt3A: N = 11, Wnt3A + sFRP1: N = 3, Wnt3A + Dkk1: N = 2. C) day 0: N = 7, day 7: N = 3, Day 14: N = 8. *p ≤ 0.01, Wilcoxon Signed Ranks Test.

    Techniques Used: In Vitro, Flow Cytometry

    Wnt3A inhibits the initial phase of stromal supported cell division of BM B progenitors . Highly purified BM CD10 + CFSE mean cells were grown on a confluent layer of MS-5 and treated with Wnt3A (25–400 ng/ml) or medium only. After three days, the cells were analyzed on a FACScan flow cytometer for the number of cell divisions of CD19 + cells. A) Tracking histograms of cell divisions of CFSE-labeled BM B progenitor cells in the presence or absence of Wnt3A (100 ng/ml) One representative experiment of six is shown. B) Dose dependent inhibition of cell division of CD34 + pro-B cells and CD34 - pre-B cells by Wnt3A (closed circles). The inhibitory effect of Wnt3A was blocked by Wnt antagonist sFRP1 (2 μg/ml) (open circle). Data are shown as percentage of cells that had gone through one or more cell divisions, as determined by cell division tracking with CFSE. One representative experiment of two is shown, except for Wnt3A (100 ng/ml) and Wnt3A + FRP1 (2 μg/ml) where one representative experiment of six is shown (*p < 0.05, Wilcoxon Signed Ranks Test, n = 6).
    Figure Legend Snippet: Wnt3A inhibits the initial phase of stromal supported cell division of BM B progenitors . Highly purified BM CD10 + CFSE mean cells were grown on a confluent layer of MS-5 and treated with Wnt3A (25–400 ng/ml) or medium only. After three days, the cells were analyzed on a FACScan flow cytometer for the number of cell divisions of CD19 + cells. A) Tracking histograms of cell divisions of CFSE-labeled BM B progenitor cells in the presence or absence of Wnt3A (100 ng/ml) One representative experiment of six is shown. B) Dose dependent inhibition of cell division of CD34 + pro-B cells and CD34 - pre-B cells by Wnt3A (closed circles). The inhibitory effect of Wnt3A was blocked by Wnt antagonist sFRP1 (2 μg/ml) (open circle). Data are shown as percentage of cells that had gone through one or more cell divisions, as determined by cell division tracking with CFSE. One representative experiment of two is shown, except for Wnt3A (100 ng/ml) and Wnt3A + FRP1 (2 μg/ml) where one representative experiment of six is shown (*p < 0.05, Wilcoxon Signed Ranks Test, n = 6).

    Techniques Used: Purification, Flow Cytometry, Labeling, Inhibition

    Primer sequences used for mRNA expression analyses of Wnt ligands, Fzd receptors and Wnt antagonists
    Figure Legend Snippet: Primer sequences used for mRNA expression analyses of Wnt ligands, Fzd receptors and Wnt antagonists

    Techniques Used: Expressing

    wnt3a cells  (ATCC)


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    Structured Review

    ATCC wnt3a cells
    ( A ) Pyrvinium inhibits TOPflash activation with an EC 50 of ∼10 nM in contrast to the structurally related compound VU-WS211. HEK 293 STF (TOPflash) luciferase reporter cells incubated with <t>Wnt3a-conditioned</t> media were treated as indicated. Graph represents mean ± SEM of TOPflash signal normalized to cell number (performed in quadruplicate). ( B ) Pyrvinium (100 nM) decreases transcript levels of endogenous Wnt target genes Myc, Dkk-1, and Axin2 as assessed by real-time PCR. GAPDH is control. ( C ) Pyrvinium binds CK1α in vitro . CK1α and GSK3 (0.5 µg each, in duplicates) were spotted on nitrocellulose, incubated with pyrvinium (10 nM), and bound pyrvinium detected based on its fluorescent property. ( D ) Pyrvinium stimulates CK1α activity. CK1α (100 nM) was incubated with recombinant casein (100 nM) plus or minus pyrvinium (10 nM) in a kinase reaction containing [γ 32 P]ATP followed by SDS-PAGE and exposure to PhosphoImager screen. ( E ) Pyrvinium decreases and increases intracellular β-catenin and Axin levels, respectively. HEK 293 cells were treated for 16 hours as indicated, and cytoplasmic preparations were immunoblotted for β-catenin and Axin. β-tubulin is loading control. ( F ) Pyrvinium decrease steady-state levels of Pygopus. HEK 293 STF cells expressing HA-tagged human Pygopus-2 were treated with pyrvinium as indicated. Lysates were immunoblotted for HA. β-galactosidase (β-gal) is transfection control.
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    1) Product Images from "Pyrvinium, a Potent Small Molecule Wnt Inhibitor, Promotes Wound Repair and Post-MI Cardiac Remodeling"

    Article Title: Pyrvinium, a Potent Small Molecule Wnt Inhibitor, Promotes Wound Repair and Post-MI Cardiac Remodeling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015521

    ( A ) Pyrvinium inhibits TOPflash activation with an EC 50 of ∼10 nM in contrast to the structurally related compound VU-WS211. HEK 293 STF (TOPflash) luciferase reporter cells incubated with Wnt3a-conditioned media were treated as indicated. Graph represents mean ± SEM of TOPflash signal normalized to cell number (performed in quadruplicate). ( B ) Pyrvinium (100 nM) decreases transcript levels of endogenous Wnt target genes Myc, Dkk-1, and Axin2 as assessed by real-time PCR. GAPDH is control. ( C ) Pyrvinium binds CK1α in vitro . CK1α and GSK3 (0.5 µg each, in duplicates) were spotted on nitrocellulose, incubated with pyrvinium (10 nM), and bound pyrvinium detected based on its fluorescent property. ( D ) Pyrvinium stimulates CK1α activity. CK1α (100 nM) was incubated with recombinant casein (100 nM) plus or minus pyrvinium (10 nM) in a kinase reaction containing [γ 32 P]ATP followed by SDS-PAGE and exposure to PhosphoImager screen. ( E ) Pyrvinium decreases and increases intracellular β-catenin and Axin levels, respectively. HEK 293 cells were treated for 16 hours as indicated, and cytoplasmic preparations were immunoblotted for β-catenin and Axin. β-tubulin is loading control. ( F ) Pyrvinium decrease steady-state levels of Pygopus. HEK 293 STF cells expressing HA-tagged human Pygopus-2 were treated with pyrvinium as indicated. Lysates were immunoblotted for HA. β-galactosidase (β-gal) is transfection control.
    Figure Legend Snippet: ( A ) Pyrvinium inhibits TOPflash activation with an EC 50 of ∼10 nM in contrast to the structurally related compound VU-WS211. HEK 293 STF (TOPflash) luciferase reporter cells incubated with Wnt3a-conditioned media were treated as indicated. Graph represents mean ± SEM of TOPflash signal normalized to cell number (performed in quadruplicate). ( B ) Pyrvinium (100 nM) decreases transcript levels of endogenous Wnt target genes Myc, Dkk-1, and Axin2 as assessed by real-time PCR. GAPDH is control. ( C ) Pyrvinium binds CK1α in vitro . CK1α and GSK3 (0.5 µg each, in duplicates) were spotted on nitrocellulose, incubated with pyrvinium (10 nM), and bound pyrvinium detected based on its fluorescent property. ( D ) Pyrvinium stimulates CK1α activity. CK1α (100 nM) was incubated with recombinant casein (100 nM) plus or minus pyrvinium (10 nM) in a kinase reaction containing [γ 32 P]ATP followed by SDS-PAGE and exposure to PhosphoImager screen. ( E ) Pyrvinium decreases and increases intracellular β-catenin and Axin levels, respectively. HEK 293 cells were treated for 16 hours as indicated, and cytoplasmic preparations were immunoblotted for β-catenin and Axin. β-tubulin is loading control. ( F ) Pyrvinium decrease steady-state levels of Pygopus. HEK 293 STF cells expressing HA-tagged human Pygopus-2 were treated with pyrvinium as indicated. Lysates were immunoblotted for HA. β-galactosidase (β-gal) is transfection control.

    Techniques Used: Activation Assay, Luciferase, Incubation, Real-time Polymerase Chain Reaction, In Vitro, Activity Assay, Recombinant, SDS Page, Expressing, Transfection

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    ATCC l wnt3a cells
    PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or <t>Wnt3a</t> for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.
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    ATCC l wnt3a crl 2647 cells
    PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or <t>Wnt3a</t> for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.
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    ATCC mouse l1 cells expressing wnt3a
    Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of <t>Wnt3a-CM</t> (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p < 0.05, ** p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.
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    ATCC l wnt3a
    ( A ) Immuno-fluorescence detection of beta-catenin in 2D monolayers of EMT6 or 4T1 cells treated with L-CM or <t>Wnt3a-CM</t> in the presence or absence of C4S or CS-E treatment. Wnt3a-CM led to a drastic increase in nuclear beta-catenin levels; concomitant treatment with CS-E, but not C4S, severely reduced nuclear beta-catenin expression levels (red arrowheads: cell membrane staining; red arrows: nuclear staining) (beta-catenin: green; DAPI: blue; scale bar = 50 micrometer). ( B, C ) Effect of CS-E on TOPFLASH reporter assays. Treatment with Wnt3a-CM for 24 hours caused a significant increase in TOPFLASH luciferase activity when compared to treatment with L-CM in both EMT6 ( B ) and 4T1 cells ( C ). Concomitant treatment with CS-E, but not C4S, significantly reduced Wnt3a-stimulated TOPFLASH reporter activity. ( D ) Western blot analysis of Wnt/beta-catenin signaling receptor activation. Treatment of EMT6 cells with Wnt3a-CM, but not L-CM for 1 hour led to phosphorylation of LRP6 (pLRP6). Concomitant treatment with CS-E (100 microgram/ml) interfered with phosphorylation of the LRP6 protein. Note that only the upper bands of the LRP6 are phosphorylated in response to Wnt3a (black arrows). Quantitation of these Western blots demonstrated that CS-E treatment significantly reduced LRP6 receptor phosphorylation by 60% in both EMT6 and 4T1 cells (*p<0.01; n≥3).
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    ATCC wnt3a l
    NCI8642 is able to revert DKK1 inhibition of the Wnt pathway. (a) Luciferase activity detected in PC12-TCF-Luc cells stimulated with <t>Wnt3a</t> alone and in presence of increasing volumes of DKK1 CM. (b) Effect of NCI8642 on Wnt3a and DKK1 treated cells. Open square: basal signal; open triangle: cells treated with Wnt3a CM; open circle: cells treated with Wnt3a, DKK1 CM, and DMSO 1%; close symbols: cells treated with serial dilutions of NCI8642 (up to 50 μ M), either in the presence (close circles) or in the absence (close triangles) of DKK1 CM.
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    ATCC wnt3a cells
    ( A ) Pyrvinium inhibits TOPflash activation with an EC 50 of ∼10 nM in contrast to the structurally related compound VU-WS211. HEK 293 STF (TOPflash) luciferase reporter cells incubated with <t>Wnt3a-conditioned</t> media were treated as indicated. Graph represents mean ± SEM of TOPflash signal normalized to cell number (performed in quadruplicate). ( B ) Pyrvinium (100 nM) decreases transcript levels of endogenous Wnt target genes Myc, Dkk-1, and Axin2 as assessed by real-time PCR. GAPDH is control. ( C ) Pyrvinium binds CK1α in vitro . CK1α and GSK3 (0.5 µg each, in duplicates) were spotted on nitrocellulose, incubated with pyrvinium (10 nM), and bound pyrvinium detected based on its fluorescent property. ( D ) Pyrvinium stimulates CK1α activity. CK1α (100 nM) was incubated with recombinant casein (100 nM) plus or minus pyrvinium (10 nM) in a kinase reaction containing [γ 32 P]ATP followed by SDS-PAGE and exposure to PhosphoImager screen. ( E ) Pyrvinium decreases and increases intracellular β-catenin and Axin levels, respectively. HEK 293 cells were treated for 16 hours as indicated, and cytoplasmic preparations were immunoblotted for β-catenin and Axin. β-tubulin is loading control. ( F ) Pyrvinium decrease steady-state levels of Pygopus. HEK 293 STF cells expressing HA-tagged human Pygopus-2 were treated with pyrvinium as indicated. Lysates were immunoblotted for HA. β-galactosidase (β-gal) is transfection control.
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    PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or Wnt3a for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.

    Journal: International Journal of Biological Sciences

    Article Title: Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/β-catenin Signaling

    doi: 10.7150/ijbs.67842

    Figure Lengend Snippet: PANK1 inhibits the Wnt/β-catenin signaling pathway. (A) The Topflash reporter was used to detect the effect of PANK1 on the transcriptional activity of β-catenin. After transfection, the cells were stimulated by lithium chloride or Wnt3a for 8h before the measurement of the reporter activity. (B) The effect of overexpression of PANK1 on the level of β-catenin protein in PVTT and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (C) The effect of knocked-down expression of PANK1 on the level of β-catenin protein in Huh7 and QGY-7701 cells was measured. After the cells were stimulated by Wnt3a for different periods of time, the β-catenin protein was measured by western blot. (D-E) The effect of overexpression of PANK1 in Huh7 and QGY-7701 cells on the half-life of β-catenin protein level was measured. After the cells were stimulated by CHX (50µg/ml) for different periods of time, the β-catenin protein was measured by western blot. The levels of β-catenin protein were quantified and analyze. (F) The effect of PANK1 overexpression in PVTT and QGY-7701 cells on the mRNA level of Axin2 was measured. After being stimulated by Wnt3a for different periods of time, the cells were collected to measure the mRNA level of Axin2. (G) The effect of knocked-down expression of PANK1 in Huh7 and QGY-7701 cells on the mRNA levels of Axin2 was measured. After the cells were stimulated by Wnt3a for different periods of time, they were collected to measure the mRNA level of Axin2. (H) RNA sequencing was performed on Huh7 cells with the knockdown of PANK1 expression, and on control cells, obtaining a volcano plot. (I) Differential expression of target genes of the Wnt/β-catenin signaling pathway from RNA sequencing, shown as a heat map. (J) As shown in the GEPIA database, PANK1 expression is negatively correlated with expression of Axin and SOX9, the target genes of Wnt/β-catenin signaling pathway. ****, P <0.0001.

    Article Snippet: The Wnt3a conditional medium was prepared from the L-Wnt3a cells according to the method from the ATCC.

    Techniques: Activity Assay, Transfection, Over Expression, Western Blot, Expressing, RNA Sequencing Assay

    PANK1 phosphorylates the N-terminal serine and threonine residues of β-catenin. (A) The effect of knockdown or overexpression of PANK1 in Huh7 and QGY-7701 cells on the phosphorylation level of β-catenin was measured. After the cells were stimulated by Wnt3a for different periods of time, the phosphorylation level of β-catenin was measured by western blot. (B) Ubiquitin assay was performed to measure the effect of PANK1 expression on the ubiquitination level of β-catenin. After being treated with MG132 for 10h, the PVTT cells with overexpressed PANK1, Huh7 cells, with down-regulated expression of PANK1, and their respective control cells were lysed for supernatant collection. The antibody against β-catenin was used for CO-IP. Then, a western blot assay was performed, and the antibody against ubiquitin was used for measurement. (C) The interaction between exogenously expressed CK1α (Myc-CK1α) and PANK1 (Flag-PANK1) was measured in 293T cells. The Myc-CK1α and Flag-PANK1 plasmids were transiently transfected into 293T cells. 48h later, CO-IP was performed with an antibody against Myc or the Flag tag. (D) The interaction between endogenous CK1α and PANK1 in Huh7 and QGY-7701 cells was measured. (E) The Wnt3a stimulation-dependent interaction between CK1α and PANK1 in QGY-7701 cells was measured. After treatment with Wnt3a for different periods of time, the QGY-7701 cells with the overexpression PANK1 were lysed and centrifuged for supernatant collection. The antibody against Flag was used for CO-IP. (F) The phosphorylation of β-catenin by PANK was measured through the in vitro kinase reaction. See “Materials and Methods” for the details on the reaction.

    Journal: International Journal of Biological Sciences

    Article Title: Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/β-catenin Signaling

    doi: 10.7150/ijbs.67842

    Figure Lengend Snippet: PANK1 phosphorylates the N-terminal serine and threonine residues of β-catenin. (A) The effect of knockdown or overexpression of PANK1 in Huh7 and QGY-7701 cells on the phosphorylation level of β-catenin was measured. After the cells were stimulated by Wnt3a for different periods of time, the phosphorylation level of β-catenin was measured by western blot. (B) Ubiquitin assay was performed to measure the effect of PANK1 expression on the ubiquitination level of β-catenin. After being treated with MG132 for 10h, the PVTT cells with overexpressed PANK1, Huh7 cells, with down-regulated expression of PANK1, and their respective control cells were lysed for supernatant collection. The antibody against β-catenin was used for CO-IP. Then, a western blot assay was performed, and the antibody against ubiquitin was used for measurement. (C) The interaction between exogenously expressed CK1α (Myc-CK1α) and PANK1 (Flag-PANK1) was measured in 293T cells. The Myc-CK1α and Flag-PANK1 plasmids were transiently transfected into 293T cells. 48h later, CO-IP was performed with an antibody against Myc or the Flag tag. (D) The interaction between endogenous CK1α and PANK1 in Huh7 and QGY-7701 cells was measured. (E) The Wnt3a stimulation-dependent interaction between CK1α and PANK1 in QGY-7701 cells was measured. After treatment with Wnt3a for different periods of time, the QGY-7701 cells with the overexpression PANK1 were lysed and centrifuged for supernatant collection. The antibody against Flag was used for CO-IP. (F) The phosphorylation of β-catenin by PANK was measured through the in vitro kinase reaction. See “Materials and Methods” for the details on the reaction.

    Article Snippet: The Wnt3a conditional medium was prepared from the L-Wnt3a cells according to the method from the ATCC.

    Techniques: Over Expression, Western Blot, Ubiquitin Assay, Expressing, Co-Immunoprecipitation Assay, Transfection, FLAG-tag, In Vitro

    Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of Wnt3a-CM (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p < 0.05, ** p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.

    Journal: BMC Medical Genetics

    Article Title: Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity

    doi: 10.1186/1471-2350-13-26

    Figure Lengend Snippet: Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of Wnt3a-CM (white bars) or L1 control medium (grey bars) . The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio between the luciferase and β-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM. * p < 0.05, ** p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.

    Article Snippet: Wnt3a-conditioned medium was produced and collected from mouse L1 cells expressing Wnt3a (L Wnt3a; ATCC CRL-2647) according to the manufacturer's instructions.

    Techniques: Luciferase, Activity Assay, Transfection, Mutagenesis

    A) Tph1 gene expression and B) 5- Htr1b gene expression in CHO cells transfected with wild type LRP5 (WT-LRP5) or mutant LRP5 and treated with Wnt3a-CM or L1-CM . Expression of the Tph1 and 5- Htr1b genes is shown as a fold change relative to the untreated control (pcDNA3.1+) and normalized to β-actin . The effects of the LRP5 mutants on the expression of these genes were examined by comparing the results for the mutants with those for WT-LRP5 using Student's t test. The statistical significance of each pair is denoted below the mutant columns; ** p < 0.01 as compared to WT-LRP5. Results are expressed as mean ± SD.

    Journal: BMC Medical Genetics

    Article Title: Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity

    doi: 10.1186/1471-2350-13-26

    Figure Lengend Snippet: A) Tph1 gene expression and B) 5- Htr1b gene expression in CHO cells transfected with wild type LRP5 (WT-LRP5) or mutant LRP5 and treated with Wnt3a-CM or L1-CM . Expression of the Tph1 and 5- Htr1b genes is shown as a fold change relative to the untreated control (pcDNA3.1+) and normalized to β-actin . The effects of the LRP5 mutants on the expression of these genes were examined by comparing the results for the mutants with those for WT-LRP5 using Student's t test. The statistical significance of each pair is denoted below the mutant columns; ** p < 0.01 as compared to WT-LRP5. Results are expressed as mean ± SD.

    Article Snippet: Wnt3a-conditioned medium was produced and collected from mouse L1 cells expressing Wnt3a (L Wnt3a; ATCC CRL-2647) according to the manufacturer's instructions.

    Techniques: Expressing, Transfection, Mutagenesis

    ( A ) Immuno-fluorescence detection of beta-catenin in 2D monolayers of EMT6 or 4T1 cells treated with L-CM or Wnt3a-CM in the presence or absence of C4S or CS-E treatment. Wnt3a-CM led to a drastic increase in nuclear beta-catenin levels; concomitant treatment with CS-E, but not C4S, severely reduced nuclear beta-catenin expression levels (red arrowheads: cell membrane staining; red arrows: nuclear staining) (beta-catenin: green; DAPI: blue; scale bar = 50 micrometer). ( B, C ) Effect of CS-E on TOPFLASH reporter assays. Treatment with Wnt3a-CM for 24 hours caused a significant increase in TOPFLASH luciferase activity when compared to treatment with L-CM in both EMT6 ( B ) and 4T1 cells ( C ). Concomitant treatment with CS-E, but not C4S, significantly reduced Wnt3a-stimulated TOPFLASH reporter activity. ( D ) Western blot analysis of Wnt/beta-catenin signaling receptor activation. Treatment of EMT6 cells with Wnt3a-CM, but not L-CM for 1 hour led to phosphorylation of LRP6 (pLRP6). Concomitant treatment with CS-E (100 microgram/ml) interfered with phosphorylation of the LRP6 protein. Note that only the upper bands of the LRP6 are phosphorylated in response to Wnt3a (black arrows). Quantitation of these Western blots demonstrated that CS-E treatment significantly reduced LRP6 receptor phosphorylation by 60% in both EMT6 and 4T1 cells (*p<0.01; n≥3).

    Journal: PLoS ONE

    Article Title: Chondroitin Sulfate-E Is a Negative Regulator of a Pro-Tumorigenic Wnt/Beta-Catenin-Collagen 1 Axis in Breast Cancer Cells

    doi: 10.1371/journal.pone.0103966

    Figure Lengend Snippet: ( A ) Immuno-fluorescence detection of beta-catenin in 2D monolayers of EMT6 or 4T1 cells treated with L-CM or Wnt3a-CM in the presence or absence of C4S or CS-E treatment. Wnt3a-CM led to a drastic increase in nuclear beta-catenin levels; concomitant treatment with CS-E, but not C4S, severely reduced nuclear beta-catenin expression levels (red arrowheads: cell membrane staining; red arrows: nuclear staining) (beta-catenin: green; DAPI: blue; scale bar = 50 micrometer). ( B, C ) Effect of CS-E on TOPFLASH reporter assays. Treatment with Wnt3a-CM for 24 hours caused a significant increase in TOPFLASH luciferase activity when compared to treatment with L-CM in both EMT6 ( B ) and 4T1 cells ( C ). Concomitant treatment with CS-E, but not C4S, significantly reduced Wnt3a-stimulated TOPFLASH reporter activity. ( D ) Western blot analysis of Wnt/beta-catenin signaling receptor activation. Treatment of EMT6 cells with Wnt3a-CM, but not L-CM for 1 hour led to phosphorylation of LRP6 (pLRP6). Concomitant treatment with CS-E (100 microgram/ml) interfered with phosphorylation of the LRP6 protein. Note that only the upper bands of the LRP6 are phosphorylated in response to Wnt3a (black arrows). Quantitation of these Western blots demonstrated that CS-E treatment significantly reduced LRP6 receptor phosphorylation by 60% in both EMT6 and 4T1 cells (*p<0.01; n≥3).

    Article Snippet: Mouse breast cancer cell lines EMT6 and 4T1, as well as L-cells and L-Wnt3a-cells were obtained from ATCC, USA.

    Techniques: Fluorescence, Expressing, Staining, Luciferase, Activity Assay, Western Blot, Activation Assay, Quantitation Assay

    ( A ) RTqPCR to quantify Col1a1 mRNA expression in response to Wnt3a stimulation, in the presence or absence of CS-E. Treatment with Wnt3a-CM led to a 2-fold (EMT6) and 4-fold (4T1) increase in Col1a1 mRNA expression. Concomitant treatment with CS-E significantly interfered with this stimulatory effect of Wnt3a and reduced Col1a1 expression levels (*p<0.05). ( B ) Wnt/beta-catenin pathway inhibition by IWR-1. IWR-1 at both 5 microMolar and 15 microMolar completely inhibited Wnt3a-stimulated TOPFLASH activity to the level of control treated cells in EMT6 cells. In 4T1 cells, an IWR-1 concentration of 40 microM led to an almost complete loss of TOPFLASH activity. ( C ) RTqPCR analysis: inhibition of Wnt/beta-catenin signaling by IWR-1 (EMT6 cells: 5 microMolar; 4T1: 40 microMolar) led to a loss of Wnt3a-mediated induction of Col1a1 mRNA expression in EMT6 and 4T1 cells.

    Journal: PLoS ONE

    Article Title: Chondroitin Sulfate-E Is a Negative Regulator of a Pro-Tumorigenic Wnt/Beta-Catenin-Collagen 1 Axis in Breast Cancer Cells

    doi: 10.1371/journal.pone.0103966

    Figure Lengend Snippet: ( A ) RTqPCR to quantify Col1a1 mRNA expression in response to Wnt3a stimulation, in the presence or absence of CS-E. Treatment with Wnt3a-CM led to a 2-fold (EMT6) and 4-fold (4T1) increase in Col1a1 mRNA expression. Concomitant treatment with CS-E significantly interfered with this stimulatory effect of Wnt3a and reduced Col1a1 expression levels (*p<0.05). ( B ) Wnt/beta-catenin pathway inhibition by IWR-1. IWR-1 at both 5 microMolar and 15 microMolar completely inhibited Wnt3a-stimulated TOPFLASH activity to the level of control treated cells in EMT6 cells. In 4T1 cells, an IWR-1 concentration of 40 microM led to an almost complete loss of TOPFLASH activity. ( C ) RTqPCR analysis: inhibition of Wnt/beta-catenin signaling by IWR-1 (EMT6 cells: 5 microMolar; 4T1: 40 microMolar) led to a loss of Wnt3a-mediated induction of Col1a1 mRNA expression in EMT6 and 4T1 cells.

    Article Snippet: Mouse breast cancer cell lines EMT6 and 4T1, as well as L-cells and L-Wnt3a-cells were obtained from ATCC, USA.

    Techniques: Expressing, Inhibition, Activity Assay, Concentration Assay

    NCI8642 is able to revert DKK1 inhibition of the Wnt pathway. (a) Luciferase activity detected in PC12-TCF-Luc cells stimulated with Wnt3a alone and in presence of increasing volumes of DKK1 CM. (b) Effect of NCI8642 on Wnt3a and DKK1 treated cells. Open square: basal signal; open triangle: cells treated with Wnt3a CM; open circle: cells treated with Wnt3a, DKK1 CM, and DMSO 1%; close symbols: cells treated with serial dilutions of NCI8642 (up to 50 μ M), either in the presence (close circles) or in the absence (close triangles) of DKK1 CM.

    Journal: ISRN Molecular Biology

    Article Title: Functional Characterization of a Small-Molecule Inhibitor of the DKK1-LRP6 Interaction

    doi: 10.5402/2012/823875

    Figure Lengend Snippet: NCI8642 is able to revert DKK1 inhibition of the Wnt pathway. (a) Luciferase activity detected in PC12-TCF-Luc cells stimulated with Wnt3a alone and in presence of increasing volumes of DKK1 CM. (b) Effect of NCI8642 on Wnt3a and DKK1 treated cells. Open square: basal signal; open triangle: cells treated with Wnt3a CM; open circle: cells treated with Wnt3a, DKK1 CM, and DMSO 1%; close symbols: cells treated with serial dilutions of NCI8642 (up to 50 μ M), either in the presence (close circles) or in the absence (close triangles) of DKK1 CM.

    Article Snippet: Wnt3a L-cells and parental L-cells were obtained from ATCC.

    Techniques: Inhibition, Luciferase, Activity Assay

    DKK1 inhibits Wnt3a-mediated β -catenin translocation. (A) Immunostaining of β -catenin in L-cells following stimulation with Wnt3a CM, either without DKK1 or in the presence of increasing volumes of DKK1 CM. Panel (a), unstimulated cells; panel (b), Wnt3a-stimulated cells; panels (c–f), Wnt3a-stimulated cells in presence of DKK1 CM dilutions. Cell nuclei counterstaining with Hoechst 33342 dye are shown for each panel. (B) Histogram representation of the nuclear/cytoplasmic intensity ratios corresponding to the cells shown in (A).

    Journal: ISRN Molecular Biology

    Article Title: Functional Characterization of a Small-Molecule Inhibitor of the DKK1-LRP6 Interaction

    doi: 10.5402/2012/823875

    Figure Lengend Snippet: DKK1 inhibits Wnt3a-mediated β -catenin translocation. (A) Immunostaining of β -catenin in L-cells following stimulation with Wnt3a CM, either without DKK1 or in the presence of increasing volumes of DKK1 CM. Panel (a), unstimulated cells; panel (b), Wnt3a-stimulated cells; panels (c–f), Wnt3a-stimulated cells in presence of DKK1 CM dilutions. Cell nuclei counterstaining with Hoechst 33342 dye are shown for each panel. (B) Histogram representation of the nuclear/cytoplasmic intensity ratios corresponding to the cells shown in (A).

    Article Snippet: Wnt3a L-cells and parental L-cells were obtained from ATCC.

    Techniques: Translocation Assay, Immunostaining

    NCI8642 is able to compete with DKK1 in β -catenin translocation assay. (A) Panel a: Wnt3a-stimulated cells; panel b: cells treated with Wnt3a and DKK1; panel c: cells treated with Wnt3a, DKK1, and 50 μ M NCI8642; panel d: cells treated with 50 μ M NCI8642 only. (B) Concentration-response curve of NCI8642 (from 1 to 50 μ M) expressed as % of activity. The activity was defined as the ratio of nuclear/cytoplasmic intensity normalized between Wnt3a + DMSO treatment (100% activity) and Wnt3a + DKK1 + DMSO treatment (0% activity).

    Journal: ISRN Molecular Biology

    Article Title: Functional Characterization of a Small-Molecule Inhibitor of the DKK1-LRP6 Interaction

    doi: 10.5402/2012/823875

    Figure Lengend Snippet: NCI8642 is able to compete with DKK1 in β -catenin translocation assay. (A) Panel a: Wnt3a-stimulated cells; panel b: cells treated with Wnt3a and DKK1; panel c: cells treated with Wnt3a, DKK1, and 50 μ M NCI8642; panel d: cells treated with 50 μ M NCI8642 only. (B) Concentration-response curve of NCI8642 (from 1 to 50 μ M) expressed as % of activity. The activity was defined as the ratio of nuclear/cytoplasmic intensity normalized between Wnt3a + DMSO treatment (100% activity) and Wnt3a + DKK1 + DMSO treatment (0% activity).

    Article Snippet: Wnt3a L-cells and parental L-cells were obtained from ATCC.

    Techniques: Translocation Assay, Concentration Assay, Activity Assay

    ( A ) Pyrvinium inhibits TOPflash activation with an EC 50 of ∼10 nM in contrast to the structurally related compound VU-WS211. HEK 293 STF (TOPflash) luciferase reporter cells incubated with Wnt3a-conditioned media were treated as indicated. Graph represents mean ± SEM of TOPflash signal normalized to cell number (performed in quadruplicate). ( B ) Pyrvinium (100 nM) decreases transcript levels of endogenous Wnt target genes Myc, Dkk-1, and Axin2 as assessed by real-time PCR. GAPDH is control. ( C ) Pyrvinium binds CK1α in vitro . CK1α and GSK3 (0.5 µg each, in duplicates) were spotted on nitrocellulose, incubated with pyrvinium (10 nM), and bound pyrvinium detected based on its fluorescent property. ( D ) Pyrvinium stimulates CK1α activity. CK1α (100 nM) was incubated with recombinant casein (100 nM) plus or minus pyrvinium (10 nM) in a kinase reaction containing [γ 32 P]ATP followed by SDS-PAGE and exposure to PhosphoImager screen. ( E ) Pyrvinium decreases and increases intracellular β-catenin and Axin levels, respectively. HEK 293 cells were treated for 16 hours as indicated, and cytoplasmic preparations were immunoblotted for β-catenin and Axin. β-tubulin is loading control. ( F ) Pyrvinium decrease steady-state levels of Pygopus. HEK 293 STF cells expressing HA-tagged human Pygopus-2 were treated with pyrvinium as indicated. Lysates were immunoblotted for HA. β-galactosidase (β-gal) is transfection control.

    Journal: PLoS ONE

    Article Title: Pyrvinium, a Potent Small Molecule Wnt Inhibitor, Promotes Wound Repair and Post-MI Cardiac Remodeling

    doi: 10.1371/journal.pone.0015521

    Figure Lengend Snippet: ( A ) Pyrvinium inhibits TOPflash activation with an EC 50 of ∼10 nM in contrast to the structurally related compound VU-WS211. HEK 293 STF (TOPflash) luciferase reporter cells incubated with Wnt3a-conditioned media were treated as indicated. Graph represents mean ± SEM of TOPflash signal normalized to cell number (performed in quadruplicate). ( B ) Pyrvinium (100 nM) decreases transcript levels of endogenous Wnt target genes Myc, Dkk-1, and Axin2 as assessed by real-time PCR. GAPDH is control. ( C ) Pyrvinium binds CK1α in vitro . CK1α and GSK3 (0.5 µg each, in duplicates) were spotted on nitrocellulose, incubated with pyrvinium (10 nM), and bound pyrvinium detected based on its fluorescent property. ( D ) Pyrvinium stimulates CK1α activity. CK1α (100 nM) was incubated with recombinant casein (100 nM) plus or minus pyrvinium (10 nM) in a kinase reaction containing [γ 32 P]ATP followed by SDS-PAGE and exposure to PhosphoImager screen. ( E ) Pyrvinium decreases and increases intracellular β-catenin and Axin levels, respectively. HEK 293 cells were treated for 16 hours as indicated, and cytoplasmic preparations were immunoblotted for β-catenin and Axin. β-tubulin is loading control. ( F ) Pyrvinium decrease steady-state levels of Pygopus. HEK 293 STF cells expressing HA-tagged human Pygopus-2 were treated with pyrvinium as indicated. Lysates were immunoblotted for HA. β-galactosidase (β-gal) is transfection control.

    Article Snippet: Wnt3a cells were purchased from ATCC.

    Techniques: Activation Assay, Luciferase, Incubation, Real-time Polymerase Chain Reaction, In Vitro, Activity Assay, Recombinant, SDS Page, Expressing, Transfection