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  • wnt3a  (ATCC)
    99
    ATCC wnt3a
    Krm2 abolished Dkk3 potentiation of <t>Wnt3a-mediated</t> signaling. (A) HEK293 cells stably expressing Dkk3 (black bars) or vector control (white bars) were transfected with GFP, Krm1, or Krm2 expression plasmids and the cells were treated with Wnt3a(+) or
    Wnt3a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc wnt 3a
    NKP608 inhibited <t>Wnt/β-catenin</t> signaling pathway in colorectal cancer cells. a Western blot revealed that NKP608 resulted in an inhibitory action of Wnt relative proteins and proteins relevant cell growth including β-catenin, <t>Wnt-3a,</t> E-Cadherin, Cyclin D1 and VEGF. b Quantitative expression levels of proteins are shown. Values are expressed as the mean ± SD (n = 3). * p
    Wnt 3a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wnt 3a/product/Cell Signaling Technology Inc
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    92
    Novus Biologicals wnt3a
    Effects of mechanical vibration stimulation on in vitro osteogenesis-related gene expression for primary rabbit osteoblasts seeded in pTi via qRT-PCR analyses, including ALP, OCN, Runx2, BMP2, OPG, <t>Wnt3a,</t> Lrp6 and β-catenin. Values are all expressed as mean ± S.D. ( n = 4), and the relative expression level of each gene was normalized to β-Actin. * Significant difference from the Control group with P
    Wnt3a, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wnt3a/product/Novus Biologicals
    Average 92 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Krm2 abolished Dkk3 potentiation of Wnt3a-mediated signaling. (A) HEK293 cells stably expressing Dkk3 (black bars) or vector control (white bars) were transfected with GFP, Krm1, or Krm2 expression plasmids and the cells were treated with Wnt3a(+) or

    Journal: Growth factors (Chur, Switzerland)

    Article Title: Analysis of Dickkopf3 interactions with Wnt signaling receptors

    doi: 10.3109/08977191003738832

    Figure Lengend Snippet: Krm2 abolished Dkk3 potentiation of Wnt3a-mediated signaling. (A) HEK293 cells stably expressing Dkk3 (black bars) or vector control (white bars) were transfected with GFP, Krm1, or Krm2 expression plasmids and the cells were treated with Wnt3a(+) or

    Article Snippet: However, when Krm1 was present, there was no potentiation of Wnt signaling by Dkk3 in the presence of Wnt3a (no significant difference between the seventh and eighth columns).

    Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection

    Dkk3 does not interact with LRP6. SH-SY5Y cells were transfected with either GFP, FLAG-Dkk1, or FLAG-Dkk3 and the LRP6 expression vector. Cells were treated with either Wnt3a-containing media or control media 48 h after transfection. Cells and media were

    Journal: Growth factors (Chur, Switzerland)

    Article Title: Analysis of Dickkopf3 interactions with Wnt signaling receptors

    doi: 10.3109/08977191003738832

    Figure Lengend Snippet: Dkk3 does not interact with LRP6. SH-SY5Y cells were transfected with either GFP, FLAG-Dkk1, or FLAG-Dkk3 and the LRP6 expression vector. Cells were treated with either Wnt3a-containing media or control media 48 h after transfection. Cells and media were

    Article Snippet: However, when Krm1 was present, there was no potentiation of Wnt signaling by Dkk3 in the presence of Wnt3a (no significant difference between the seventh and eighth columns).

    Techniques: Transfection, Expressing, Plasmid Preparation

    Morphological analysis of the impact of Wnt3a on BCG-infected RAW264.7 cells necrosis. RAW264.7 cells were exposed to Wnt3a-CM or control-CM, followed by infection of BCG at MOI of 10 for 24 h prior to be employed for EM analysis. (A) Representative images of TEM (top panel) and SEM (bottom panel) of healthy RAM264.7 cells (left panel) and necrotic cells (right panel). (B) Quantitative analysis of cells with a necrotic phenotype as determined by morphology using EM images. Control-CM treated cells compared to its corresponding Wnt3a-CM treated cells, #: p

    Journal: BMC Immunology

    Article Title: Wnt/β-Catenin signaling reduces Bacillus Calmette-Guerin-induced macrophage necrosis through a ROS -mediated PARP/AIF-dependent pathway

    doi: 10.1186/s12865-015-0080-5

    Figure Lengend Snippet: Morphological analysis of the impact of Wnt3a on BCG-infected RAW264.7 cells necrosis. RAW264.7 cells were exposed to Wnt3a-CM or control-CM, followed by infection of BCG at MOI of 10 for 24 h prior to be employed for EM analysis. (A) Representative images of TEM (top panel) and SEM (bottom panel) of healthy RAM264.7 cells (left panel) and necrotic cells (right panel). (B) Quantitative analysis of cells with a necrotic phenotype as determined by morphology using EM images. Control-CM treated cells compared to its corresponding Wnt3a-CM treated cells, #: p

    Article Snippet: Cell lines and Wnt3a conditioned medium Murine macrophage RAW264.7 cell line was purchased from shanghai Institute of Biochemistry and Cell Biology (Shanghai, China); the Wnt3a producing cell line, L Wnt3a (overexpressing mouse Wnt3a, ATCC #CRL-2647) and its control L cell line (ATCC #ATCC #CRL-2648) were purchased from American Type Culture Collection (ATCC) (Masassas, VA, USA).

    Techniques: Infection, Transmission Electron Microscopy

    Impacts of BCG and/or Wnt/β-catenin signaling on ROS production in RAW264.7 cells. (A) RAW264.7 cells were infected with BCG at MOI of 10 for indicated time, then they were used for intracellular ROS measurement by a flow cytometry assay. A time-dependent ROS production was observed within 6 h. (B) RAW264.7 cells were infected with indicated doses of BCG for 6 h prior to be used for examination of intracellular ROS by a flow cytometry assay. A BCG dose-dependent ROS production was observed. (C) Impact of Wnt/β-catenin signaling on BCG-induced ROS production in RAW264.7 cells. RAW264.7 cells were infected with BCG at MOI of 10 for 6 h prior to be used for measuring intracellular ROS by a flow cytometry assay. An activation of Wnt/β-catenin signaling exhibited an ability to reduce BCG-induced ROS production. (D) Impact of Wnt/β-catenin signaling on oxidative stress in RAW264.7 cells. RAW264.7 cells were exposed to 500 μmol/L of H 2 O 2 for 6 h before they were harvested for intracellular ROS measurement. The addition of Wnt3a showed a capacity to scavenge oxidative stressed-ROS accumulation.

    Journal: BMC Immunology

    Article Title: Wnt/β-Catenin signaling reduces Bacillus Calmette-Guerin-induced macrophage necrosis through a ROS -mediated PARP/AIF-dependent pathway

    doi: 10.1186/s12865-015-0080-5

    Figure Lengend Snippet: Impacts of BCG and/or Wnt/β-catenin signaling on ROS production in RAW264.7 cells. (A) RAW264.7 cells were infected with BCG at MOI of 10 for indicated time, then they were used for intracellular ROS measurement by a flow cytometry assay. A time-dependent ROS production was observed within 6 h. (B) RAW264.7 cells were infected with indicated doses of BCG for 6 h prior to be used for examination of intracellular ROS by a flow cytometry assay. A BCG dose-dependent ROS production was observed. (C) Impact of Wnt/β-catenin signaling on BCG-induced ROS production in RAW264.7 cells. RAW264.7 cells were infected with BCG at MOI of 10 for 6 h prior to be used for measuring intracellular ROS by a flow cytometry assay. An activation of Wnt/β-catenin signaling exhibited an ability to reduce BCG-induced ROS production. (D) Impact of Wnt/β-catenin signaling on oxidative stress in RAW264.7 cells. RAW264.7 cells were exposed to 500 μmol/L of H 2 O 2 for 6 h before they were harvested for intracellular ROS measurement. The addition of Wnt3a showed a capacity to scavenge oxidative stressed-ROS accumulation.

    Article Snippet: Cell lines and Wnt3a conditioned medium Murine macrophage RAW264.7 cell line was purchased from shanghai Institute of Biochemistry and Cell Biology (Shanghai, China); the Wnt3a producing cell line, L Wnt3a (overexpressing mouse Wnt3a, ATCC #CRL-2647) and its control L cell line (ATCC #ATCC #CRL-2648) were purchased from American Type Culture Collection (ATCC) (Masassas, VA, USA).

    Techniques: Infection, Flow Cytometry, Cytometry, Activation Assay

    Activation of Wnt/β-catenin signaling increases GSH concentration in RAW264.7 cells. RAW264.7 cells were treated with BCG, Wnt3a, DKK1, H 2 O 2 or their combination as indicated, they were then used for determination of intracellular GSH levels by an ELISA for GSH. (A) Impact of Wnt/β-catenin signaling on GSH production of RAW264.7 cells infected with BCG at MOI of 10 for 6 h. An activation of Wnt signaling by addition of Wnt3a-CM could induce GSH generation; in contrast, overexpression of Wnt inhibitor DKK1 reduced GSH levels. (B) Impact of Wnt/β-catenin signaling on GSH production of RAW264.7 cells exposed to oxidative stress H 2 O 2 (500 μmol/L) or LPS (100 ng/mL) stimulation for 6 h. The Wnt signaling could increase intracellular reduced GSH concentration in cells stressed by H 2 O 2 and LPS. Compared to a control-CM treated cells, *: p

    Journal: BMC Immunology

    Article Title: Wnt/β-Catenin signaling reduces Bacillus Calmette-Guerin-induced macrophage necrosis through a ROS -mediated PARP/AIF-dependent pathway

    doi: 10.1186/s12865-015-0080-5

    Figure Lengend Snippet: Activation of Wnt/β-catenin signaling increases GSH concentration in RAW264.7 cells. RAW264.7 cells were treated with BCG, Wnt3a, DKK1, H 2 O 2 or their combination as indicated, they were then used for determination of intracellular GSH levels by an ELISA for GSH. (A) Impact of Wnt/β-catenin signaling on GSH production of RAW264.7 cells infected with BCG at MOI of 10 for 6 h. An activation of Wnt signaling by addition of Wnt3a-CM could induce GSH generation; in contrast, overexpression of Wnt inhibitor DKK1 reduced GSH levels. (B) Impact of Wnt/β-catenin signaling on GSH production of RAW264.7 cells exposed to oxidative stress H 2 O 2 (500 μmol/L) or LPS (100 ng/mL) stimulation for 6 h. The Wnt signaling could increase intracellular reduced GSH concentration in cells stressed by H 2 O 2 and LPS. Compared to a control-CM treated cells, *: p

    Article Snippet: Cell lines and Wnt3a conditioned medium Murine macrophage RAW264.7 cell line was purchased from shanghai Institute of Biochemistry and Cell Biology (Shanghai, China); the Wnt3a producing cell line, L Wnt3a (overexpressing mouse Wnt3a, ATCC #CRL-2647) and its control L cell line (ATCC #ATCC #CRL-2648) were purchased from American Type Culture Collection (ATCC) (Masassas, VA, USA).

    Techniques: Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, Over Expression

    PARP-1/AIF pathway is involved in BCG-induced RAW264.7 cell necrosis. RAW264.7 cells were treated with indicated conditions for 24 h, the cells were then collected for immunobotting analysis or flow cytometric analysis. (A) Immunoblotting analysis for PARP-1 and its downstream mediator AIF proteins in RAW264.7 cells treated with indicated conditions for 24 h. Both Wnt3a-CM and PARP-1 inhibitor 3-AB displayed an ability to down-regulate PARP-1 and AIF. (B) NAD + analysis for NAD + in RAW264.7 cells treated with indicated conditions for 12 h. Wnt3a could inhibit the NAD + depletion of RAW264.7 cells infected with BCG. (C) Flow cytometric analysis showed a time-dependent inhibition of BCG-induced necrosis mediated by Wnt3a or PARP-1 inhibitor 3-AB (2.5 mmol/L), suggesting the PARP-1 was involved in BCG-induced necrosis for RAW264.7 cells, and the Wnt signaling could inhibit RAW264.7 cell necrosis by down-regulation of PARP-1 activity. (D) Representatives of scatter dot plot from three independent experiments of flow cytometric analysis of the necrotic fraction of RAW264.7 cells treated with indicated conditions for 6 h. Compared to a control-CM treated cells * or #: p

    Journal: BMC Immunology

    Article Title: Wnt/β-Catenin signaling reduces Bacillus Calmette-Guerin-induced macrophage necrosis through a ROS -mediated PARP/AIF-dependent pathway

    doi: 10.1186/s12865-015-0080-5

    Figure Lengend Snippet: PARP-1/AIF pathway is involved in BCG-induced RAW264.7 cell necrosis. RAW264.7 cells were treated with indicated conditions for 24 h, the cells were then collected for immunobotting analysis or flow cytometric analysis. (A) Immunoblotting analysis for PARP-1 and its downstream mediator AIF proteins in RAW264.7 cells treated with indicated conditions for 24 h. Both Wnt3a-CM and PARP-1 inhibitor 3-AB displayed an ability to down-regulate PARP-1 and AIF. (B) NAD + analysis for NAD + in RAW264.7 cells treated with indicated conditions for 12 h. Wnt3a could inhibit the NAD + depletion of RAW264.7 cells infected with BCG. (C) Flow cytometric analysis showed a time-dependent inhibition of BCG-induced necrosis mediated by Wnt3a or PARP-1 inhibitor 3-AB (2.5 mmol/L), suggesting the PARP-1 was involved in BCG-induced necrosis for RAW264.7 cells, and the Wnt signaling could inhibit RAW264.7 cell necrosis by down-regulation of PARP-1 activity. (D) Representatives of scatter dot plot from three independent experiments of flow cytometric analysis of the necrotic fraction of RAW264.7 cells treated with indicated conditions for 6 h. Compared to a control-CM treated cells * or #: p

    Article Snippet: Cell lines and Wnt3a conditioned medium Murine macrophage RAW264.7 cell line was purchased from shanghai Institute of Biochemistry and Cell Biology (Shanghai, China); the Wnt3a producing cell line, L Wnt3a (overexpressing mouse Wnt3a, ATCC #CRL-2647) and its control L cell line (ATCC #ATCC #CRL-2648) were purchased from American Type Culture Collection (ATCC) (Masassas, VA, USA).

    Techniques: Flow Cytometry, Infection, Inhibition, Activity Assay

    D2R modulates β-catenin phosphorylation and TCF/LEF activity. ( a ) siRNA knockdown of D2R (D2R siRNA) in mouse renal proximal tubule cells (mRPTCs) caused a 20% decrease in phosphorylated β-catenin (P-β-catenin, left panel) compared with the non-silencing (NS) siRNA control (72 hr). Acute sulpiride (D2R antagonist) treatment (1 µM, 6 hr) similarly reduced β-catenin phosphorylation by 25% compared with the vehicle control. Conversely, D2R agonism by quinpirole (1 µM, 24 hr) increased β-catenin phosphorylation, relative to the vehicle control (right panel). Results are represented as the ratio of P-β-catenin/total β-catenin, and then normalized as the percentage of NS siRNA or vehicle (% of control). (b) β-catenin-mediated TCF/LEF luciferase reporter activity was significantly increased by 40% via siRNA-mediated D2R knockdown and decreased by 36% via 1 µM quinpirole treatment to stimulate D2R activity; results are normalized to the respective controls. (c) Acute sulpiride treatment significantly increased TCF/LEF promoter activity by 80%, while direct stimulation by Wnt3a (100 ng/ml, 2 hr) more than doubled (224%) TCF/LEF promoter activity, relative to vehicle control. All data are represented as the mean ± SEM conducted on n ≥ 3 separate experimental dates and performed in triplicate. *P

    Journal: Scientific Reports

    Article Title: Dopamine D2 receptor modulates Wnt expression and control of cell proliferation

    doi: 10.1038/s41598-019-52528-4

    Figure Lengend Snippet: D2R modulates β-catenin phosphorylation and TCF/LEF activity. ( a ) siRNA knockdown of D2R (D2R siRNA) in mouse renal proximal tubule cells (mRPTCs) caused a 20% decrease in phosphorylated β-catenin (P-β-catenin, left panel) compared with the non-silencing (NS) siRNA control (72 hr). Acute sulpiride (D2R antagonist) treatment (1 µM, 6 hr) similarly reduced β-catenin phosphorylation by 25% compared with the vehicle control. Conversely, D2R agonism by quinpirole (1 µM, 24 hr) increased β-catenin phosphorylation, relative to the vehicle control (right panel). Results are represented as the ratio of P-β-catenin/total β-catenin, and then normalized as the percentage of NS siRNA or vehicle (% of control). (b) β-catenin-mediated TCF/LEF luciferase reporter activity was significantly increased by 40% via siRNA-mediated D2R knockdown and decreased by 36% via 1 µM quinpirole treatment to stimulate D2R activity; results are normalized to the respective controls. (c) Acute sulpiride treatment significantly increased TCF/LEF promoter activity by 80%, while direct stimulation by Wnt3a (100 ng/ml, 2 hr) more than doubled (224%) TCF/LEF promoter activity, relative to vehicle control. All data are represented as the mean ± SEM conducted on n ≥ 3 separate experimental dates and performed in triplicate. *P

    Article Snippet: Preparation of Wnt3a-conditioned and L-cell control medium Mouse fibroblast-derived L-Wnt3A cells (CRL-2647; ATCC, Manassas, VA) that stably express and secrete Wnt3a and the control untransfected parental L-Cell line (CRL-2648, ATCC) were purchased from ATCC (Manassas, VA).

    Techniques: Activity Assay, Luciferase

    Model of cross-talk between D2R and Wnt/β-catenin pathways, and Wnt3a transcriptional regulation. We propose the following mechanism by which D2R and Wnt3a reciprocally regulate their expression: (a) Wnt3a stimulation of the Frizzled (FZD) receptor/LRP5/6 complex leads to less β-catenin degradation via the destruction complex and proteasomal pathway. Instead, more β-catenin is available for translocation into the nucleus where it increases Wnt3a transcription and expression via a TCF/LEF site within the Wnt3a promoter. This leads to a positive feedback loop that amplifies further Wnt3a signaling. (b) Increased nuclear translocation of β-catenin also upregulates D2R expression through TCF/LEF sites within the D2R promoter. Increased D2R expression leads to more β-catenin phosphorylation, resulting in enhanced β-catenin proteasomal degradation and therefore less β-catenin nuclear translocation. This ultimately inhibits further β-catenin-mediated Wnt3a transcription to maintain cellular homeostasis.

    Journal: Scientific Reports

    Article Title: Dopamine D2 receptor modulates Wnt expression and control of cell proliferation

    doi: 10.1038/s41598-019-52528-4

    Figure Lengend Snippet: Model of cross-talk between D2R and Wnt/β-catenin pathways, and Wnt3a transcriptional regulation. We propose the following mechanism by which D2R and Wnt3a reciprocally regulate their expression: (a) Wnt3a stimulation of the Frizzled (FZD) receptor/LRP5/6 complex leads to less β-catenin degradation via the destruction complex and proteasomal pathway. Instead, more β-catenin is available for translocation into the nucleus where it increases Wnt3a transcription and expression via a TCF/LEF site within the Wnt3a promoter. This leads to a positive feedback loop that amplifies further Wnt3a signaling. (b) Increased nuclear translocation of β-catenin also upregulates D2R expression through TCF/LEF sites within the D2R promoter. Increased D2R expression leads to more β-catenin phosphorylation, resulting in enhanced β-catenin proteasomal degradation and therefore less β-catenin nuclear translocation. This ultimately inhibits further β-catenin-mediated Wnt3a transcription to maintain cellular homeostasis.

    Article Snippet: Preparation of Wnt3a-conditioned and L-cell control medium Mouse fibroblast-derived L-Wnt3A cells (CRL-2647; ATCC, Manassas, VA) that stably express and secrete Wnt3a and the control untransfected parental L-Cell line (CRL-2648, ATCC) were purchased from ATCC (Manassas, VA).

    Techniques: Expressing, Translocation Assay

    Wnt3a expression and cell proliferation increase in an ischemia/reperfusion-injury disease model. ( a ) Ischemia/reperfusion-injury (I/R) caused a 200% increase in Wnt3a mRNA levels (left panel) and 50% increase in Wnt3a protein levels (middle panel) in mouse renal cortex. There was an accompanying 55% increase in expression of the Ki-67 cell proliferation marker compared with the control (right panel). Alongside is the schedule for the I/R treatment. ( b ) Representative confocal immunofluorescence images of mouse renal cortex following I/R demonstrated increased nuclear Ki-67 (red), nuclear β-catenin (green), and increased nuclear co-localization of Ki-67 and β-catenin with DAPI (blue). Merged images: blue = DAPI, red = Ki-67, green = β-catenin, overlap = white); arrowheads indicate nuclear overlap among Ki-67, β-catenin, and DAPI signals. Scale bar = 50 µm. All data are represented as the mean ± SEM using 4 animals per group. All measurements were performed in triplicate. *P

    Journal: Scientific Reports

    Article Title: Dopamine D2 receptor modulates Wnt expression and control of cell proliferation

    doi: 10.1038/s41598-019-52528-4

    Figure Lengend Snippet: Wnt3a expression and cell proliferation increase in an ischemia/reperfusion-injury disease model. ( a ) Ischemia/reperfusion-injury (I/R) caused a 200% increase in Wnt3a mRNA levels (left panel) and 50% increase in Wnt3a protein levels (middle panel) in mouse renal cortex. There was an accompanying 55% increase in expression of the Ki-67 cell proliferation marker compared with the control (right panel). Alongside is the schedule for the I/R treatment. ( b ) Representative confocal immunofluorescence images of mouse renal cortex following I/R demonstrated increased nuclear Ki-67 (red), nuclear β-catenin (green), and increased nuclear co-localization of Ki-67 and β-catenin with DAPI (blue). Merged images: blue = DAPI, red = Ki-67, green = β-catenin, overlap = white); arrowheads indicate nuclear overlap among Ki-67, β-catenin, and DAPI signals. Scale bar = 50 µm. All data are represented as the mean ± SEM using 4 animals per group. All measurements were performed in triplicate. *P

    Article Snippet: Preparation of Wnt3a-conditioned and L-cell control medium Mouse fibroblast-derived L-Wnt3A cells (CRL-2647; ATCC, Manassas, VA) that stably express and secrete Wnt3a and the control untransfected parental L-Cell line (CRL-2648, ATCC) were purchased from ATCC (Manassas, VA).

    Techniques: Expressing, Marker, Immunofluorescence

    D2R overexpression partially reverses increased Wnt3a expression and cell proliferation in response to ischemia/reperfusion injury. ( a ) AAV-mediated D2R overexpression (D2R AAV) in mouse kidney prior to reperfusion caused a 70% decrease in Wnt3a mRNA levels (left panel) and a 40% decrease in Wnt3a protein levels (middle panel). There was also a 30% decrease in Ki-67 mRNA in the renal cortex relative to the control AAV vector (right panel). Alongside is the schedule for the respective ischemia/reperfusion (I/R) and AAV treatments. ( b ) Representative confocal immunofluorescence images of mouse renal cortex following AAV-mediated D2R overexpression in an I/R model of acute renal injury. There was decreased nuclear Ki-67 (red), nuclear β-catenin (green) and decreased nuclear co-localization of Ki-67 and β-catenin with DAPI (blue). Merged images: blue = DAPI, red = Ki-67, green = β-catenin, overlap = white); arrowheads indicate nuclear overlap among Ki-67, β-catenin, and DAPI signals. Scale bar = 50 µm. All data are represented as the mean ± SEM using 4 animals per group. All measurements were performed in triplicate. *P

    Journal: Scientific Reports

    Article Title: Dopamine D2 receptor modulates Wnt expression and control of cell proliferation

    doi: 10.1038/s41598-019-52528-4

    Figure Lengend Snippet: D2R overexpression partially reverses increased Wnt3a expression and cell proliferation in response to ischemia/reperfusion injury. ( a ) AAV-mediated D2R overexpression (D2R AAV) in mouse kidney prior to reperfusion caused a 70% decrease in Wnt3a mRNA levels (left panel) and a 40% decrease in Wnt3a protein levels (middle panel). There was also a 30% decrease in Ki-67 mRNA in the renal cortex relative to the control AAV vector (right panel). Alongside is the schedule for the respective ischemia/reperfusion (I/R) and AAV treatments. ( b ) Representative confocal immunofluorescence images of mouse renal cortex following AAV-mediated D2R overexpression in an I/R model of acute renal injury. There was decreased nuclear Ki-67 (red), nuclear β-catenin (green) and decreased nuclear co-localization of Ki-67 and β-catenin with DAPI (blue). Merged images: blue = DAPI, red = Ki-67, green = β-catenin, overlap = white); arrowheads indicate nuclear overlap among Ki-67, β-catenin, and DAPI signals. Scale bar = 50 µm. All data are represented as the mean ± SEM using 4 animals per group. All measurements were performed in triplicate. *P

    Article Snippet: Preparation of Wnt3a-conditioned and L-cell control medium Mouse fibroblast-derived L-Wnt3A cells (CRL-2647; ATCC, Manassas, VA) that stably express and secrete Wnt3a and the control untransfected parental L-Cell line (CRL-2648, ATCC) were purchased from ATCC (Manassas, VA).

    Techniques: Over Expression, Expressing, Plasmid Preparation, Immunofluorescence

    D2R modulates Wnt3a expression. ( a ) D2R siRNA (72 hr) increased Wnt3a mRNA levels 2.6-fold (left panel) in mouse renal proximal tubule cells. Conversely, the D2R agonist quinpirole (1 µM, 24 hr) decreased Wnt3a expression by 70% in these cells (right panel). ( b ) Kidney-selective D2R knockdown in vivo via renal subcapsular D2R siRNA infusion similarly increased Wnt3a expression 4-fold in mouse renal cortex (left panel). ( c ) Human renal proximal tubule cells with D2R expression-reducing SNPs rs6276 and rs6277 had increased Wnt3a mRNA levels (2.9-fold, left panel) and protein expression (1.5-fold, right panel) in the setting of diminished D2R expression, relative to cells without these SNPs (wild-type, WT). (d) D2R agonist quinpirole (1 µM, 24 hr) decreased Wnt3a protein by 44% in the SNP-free wild-type (WT) human renal proximal tubule cells compared with vehicle. Acute treatment with D2R antagonist sulpiride (1 µM, 6 hr) significantly increased Wnt3a protein by 180% in the WT human renal proximal tubule cells compared with vehicle. (e) Wnt3a knockdown via siRNA (Wnt3a siRNA) significantly decreased Wnt3a protein levels (right panel) in human renal proximal tubule cells expressing SNP cluster rs6276 and 6277 and partially attenuated the increased levels of proliferation in these cells, as indicated by the decrease in Ki-67 mRNA (right panel). All data are represented as the mean ± SEM. For Panels (a,c–e): all cell experiments were conducted on n ≥ 3 separate experimental dates and performed in triplicate. For Panel (b): all animal experiments used n = 4–5 animals per group; measurements were performed in triplicate. *P

    Journal: Scientific Reports

    Article Title: Dopamine D2 receptor modulates Wnt expression and control of cell proliferation

    doi: 10.1038/s41598-019-52528-4

    Figure Lengend Snippet: D2R modulates Wnt3a expression. ( a ) D2R siRNA (72 hr) increased Wnt3a mRNA levels 2.6-fold (left panel) in mouse renal proximal tubule cells. Conversely, the D2R agonist quinpirole (1 µM, 24 hr) decreased Wnt3a expression by 70% in these cells (right panel). ( b ) Kidney-selective D2R knockdown in vivo via renal subcapsular D2R siRNA infusion similarly increased Wnt3a expression 4-fold in mouse renal cortex (left panel). ( c ) Human renal proximal tubule cells with D2R expression-reducing SNPs rs6276 and rs6277 had increased Wnt3a mRNA levels (2.9-fold, left panel) and protein expression (1.5-fold, right panel) in the setting of diminished D2R expression, relative to cells without these SNPs (wild-type, WT). (d) D2R agonist quinpirole (1 µM, 24 hr) decreased Wnt3a protein by 44% in the SNP-free wild-type (WT) human renal proximal tubule cells compared with vehicle. Acute treatment with D2R antagonist sulpiride (1 µM, 6 hr) significantly increased Wnt3a protein by 180% in the WT human renal proximal tubule cells compared with vehicle. (e) Wnt3a knockdown via siRNA (Wnt3a siRNA) significantly decreased Wnt3a protein levels (right panel) in human renal proximal tubule cells expressing SNP cluster rs6276 and 6277 and partially attenuated the increased levels of proliferation in these cells, as indicated by the decrease in Ki-67 mRNA (right panel). All data are represented as the mean ± SEM. For Panels (a,c–e): all cell experiments were conducted on n ≥ 3 separate experimental dates and performed in triplicate. For Panel (b): all animal experiments used n = 4–5 animals per group; measurements were performed in triplicate. *P

    Article Snippet: Preparation of Wnt3a-conditioned and L-cell control medium Mouse fibroblast-derived L-Wnt3A cells (CRL-2647; ATCC, Manassas, VA) that stably express and secrete Wnt3a and the control untransfected parental L-Cell line (CRL-2648, ATCC) were purchased from ATCC (Manassas, VA).

    Techniques: Expressing, In Vivo

    Modulation of Wnt3a expression via a conserved TCF/LEF promoter site. ( a ) Phylogenetic tree of WNT3A across multiple species: human, mouse, rat, chicken, and zebrafish. The WNT3A exons were used to construct the phylogenetic tree. Evolutionary distances in the units of base substitutions per site were calculated using the maximum composite likelihood method. The phylogenetic tree was then constructed using the Neighbor-Joining method with the sum of branch lengths = 0.645. (b) Binding site analysis of aligned human, mouse, rat, chicken, and zebrafish WNT3A promoter sequences highlighting conserved β-catenin-binding TCF/LEF sites within the 5 kb region upstream of the transcriptional start site (TSS). (c) Schematic representation of our luciferase reporter of TCF/LEF-dependent WNT3A transcription (left panel). The reporter construct consists of a selected segment of the human WNT3A promoter region containing a highly conserved TCF/LEF site upstream of the Gaussia luciferase gene. We show two variants of the reporter: one containing the wild-type (WT) TCF/LEF site (Human-WT) and another where the TCF/LEF site was inactivated by mutation (Human-Mut). Upon expression of a WNT3A transcriptional reporter in human renal proximal tubule cells, the Human-Mut reporter demonstrated significantly decreased WNT3A promoter activity compared with the WT control (right panel). The data were normalized to alkaline phosphatase expression. *P

    Journal: Scientific Reports

    Article Title: Dopamine D2 receptor modulates Wnt expression and control of cell proliferation

    doi: 10.1038/s41598-019-52528-4

    Figure Lengend Snippet: Modulation of Wnt3a expression via a conserved TCF/LEF promoter site. ( a ) Phylogenetic tree of WNT3A across multiple species: human, mouse, rat, chicken, and zebrafish. The WNT3A exons were used to construct the phylogenetic tree. Evolutionary distances in the units of base substitutions per site were calculated using the maximum composite likelihood method. The phylogenetic tree was then constructed using the Neighbor-Joining method with the sum of branch lengths = 0.645. (b) Binding site analysis of aligned human, mouse, rat, chicken, and zebrafish WNT3A promoter sequences highlighting conserved β-catenin-binding TCF/LEF sites within the 5 kb region upstream of the transcriptional start site (TSS). (c) Schematic representation of our luciferase reporter of TCF/LEF-dependent WNT3A transcription (left panel). The reporter construct consists of a selected segment of the human WNT3A promoter region containing a highly conserved TCF/LEF site upstream of the Gaussia luciferase gene. We show two variants of the reporter: one containing the wild-type (WT) TCF/LEF site (Human-WT) and another where the TCF/LEF site was inactivated by mutation (Human-Mut). Upon expression of a WNT3A transcriptional reporter in human renal proximal tubule cells, the Human-Mut reporter demonstrated significantly decreased WNT3A promoter activity compared with the WT control (right panel). The data were normalized to alkaline phosphatase expression. *P

    Article Snippet: Preparation of Wnt3a-conditioned and L-cell control medium Mouse fibroblast-derived L-Wnt3A cells (CRL-2647; ATCC, Manassas, VA) that stably express and secrete Wnt3a and the control untransfected parental L-Cell line (CRL-2648, ATCC) were purchased from ATCC (Manassas, VA).

    Techniques: Expressing, Construct, Binding Assay, Luciferase, Mutagenesis, Activity Assay

    Differentiation of TkDN4-M into Human Intestinal Organoids (A) Schematic procedures for intestinal organoid differentiation from human iPSC lines. (B) Images of the human iPSC line TkDN4-M that had been differentiated into spheroids (upper panels) and organoids (lower panels) with (right panels) or without (left panels) recombinant hWNT3A and human FGF2 (hFGF2). Arrowheads indicate spheroid- and organoid-like cell clumps. Scale bars, 200 μm. (C) Magnified images of the spheroids and organoids. Scale bars, 40 μm. (D) The numbers of spheroids per microscopic bright field in the presence or absence of WNT3A/FGF2 supplementation. The assays were performed in three independent experiments. Data are presented as mean ± SEM. ∗ p

    Journal: Stem Cell Reports

    Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

    doi: 10.1016/j.stemcr.2017.11.004

    Figure Lengend Snippet: Differentiation of TkDN4-M into Human Intestinal Organoids (A) Schematic procedures for intestinal organoid differentiation from human iPSC lines. (B) Images of the human iPSC line TkDN4-M that had been differentiated into spheroids (upper panels) and organoids (lower panels) with (right panels) or without (left panels) recombinant hWNT3A and human FGF2 (hFGF2). Arrowheads indicate spheroid- and organoid-like cell clumps. Scale bars, 200 μm. (C) Magnified images of the spheroids and organoids. Scale bars, 40 μm. (D) The numbers of spheroids per microscopic bright field in the presence or absence of WNT3A/FGF2 supplementation. The assays were performed in three independent experiments. Data are presented as mean ± SEM. ∗ p

    Article Snippet: Although a cell line simultaneously expressing mouse WNT3A (mWNT3A), mouse RSPO3, and mouse NOG has already been established and deposited to the American Type Culture Collection , it was originally developed for using mouse organoid culture ( ).

    Techniques: Recombinant

    Preparation of WRN CM by Lentiviral Infection (A and B) Wnt activities of recombinant hWNT3A or mWNT3A (A) and recombinant hRSPO1 or mRSPO1 (B). Activities in the absence of the hRSPO1 expression plasmid and recombinant proteins were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. n.s., not significant (Student’s t test). (C–H) Mouse L cells stably expressing WRN were generated by lentiviral infection of each gene. (C–E) Wnt signaling activities of CM from L-WRN cells at densities of 2.8, 1.4, 0.7, and 0.35 × 10 6 cells/35-mm dish after 48 hr of culture (C); at a density of 1.4 × 10 6 cells/dish cultured between 24 and 96 hr (D); and at 1.4 × 10 6 cells/dish cultured for 72 hr in diluted CM or 300 ng/mL recombinant mWNT3A, with or without an hRSPO1 expression plasmid (E). Activities in the absence of WRN CM were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. (F) Western blot analysis of the supernatants used in (C) undiluted (8 μL) and 4-fold diluted, and the recombinant proteins mWNT3A (1 and 0.3 ng), hRSPO1 (30 and 10 ng), and hNOG (10 and 3 ng) using anti-WNT3A, anti-RSPO1, and anti-NOG antibodies. (G) Images of human primary ileal organoids cultured in 25% WRN CM or the corresponding recombinant proteins for 7 days after a passage. Scale bars, 200 μm. (H) Wnt signaling activities of WRN CM (×1, ×1/3, ×1/10) stored in the deep freezer for 0, 6, or 12 months. The activities in the absence of CM were considered as “1.” The assay was performed in three independent biological replicates. Data expressed as mean ± SEM.

    Journal: Stem Cell Reports

    Article Title: A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

    doi: 10.1016/j.stemcr.2017.11.004

    Figure Lengend Snippet: Preparation of WRN CM by Lentiviral Infection (A and B) Wnt activities of recombinant hWNT3A or mWNT3A (A) and recombinant hRSPO1 or mRSPO1 (B). Activities in the absence of the hRSPO1 expression plasmid and recombinant proteins were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. n.s., not significant (Student’s t test). (C–H) Mouse L cells stably expressing WRN were generated by lentiviral infection of each gene. (C–E) Wnt signaling activities of CM from L-WRN cells at densities of 2.8, 1.4, 0.7, and 0.35 × 10 6 cells/35-mm dish after 48 hr of culture (C); at a density of 1.4 × 10 6 cells/dish cultured between 24 and 96 hr (D); and at 1.4 × 10 6 cells/dish cultured for 72 hr in diluted CM or 300 ng/mL recombinant mWNT3A, with or without an hRSPO1 expression plasmid (E). Activities in the absence of WRN CM were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. (F) Western blot analysis of the supernatants used in (C) undiluted (8 μL) and 4-fold diluted, and the recombinant proteins mWNT3A (1 and 0.3 ng), hRSPO1 (30 and 10 ng), and hNOG (10 and 3 ng) using anti-WNT3A, anti-RSPO1, and anti-NOG antibodies. (G) Images of human primary ileal organoids cultured in 25% WRN CM or the corresponding recombinant proteins for 7 days after a passage. Scale bars, 200 μm. (H) Wnt signaling activities of WRN CM (×1, ×1/3, ×1/10) stored in the deep freezer for 0, 6, or 12 months. The activities in the absence of CM were considered as “1.” The assay was performed in three independent biological replicates. Data expressed as mean ± SEM.

    Article Snippet: Although a cell line simultaneously expressing mouse WNT3A (mWNT3A), mouse RSPO3, and mouse NOG has already been established and deposited to the American Type Culture Collection , it was originally developed for using mouse organoid culture ( ).

    Techniques: Infection, Recombinant, Expressing, Plasmid Preparation, Stable Transfection, Generated, Cell Culture, Western Blot

    Pyrvinium inhibits Wnt signaling. ( 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: Pyrvinium inhibits Wnt signaling. ( 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

    WIKI4 increases the steady-state abundance of the Wnt/ß-catenin inhibitory protein, AXIN1. ( A ) WIKI4 prevents degradation of AXIN1 following stimulation with Wnt3A. A375 melanoma cells were stimulated with 10% (vol/vol) Wnt3A CM for the indicated time periods with or without WIKI4 treatment, lysed and analyzed by western blot using the indicated antibodies. ( B ) WIKI4 increases the steady-state abundance of AXIN1 and AXIN2 protein. DLD1 colorectal carcinoma cells were incubated with DMSO, WIKI4 or XAV-939 for the indicated times, lysed and analyzed by western blot. ( C ) WIKI4 does not significantly affect the steady-state RNA abundance of AXIN1. DLD1 colorectal carcinoma cells were incubated with WIK4 for the indicated times, and processed for qPCR to assess changes in the steady-state abundance of AXIN1 transcript. This data is representative of two independent experiments and the error bars represent standard deviation. ( D ) WIKI4-dependent increases in AXIN1 protein abundance can be maintained by treatment with a proteasome inhibitor. DLD1 colorectal carcinoma cells were treated overnight with WIKI4, and after washing were then incubated for two hours with DMSO (D), WIKI4 (W), or the proteasome inhibitor MG132 (M). The cells were lysed and analyzed by western blotting for the indicated antibodies.

    Journal: PLoS ONE

    Article Title: WIKI4, a Novel Inhibitor of Tankyrase and Wnt/ss-Catenin Signaling

    doi: 10.1371/journal.pone.0050457

    Figure Lengend Snippet: WIKI4 increases the steady-state abundance of the Wnt/ß-catenin inhibitory protein, AXIN1. ( A ) WIKI4 prevents degradation of AXIN1 following stimulation with Wnt3A. A375 melanoma cells were stimulated with 10% (vol/vol) Wnt3A CM for the indicated time periods with or without WIKI4 treatment, lysed and analyzed by western blot using the indicated antibodies. ( B ) WIKI4 increases the steady-state abundance of AXIN1 and AXIN2 protein. DLD1 colorectal carcinoma cells were incubated with DMSO, WIKI4 or XAV-939 for the indicated times, lysed and analyzed by western blot. ( C ) WIKI4 does not significantly affect the steady-state RNA abundance of AXIN1. DLD1 colorectal carcinoma cells were incubated with WIK4 for the indicated times, and processed for qPCR to assess changes in the steady-state abundance of AXIN1 transcript. This data is representative of two independent experiments and the error bars represent standard deviation. ( D ) WIKI4-dependent increases in AXIN1 protein abundance can be maintained by treatment with a proteasome inhibitor. DLD1 colorectal carcinoma cells were treated overnight with WIKI4, and after washing were then incubated for two hours with DMSO (D), WIKI4 (W), or the proteasome inhibitor MG132 (M). The cells were lysed and analyzed by western blotting for the indicated antibodies.

    Article Snippet: Wnt3A conditioned medium (CM) and control L CM were generated as previously described from L cells and L-Wnt3A cells (ATCC).

    Techniques: Western Blot, Incubation, Real-time Polymerase Chain Reaction, Standard Deviation

    WIKI4 inhibits the functional outcomes of Wnt/ß-catenin signaling. ( A ) WIKI4 inhibits colony formation of DLD1 colorectal cancer cells. DLD1 cells were plated individually in 0.5% serum containing medium, and treated with the indicated concentrations of WIKI4 and XAV-939. This experiment is representative of three independent experiments and the error bars represent standard deviation of three technical replicates. ( B-F ) WIKI4 prevents Wnt3A-dependent differentiation of H1 human embryonic stem cells (hESCs). ( B ) Culturing hESCs for six days with Wnt3A causes marked morphological changes that are rescued by treatment with WIKI4. Scalebar = 500 µm. ( C ) Treatment with WIKI4 prevents the decrease in co-expression of markers of undifferentiated hESCs following Wnt3A stimulus. hESCs were stimulated with the indicated treatments and expression of GCTM2 and CD9 was assessed by flow cytometry following six days of treatment. ( D-F ) The effect of WIKI4 treatment on the expression of genes that are altered during Wnt3A-dependent differentiation of hESCs was assessed by qPCR. hESCs were treated for the indicated conditions for six days, and then analyzed by qPCR for markers of undifferentiated stem cells ( NANOG, POU5F1 ) ( D ), endoderm ( SOX17, GATA6 ) ( E ), and mesoderm ( T, KDR ) ( F ). The data was normalized to 100,000 copies of GAPDH and plotted as a ratio to the untreated hESCs (cultured in KSR media). The data in the experiments presented in B-F are representative of three independent experiments and the error represents standard deviation of technical replicates. In B-F , LCM = control L cell CM, WNT3A = Wnt3a CM; both 50% (vol/vol) in KSR medium.

    Journal: PLoS ONE

    Article Title: WIKI4, a Novel Inhibitor of Tankyrase and Wnt/ss-Catenin Signaling

    doi: 10.1371/journal.pone.0050457

    Figure Lengend Snippet: WIKI4 inhibits the functional outcomes of Wnt/ß-catenin signaling. ( A ) WIKI4 inhibits colony formation of DLD1 colorectal cancer cells. DLD1 cells were plated individually in 0.5% serum containing medium, and treated with the indicated concentrations of WIKI4 and XAV-939. This experiment is representative of three independent experiments and the error bars represent standard deviation of three technical replicates. ( B-F ) WIKI4 prevents Wnt3A-dependent differentiation of H1 human embryonic stem cells (hESCs). ( B ) Culturing hESCs for six days with Wnt3A causes marked morphological changes that are rescued by treatment with WIKI4. Scalebar = 500 µm. ( C ) Treatment with WIKI4 prevents the decrease in co-expression of markers of undifferentiated hESCs following Wnt3A stimulus. hESCs were stimulated with the indicated treatments and expression of GCTM2 and CD9 was assessed by flow cytometry following six days of treatment. ( D-F ) The effect of WIKI4 treatment on the expression of genes that are altered during Wnt3A-dependent differentiation of hESCs was assessed by qPCR. hESCs were treated for the indicated conditions for six days, and then analyzed by qPCR for markers of undifferentiated stem cells ( NANOG, POU5F1 ) ( D ), endoderm ( SOX17, GATA6 ) ( E ), and mesoderm ( T, KDR ) ( F ). The data was normalized to 100,000 copies of GAPDH and plotted as a ratio to the untreated hESCs (cultured in KSR media). The data in the experiments presented in B-F are representative of three independent experiments and the error represents standard deviation of technical replicates. In B-F , LCM = control L cell CM, WNT3A = Wnt3a CM; both 50% (vol/vol) in KSR medium.

    Article Snippet: Wnt3A conditioned medium (CM) and control L CM were generated as previously described from L cells and L-Wnt3A cells (ATCC).

    Techniques: Functional Assay, Standard Deviation, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Cell Culture, Laser Capture Microdissection

    DPY19L3-mediated C -mannosylation of Rspo1 at W 156 regulates its secretion. (A) Effect of knockdown of DPY19L1, DPY19L3, or DPY19L4 on Rspo1 secretion. HT1080-Rspo1-MH cells were treated with the indicated siRNAs and cultured in serum-free medium with 50 μg/ml soluble heparin, and cell lysates and conditioned media were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin. Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (siCtrl) was defined as 1.0. (B, C) Effect of knockdown of DPY19L3 on Rspo1 secretion. HT1080-Rspo1-MH cells were treated with the indicated siRNAs. Total RNA was isolated from each cell line, and quantitative RT-PCR was performed (B). Each cell line was cultured in serum-free medium with 50 μg/ml soluble heparin, and cell lysates and conditioned media were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin (C). Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (siCtrl) was defined as 1.0. (D) Effect of C -mannosylation of Rspo1 at W 153 on Wnt signaling enhancing activity. HT1080-Rspo1-MH cells were treated with siGFP or siDPY19L3, and conditioned medium from each cell was collected. Rspo1 proteins were purified from the conditioned medium of siGFP- or siDPY19L3-treated cells, and the amounts of proteins were equalized by Western blot (inset). 293T cells were transfected with TOPFlash or FOPFlash in the presence of 10% Wnt3a-conditioned medium and treated with equal amounts of purified Rspo1 proteins. After 24 h, luciferase activities were measured and normalized to Renilla luciferase. W 153 - C -mannosylated Rspo1 (produced by siDPY19L3-treated cells) had almost same activity as wild-type Rspo1 (produced by siGFP-treated cells). Data shown are means ± SD. * p

    Journal: Molecular Biology of the Cell

    Article Title: Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

    doi: 10.1091/mbc.E15-06-0373

    Figure Lengend Snippet: DPY19L3-mediated C -mannosylation of Rspo1 at W 156 regulates its secretion. (A) Effect of knockdown of DPY19L1, DPY19L3, or DPY19L4 on Rspo1 secretion. HT1080-Rspo1-MH cells were treated with the indicated siRNAs and cultured in serum-free medium with 50 μg/ml soluble heparin, and cell lysates and conditioned media were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin. Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (siCtrl) was defined as 1.0. (B, C) Effect of knockdown of DPY19L3 on Rspo1 secretion. HT1080-Rspo1-MH cells were treated with the indicated siRNAs. Total RNA was isolated from each cell line, and quantitative RT-PCR was performed (B). Each cell line was cultured in serum-free medium with 50 μg/ml soluble heparin, and cell lysates and conditioned media were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin (C). Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (siCtrl) was defined as 1.0. (D) Effect of C -mannosylation of Rspo1 at W 153 on Wnt signaling enhancing activity. HT1080-Rspo1-MH cells were treated with siGFP or siDPY19L3, and conditioned medium from each cell was collected. Rspo1 proteins were purified from the conditioned medium of siGFP- or siDPY19L3-treated cells, and the amounts of proteins were equalized by Western blot (inset). 293T cells were transfected with TOPFlash or FOPFlash in the presence of 10% Wnt3a-conditioned medium and treated with equal amounts of purified Rspo1 proteins. After 24 h, luciferase activities were measured and normalized to Renilla luciferase. W 153 - C -mannosylated Rspo1 (produced by siDPY19L3-treated cells) had almost same activity as wild-type Rspo1 (produced by siGFP-treated cells). Data shown are means ± SD. * p

    Article Snippet: After 24 h, the cells were transiently transfected with 400 ng of canonical Wnt signaling reporter Super 8×TopFlash (Addgene plasmid 12456) or mutant reporter Super 8×FopFlash (Addgene plasmid 12457; ) and 20 ng of phRL-TK vector (Promega) in the presence of 10% (vol/vol) Wnt3a-conditioned medium ( ) from L-Wnt3a cells (ATCC CRL-2647) as described ( ).

    Techniques: Cell Culture, Expressing, Software, Isolation, Quantitative RT-PCR, Activity Assay, Purification, Western Blot, Transfection, Luciferase, Produced

    DPY19L3 is the C -mannosyltransferase of Rspo1 at W 156 . (A, B) Identification of C -mannosyltransferase of Rspo1. Human DPY19L1-L4– or empty vector (mock)–expressing Drosophila S2 cells were transiently transfected with pMT-Rspo1-MH, and protein expression was induced by 200 μM CuSO 4 for 72 h. Rspo1-MH protein was purified by tandem affinity chromatography, heparin–Sepharose, and Ni-NTA agarose. The samples were digested with trypsin and Asp-N, and the resulting peptides were analyzed by LC-MS/MS. Monomannosylated peptide was observed only when the protein was produced in DPY19L3-expressing S2 cells (A). Unmannosylated and monomannosylated peptides derived from DPY19L3-expressing S2 cells were further analyzed by LC-MS/MS. Indicated y ions were detected, and signals resulting from the characteristic cross-ring cleavages were observed at the y 7 and y 8 ions in monomannosylated peptide (B). # W, C -mannosyltryptophan; *C, propionamide cysteine. (C) Effect of C -mannosylation of Rspo1 at W 156 on Wnt signaling–enhancing activity. Recombinant Rspo1 proteins produced by mock- or DPY19L3-transfected S2 cells were purified, and the amounts of proteins were equalized by Western blot (inset). 293T cells were transfected with TOPFlash or FOPFlash in the presence of 10% Wnt3a-conditioned medium and treated with equal amounts of purified Rspo1 proteins. After 24 h, luciferase activities were measured and normalized to Renilla luciferase. C -mannosylated Rspo1 at W 156 (produced by DPY19L3-expressing S2 cells) had increased activity compared with non– C -mannosylated Rspo1 (produced by mock-transfected S2 cells). Data shown are means ± SD. * p

    Journal: Molecular Biology of the Cell

    Article Title: Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

    doi: 10.1091/mbc.E15-06-0373

    Figure Lengend Snippet: DPY19L3 is the C -mannosyltransferase of Rspo1 at W 156 . (A, B) Identification of C -mannosyltransferase of Rspo1. Human DPY19L1-L4– or empty vector (mock)–expressing Drosophila S2 cells were transiently transfected with pMT-Rspo1-MH, and protein expression was induced by 200 μM CuSO 4 for 72 h. Rspo1-MH protein was purified by tandem affinity chromatography, heparin–Sepharose, and Ni-NTA agarose. The samples were digested with trypsin and Asp-N, and the resulting peptides were analyzed by LC-MS/MS. Monomannosylated peptide was observed only when the protein was produced in DPY19L3-expressing S2 cells (A). Unmannosylated and monomannosylated peptides derived from DPY19L3-expressing S2 cells were further analyzed by LC-MS/MS. Indicated y ions were detected, and signals resulting from the characteristic cross-ring cleavages were observed at the y 7 and y 8 ions in monomannosylated peptide (B). # W, C -mannosyltryptophan; *C, propionamide cysteine. (C) Effect of C -mannosylation of Rspo1 at W 156 on Wnt signaling–enhancing activity. Recombinant Rspo1 proteins produced by mock- or DPY19L3-transfected S2 cells were purified, and the amounts of proteins were equalized by Western blot (inset). 293T cells were transfected with TOPFlash or FOPFlash in the presence of 10% Wnt3a-conditioned medium and treated with equal amounts of purified Rspo1 proteins. After 24 h, luciferase activities were measured and normalized to Renilla luciferase. C -mannosylated Rspo1 at W 156 (produced by DPY19L3-expressing S2 cells) had increased activity compared with non– C -mannosylated Rspo1 (produced by mock-transfected S2 cells). Data shown are means ± SD. * p

    Article Snippet: After 24 h, the cells were transiently transfected with 400 ng of canonical Wnt signaling reporter Super 8×TopFlash (Addgene plasmid 12456) or mutant reporter Super 8×FopFlash (Addgene plasmid 12457; ) and 20 ng of phRL-TK vector (Promega) in the presence of 10% (vol/vol) Wnt3a-conditioned medium ( ) from L-Wnt3a cells (ATCC CRL-2647) as described ( ).

    Techniques: Plasmid Preparation, Expressing, Transfection, Purification, Affinity Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Produced, Derivative Assay, Activity Assay, Recombinant, Western Blot, Luciferase

    Effect of C -mannosylation on Rspo1 function. (A) Effect of C -mannosylation on Rspo1 secretion using CHO-K1 and Lec15.2 cells. CHO-K1 and Lec15.2 cells were transiently transfected with pCI-neo-Rspo1/N137Q-MH vector for 6 h and then cultured in serum-free medium with 50 μg/ml soluble heparin for 18 h. The protein samples were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin. Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (CHO-K1) was defined as 1.0. (B, C) Establishment of mutant form of Rspo1 (W 153 and W 156 replaced by alanine residues; W153A/W156A: 2WA)–overexpressing HT1080 cell line, HT1080-Rspo1/2WA-MH. HT1080-neo (neo), HT1080-Rspo1-MH (wt), and HT1080-Rspo1/2WA-MH (2WA) cells were lysed, and aliquots of the cell lysates were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin (B). Total RNA was isolated from each cell line, and semiquantitative (left) and quantitative (right) RT-PCR was performed (C). Equal amounts of exogenous Rspo1 in these cells were confirmed. ns, not significant. (D, E) Effect of C -mannosylation on intracellular trafficking. Cells were cultured with 50 μg/ml soluble heparin, stained with Hoechst 33258 (blue), anti–c-myc (green), and anti-KDEL (red; D) or anti-GRASP65 (red; E), and examined by fluorescence microscopy. Areas of overlapping stains are represented in yellow in the superimposed images. Bars, 10 μm. (F) Effect of C -mannosylation on Rspo1 secretion using a mutant Rspo1-overexpressing cell line. Cells were cultured with 50 μg/ml soluble heparin, and cell lysates and conditioned media were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin. Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (wt) was defined as 1.0. (G) Effect of C -mannosylation on the kinetics of Rspo1 secretion. Cells were cultured with 50 μg/ml soluble heparin, and conditioned media were collected at the indicated times, electrophoresed, and immunoblotted with anti–c-myc (top). Protein bands were quantified by using ImageJ software (bottom). The secreted amount of wild-type Rspo1 at 24 h was defined as 100%. (H) Effect of C -mannosylation on Rspo1-mediated enhancement of Wnt signaling. Recombinant Rspo1 and Rspo1/2WA were purified from each cell line, and the amounts of proteins were equalized by Western blot (inset). 293T cells were transfected with TOPFlash or FOPFlash in the presence of 10% Wnt3a-conditioned medium and treated with equal amounts of purified Rspo1. After 24 h, luciferase activities were measured and normalized to Renilla luciferase. Non– C -mannosylated Rspo1 slightly enhanced Wnt signaling activity. Data shown are means ± SD. * p

    Journal: Molecular Biology of the Cell

    Article Title: Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

    doi: 10.1091/mbc.E15-06-0373

    Figure Lengend Snippet: Effect of C -mannosylation on Rspo1 function. (A) Effect of C -mannosylation on Rspo1 secretion using CHO-K1 and Lec15.2 cells. CHO-K1 and Lec15.2 cells were transiently transfected with pCI-neo-Rspo1/N137Q-MH vector for 6 h and then cultured in serum-free medium with 50 μg/ml soluble heparin for 18 h. The protein samples were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin. Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (CHO-K1) was defined as 1.0. (B, C) Establishment of mutant form of Rspo1 (W 153 and W 156 replaced by alanine residues; W153A/W156A: 2WA)–overexpressing HT1080 cell line, HT1080-Rspo1/2WA-MH. HT1080-neo (neo), HT1080-Rspo1-MH (wt), and HT1080-Rspo1/2WA-MH (2WA) cells were lysed, and aliquots of the cell lysates were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin (B). Total RNA was isolated from each cell line, and semiquantitative (left) and quantitative (right) RT-PCR was performed (C). Equal amounts of exogenous Rspo1 in these cells were confirmed. ns, not significant. (D, E) Effect of C -mannosylation on intracellular trafficking. Cells were cultured with 50 μg/ml soluble heparin, stained with Hoechst 33258 (blue), anti–c-myc (green), and anti-KDEL (red; D) or anti-GRASP65 (red; E), and examined by fluorescence microscopy. Areas of overlapping stains are represented in yellow in the superimposed images. Bars, 10 μm. (F) Effect of C -mannosylation on Rspo1 secretion using a mutant Rspo1-overexpressing cell line. Cells were cultured with 50 μg/ml soluble heparin, and cell lysates and conditioned media were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin. Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (wt) was defined as 1.0. (G) Effect of C -mannosylation on the kinetics of Rspo1 secretion. Cells were cultured with 50 μg/ml soluble heparin, and conditioned media were collected at the indicated times, electrophoresed, and immunoblotted with anti–c-myc (top). Protein bands were quantified by using ImageJ software (bottom). The secreted amount of wild-type Rspo1 at 24 h was defined as 100%. (H) Effect of C -mannosylation on Rspo1-mediated enhancement of Wnt signaling. Recombinant Rspo1 and Rspo1/2WA were purified from each cell line, and the amounts of proteins were equalized by Western blot (inset). 293T cells were transfected with TOPFlash or FOPFlash in the presence of 10% Wnt3a-conditioned medium and treated with equal amounts of purified Rspo1. After 24 h, luciferase activities were measured and normalized to Renilla luciferase. Non– C -mannosylated Rspo1 slightly enhanced Wnt signaling activity. Data shown are means ± SD. * p

    Article Snippet: After 24 h, the cells were transiently transfected with 400 ng of canonical Wnt signaling reporter Super 8×TopFlash (Addgene plasmid 12456) or mutant reporter Super 8×FopFlash (Addgene plasmid 12457; ) and 20 ng of phRL-TK vector (Promega) in the presence of 10% (vol/vol) Wnt3a-conditioned medium ( ) from L-Wnt3a cells (ATCC CRL-2647) as described ( ).

    Techniques: Transfection, Plasmid Preparation, Cell Culture, Expressing, Software, Mutagenesis, Isolation, Reverse Transcription Polymerase Chain Reaction, Staining, Fluorescence, Microscopy, Recombinant, Purification, Western Blot, Luciferase, Activity Assay

    hALFY protein co-localizes with aggregated DVL3 and facilitates its removal. A. Cloning of the different DVLs. The three different DVLs were PCR-amplified from brain cDNA and cloned in frame with an N-terminal FLAG tag and a C-terminal EGFP. B. Co-localization of hALFY with the different DVLs. Neuroblastoma cells were co-transfected with the different DVLs (1, 2 3) as well as tdTomato hALFY wild type/mutant constructs. Both wild type and mutant hALFY co-localize specifically with DVL3 (c-i, c-ii) and not DVL1 (a-i, a-ii) or DVL2 (b-i,b-ii). Minimal co-localization with DVL1 was visible (a-i, a-ii), and could be due to the strong over expression. C. Western blot analysis of DVL over expression. Neuroblastoma cells were co-transfected with the different DVLs and FLAG hALFY (wild type, mutant, Δ-FYVE and mock). After 24 hours, cells were treated with cycloheximide to arrest further de-novo protein synthesis. WB analysis reveals that hALFY wild type removes aggregated DVL3 (student t test, n = 3, P value = 0.04) yet not DVL1 or DVL2. DVL3 protein accumulated in hALFY mutant expressing cells. D. Western blot analysis of endogenous DVLs. Following transfection of the different FLAG hALFY constructs, neuroblastoma cells were incubated with Wnt3a conditioned medium for 8 hours and harvested for WB analysis. hALFY wild type specifically removed endogenous DVL3 aggregates, yet not DVL1 or DVL2. This effect was inhibited by wortmannin, indicating that hALFY removes DVL3 aggregates presumably in an autophagy dependent manner (student t test, n = 3, P value = 0.03).

    Journal: PLoS Genetics

    Article Title: ALFY-Controlled DVL3 Autophagy Regulates Wnt Signaling, Determining Human Brain Size

    doi: 10.1371/journal.pgen.1005919

    Figure Lengend Snippet: hALFY protein co-localizes with aggregated DVL3 and facilitates its removal. A. Cloning of the different DVLs. The three different DVLs were PCR-amplified from brain cDNA and cloned in frame with an N-terminal FLAG tag and a C-terminal EGFP. B. Co-localization of hALFY with the different DVLs. Neuroblastoma cells were co-transfected with the different DVLs (1, 2 3) as well as tdTomato hALFY wild type/mutant constructs. Both wild type and mutant hALFY co-localize specifically with DVL3 (c-i, c-ii) and not DVL1 (a-i, a-ii) or DVL2 (b-i,b-ii). Minimal co-localization with DVL1 was visible (a-i, a-ii), and could be due to the strong over expression. C. Western blot analysis of DVL over expression. Neuroblastoma cells were co-transfected with the different DVLs and FLAG hALFY (wild type, mutant, Δ-FYVE and mock). After 24 hours, cells were treated with cycloheximide to arrest further de-novo protein synthesis. WB analysis reveals that hALFY wild type removes aggregated DVL3 (student t test, n = 3, P value = 0.04) yet not DVL1 or DVL2. DVL3 protein accumulated in hALFY mutant expressing cells. D. Western blot analysis of endogenous DVLs. Following transfection of the different FLAG hALFY constructs, neuroblastoma cells were incubated with Wnt3a conditioned medium for 8 hours and harvested for WB analysis. hALFY wild type specifically removed endogenous DVL3 aggregates, yet not DVL1 or DVL2. This effect was inhibited by wortmannin, indicating that hALFY removes DVL3 aggregates presumably in an autophagy dependent manner (student t test, n = 3, P value = 0.03).

    Article Snippet: Wnt3a conditioned medium Wnt3a conditioned medium was obtained by collecting growth medium from mouse fibroblast secreting the active form of Wnt3a glycoprotein (ATCC CRL-2647) according to the manufacturer protocol.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, FLAG-tag, Transfection, Mutagenesis, Construct, Over Expression, Western Blot, Expressing, Incubation

    E-selectin-induced MET activates Wnt signaling. (a) BM2 cells stably expressing the 7x TCF-GFP reporter and SV40-mCherry as internal control (BM2-TGC) were plated on either E-selectin or IgG coated (10 μg/mL) plates with or without recombinant Wnt3a (100 ng/mL). Fluorescence was assessed by confocal microscopy after 48 h and quantified by flow cytometry. Scale bars represent 100 μm. Data representative of > 5 independent experiments. (b) qPCR analysis of EMT- or Wnt-associated genes from the BM2 cells plated on E-selectin or IgG (10 μg/mL) for 48 h. n= 3 technical replicates. (c) Ex vivo confocal images of bone metastasis in Nu/Nu mice injected with BM2-TGC. Live animal labeling with anti-CD31 was conducted to visualize vasculature. Scale bar represents 100 μm. Data representative of 4 biological replicates. (d) qPCR of Glg1-variant 3 , Fut3 and Fut6 levels in BM2 cells seeded on either IgG or E-selectin (10 μg/mL) for 48 h. n = 4 technical replicates. (e) BM2 cells stably-expressing the SORE6-mCherry stemness reporter or control mCherry reporter were plated on E-selectin or control IgG (10 μg/mL) for 48 h. mCherry fluorescence was assessed by flow cytometry after 48 h. Data representative of 3 independent biological replicates. (f) Proposed model of E-selectin function during bone metastasis. Data represent mean ± SEM.

    Journal: Nature cell biology

    Article Title: Bone Vascular Niche E-selectin Induces Mesenchymal-Epithelial Transition and Wnt Activation in Cancer Cells to Promote Bone Metastasis

    doi: 10.1038/s41556-019-0309-2

    Figure Lengend Snippet: E-selectin-induced MET activates Wnt signaling. (a) BM2 cells stably expressing the 7x TCF-GFP reporter and SV40-mCherry as internal control (BM2-TGC) were plated on either E-selectin or IgG coated (10 μg/mL) plates with or without recombinant Wnt3a (100 ng/mL). Fluorescence was assessed by confocal microscopy after 48 h and quantified by flow cytometry. Scale bars represent 100 μm. Data representative of > 5 independent experiments. (b) qPCR analysis of EMT- or Wnt-associated genes from the BM2 cells plated on E-selectin or IgG (10 μg/mL) for 48 h. n= 3 technical replicates. (c) Ex vivo confocal images of bone metastasis in Nu/Nu mice injected with BM2-TGC. Live animal labeling with anti-CD31 was conducted to visualize vasculature. Scale bar represents 100 μm. Data representative of 4 biological replicates. (d) qPCR of Glg1-variant 3 , Fut3 and Fut6 levels in BM2 cells seeded on either IgG or E-selectin (10 μg/mL) for 48 h. n = 4 technical replicates. (e) BM2 cells stably-expressing the SORE6-mCherry stemness reporter or control mCherry reporter were plated on E-selectin or control IgG (10 μg/mL) for 48 h. mCherry fluorescence was assessed by flow cytometry after 48 h. Data representative of 3 independent biological replicates. (f) Proposed model of E-selectin function during bone metastasis. Data represent mean ± SEM.

    Article Snippet: Wnt3a and control-conditioned media were generated by collecting 72 h conditioned media from L cells with or without Wnt3a expression (ATCC CRL-2647 and CRL-2648) followed by filtration at 0.45 μm.

    Techniques: Stable Transfection, Expressing, Recombinant, Fluorescence, Confocal Microscopy, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Ex Vivo, Mouse Assay, Injection, Labeling, Variant Assay

    SOXC proteins amplify canonical WNT/signaling by inhibiting GSK3-dependent phosphorylation of β-catenin. (A) TOP-Flash reporter activity in HEK293 cells transfected with empty (none) or FLAG-SOX11 expression plasmid and treated with various dilutions of WNT3A medium for the last 6 h of culture. Each dot represents one sample. The equations of linear fits are indicated. (B) β-Catenin level in SoxC fl/fl primary limb bud cells infected with GFP or CRE adenovirus for 24 h. Cells were treated with 20% WNT3A medium for the last 8 h. Representative blots are shown. (C) TOP-Flash reporter activity in 10T1/2 cells transfected for 24 h with empty or 3FLAG-SOX11 expression plasmid. None (−) or DKK1 protein was added 1 h before transfection, and 20% WNT3A medium was added for the last 6 h. Normalized reporter activities are presented as means with standard deviation for triplicates in a typical experiment. Fold increases caused by SOX11 are indicated. *, P

    Journal: The Journal of Cell Biology

    Article Title: SOXC proteins amplify canonical WNT signaling to secure nonchondrocytic fates in skeletogenesis

    doi: 10.1083/jcb.201405098

    Figure Lengend Snippet: SOXC proteins amplify canonical WNT/signaling by inhibiting GSK3-dependent phosphorylation of β-catenin. (A) TOP-Flash reporter activity in HEK293 cells transfected with empty (none) or FLAG-SOX11 expression plasmid and treated with various dilutions of WNT3A medium for the last 6 h of culture. Each dot represents one sample. The equations of linear fits are indicated. (B) β-Catenin level in SoxC fl/fl primary limb bud cells infected with GFP or CRE adenovirus for 24 h. Cells were treated with 20% WNT3A medium for the last 8 h. Representative blots are shown. (C) TOP-Flash reporter activity in 10T1/2 cells transfected for 24 h with empty or 3FLAG-SOX11 expression plasmid. None (−) or DKK1 protein was added 1 h before transfection, and 20% WNT3A medium was added for the last 6 h. Normalized reporter activities are presented as means with standard deviation for triplicates in a typical experiment. Fold increases caused by SOX11 are indicated. *, P

    Article Snippet: WNT3A medium was produced using LWNT-3A cells (ATCC).

    Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Infection, Standard Deviation

    Triptonide effectively inhibits Wnt/β-catenin signaling through a mechanism different from triptolide. ( A ) Chemical structures of Triptonide and ( B ) troptolide are displayed. ( C ) Triptonide (TN in the figure) effectively inhibits Wnt/β-catenin signaling induced by Wnt3a conditional media (Wnt3a-CM) in STF293 cells that were stably transfected with TOPFLASH luciferase plasmid. The data was represented as mean relative luciferase activities (RLA) + SEM (n = 3). All the P values are compared to the luciferase activity induced by Wnt3a-CM (*P

    Journal: Scientific Reports

    Article Title: Triptonide Effectively Inhibits Wnt/β-Catenin Signaling via C-terminal Transactivation Domain of β-catenin

    doi: 10.1038/srep32779

    Figure Lengend Snippet: Triptonide effectively inhibits Wnt/β-catenin signaling through a mechanism different from triptolide. ( A ) Chemical structures of Triptonide and ( B ) troptolide are displayed. ( C ) Triptonide (TN in the figure) effectively inhibits Wnt/β-catenin signaling induced by Wnt3a conditional media (Wnt3a-CM) in STF293 cells that were stably transfected with TOPFLASH luciferase plasmid. The data was represented as mean relative luciferase activities (RLA) + SEM (n = 3). All the P values are compared to the luciferase activity induced by Wnt3a-CM (*P

    Article Snippet: Wnt3a-conditioned media (Wnt3a-CM) was made from Wnt3A cell line (ATCC® CRL2647™) according to instructions of the American Type Culture Collection (Manassas, VA), USA.

    Techniques: Stable Transfection, Transfection, Luciferase, Plasmid Preparation, Activity Assay

    Enforced expression of Sox7 inhibits not only Wnt/β-catenin signaling activity but also cell growth of endometrial cancer cells (A) Western blotting showed Sox7 could suppress the expressions of C-myc and CyclinD1 in Sox7 stable expression clones in HEC1A and TOV112D cultured in normal and Wnt3A condition media. (B) XTT cell proliferation assay demonstrated that Sox7 could significantly inhibit cell growth of Sox7 stable expression clones in HEC1A and TOV112D cultured in normal and Wnt3A condition media.

    Journal: Oncotarget

    Article Title: Down-regulation of Sox7 is associated with aberrant activation of Wnt/?-catenin signaling in endometrial cancer

    doi:

    Figure Lengend Snippet: Enforced expression of Sox7 inhibits not only Wnt/β-catenin signaling activity but also cell growth of endometrial cancer cells (A) Western blotting showed Sox7 could suppress the expressions of C-myc and CyclinD1 in Sox7 stable expression clones in HEC1A and TOV112D cultured in normal and Wnt3A condition media. (B) XTT cell proliferation assay demonstrated that Sox7 could significantly inhibit cell growth of Sox7 stable expression clones in HEC1A and TOV112D cultured in normal and Wnt3A condition media.

    Article Snippet: Human Embryonic Kidney 293 cells (HEK 293) (ATCC) was used for TOP/FOP luciferase reporter assay, and L Wnt3A cell line (CRL-2647) was used for Wnt3A conditioned medium (ATCC).

    Techniques: Expressing, Activity Assay, Western Blot, Clone Assay, Cell Culture, Proliferation Assay

    NKP608 inhibited Wnt/β-catenin signaling pathway in colorectal cancer cells. a Western blot revealed that NKP608 resulted in an inhibitory action of Wnt relative proteins and proteins relevant cell growth including β-catenin, Wnt-3a, E-Cadherin, Cyclin D1 and VEGF. b Quantitative expression levels of proteins are shown. Values are expressed as the mean ± SD (n = 3). * p

    Journal: Biological Research

    Article Title: The NK1 receptor antagonist NKP608 inhibits proliferation of human colorectal cancer cells via Wnt signaling pathway

    doi: 10.1186/s40659-018-0163-x

    Figure Lengend Snippet: NKP608 inhibited Wnt/β-catenin signaling pathway in colorectal cancer cells. a Western blot revealed that NKP608 resulted in an inhibitory action of Wnt relative proteins and proteins relevant cell growth including β-catenin, Wnt-3a, E-Cadherin, Cyclin D1 and VEGF. b Quantitative expression levels of proteins are shown. Values are expressed as the mean ± SD (n = 3). * p

    Article Snippet: Antibodies to Active-Caspase-3, Wnt-3a, β-catenin, E-Cadherin, (vascular endothelial growth factor) VEGF were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Western Blot, Expressing

    The expression of C4ST-1 gene was suppressed in L-Wnt-3a cells. A , the disaccharide composition of HS chains produced in L and L-Wnt-3a cells was analyzed. GAG chains isolated from L and L-Wnt-3a cells were digested with a mixture of heparitinase and

    Journal: The Journal of Biological Chemistry

    Article Title: Down-regulation of Chondroitin 4-O-Sulfotransferase-1 by Wnt Signaling Triggers Diffusion of Wnt-3a *

    doi: 10.1074/jbc.M110.155093

    Figure Lengend Snippet: The expression of C4ST-1 gene was suppressed in L-Wnt-3a cells. A , the disaccharide composition of HS chains produced in L and L-Wnt-3a cells was analyzed. GAG chains isolated from L and L-Wnt-3a cells were digested with a mixture of heparitinase and

    Article Snippet: Anti-Wnt-3a antibody (#2391), the anti-β-catenin antibody (#610154), and the monoclonal anti-actin antibody (clone AC-40) (#A3853) were purchased from Cell Signaling Technology (Boston, MA), BD Transduction Laboratories, and Sigma, respectively.

    Techniques: Expressing, Produced, Isolation

    C4ST-1 expression levels are positively correlated with E-disaccharide content and negatively correlated with Wnt-3a secretion. A , the amount of secreted Wnt-3a ( top panel ) and the content of E-disaccharide units in cellular CS chains ( bottom panel ) in

    Journal: The Journal of Biological Chemistry

    Article Title: Down-regulation of Chondroitin 4-O-Sulfotransferase-1 by Wnt Signaling Triggers Diffusion of Wnt-3a *

    doi: 10.1074/jbc.M110.155093

    Figure Lengend Snippet: C4ST-1 expression levels are positively correlated with E-disaccharide content and negatively correlated with Wnt-3a secretion. A , the amount of secreted Wnt-3a ( top panel ) and the content of E-disaccharide units in cellular CS chains ( bottom panel ) in

    Article Snippet: Anti-Wnt-3a antibody (#2391), the anti-β-catenin antibody (#610154), and the monoclonal anti-actin antibody (clone AC-40) (#A3853) were purchased from Cell Signaling Technology (Boston, MA), BD Transduction Laboratories, and Sigma, respectively.

    Techniques: Expressing

    Sustained inhibition of Wnt signaling can recover C4ST-1 expression. A , L and L-Wnt-3a cells were incubated in the presence or absence of 5 μ m XAV939 for 7 days, and cytosolic β-catenin accumulation was examined in each cell and treatment

    Journal: The Journal of Biological Chemistry

    Article Title: Down-regulation of Chondroitin 4-O-Sulfotransferase-1 by Wnt Signaling Triggers Diffusion of Wnt-3a *

    doi: 10.1074/jbc.M110.155093

    Figure Lengend Snippet: Sustained inhibition of Wnt signaling can recover C4ST-1 expression. A , L and L-Wnt-3a cells were incubated in the presence or absence of 5 μ m XAV939 for 7 days, and cytosolic β-catenin accumulation was examined in each cell and treatment

    Article Snippet: Anti-Wnt-3a antibody (#2391), the anti-β-catenin antibody (#610154), and the monoclonal anti-actin antibody (clone AC-40) (#A3853) were purchased from Cell Signaling Technology (Boston, MA), BD Transduction Laboratories, and Sigma, respectively.

    Techniques: Inhibition, Expressing, Incubation

    Wnt-3a preferentially binds to E-disaccharide units. A , L cells were treated with rWnt-3a preincubated with or without competitor GAGs prepared from the indicated cells. Accumulated β-catenin in the cytosol was examined by immunoblotting. Each

    Journal: The Journal of Biological Chemistry

    Article Title: Down-regulation of Chondroitin 4-O-Sulfotransferase-1 by Wnt Signaling Triggers Diffusion of Wnt-3a *

    doi: 10.1074/jbc.M110.155093

    Figure Lengend Snippet: Wnt-3a preferentially binds to E-disaccharide units. A , L cells were treated with rWnt-3a preincubated with or without competitor GAGs prepared from the indicated cells. Accumulated β-catenin in the cytosol was examined by immunoblotting. Each

    Article Snippet: Anti-Wnt-3a antibody (#2391), the anti-β-catenin antibody (#610154), and the monoclonal anti-actin antibody (clone AC-40) (#A3853) were purchased from Cell Signaling Technology (Boston, MA), BD Transduction Laboratories, and Sigma, respectively.

    Techniques:

    The expression of C4ST-1 gene was suppressed in C2C12 cells expressing Wnt-3a. A , two cell lines of C2C12 cells expressing FLAG-tagged Wnt-3a proteins were established and named clone 1 ( panels a and b ) and clone 2 ( panels c and d ). C4ST-1 mRNA expression

    Journal: The Journal of Biological Chemistry

    Article Title: Down-regulation of Chondroitin 4-O-Sulfotransferase-1 by Wnt Signaling Triggers Diffusion of Wnt-3a *

    doi: 10.1074/jbc.M110.155093

    Figure Lengend Snippet: The expression of C4ST-1 gene was suppressed in C2C12 cells expressing Wnt-3a. A , two cell lines of C2C12 cells expressing FLAG-tagged Wnt-3a proteins were established and named clone 1 ( panels a and b ) and clone 2 ( panels c and d ). C4ST-1 mRNA expression

    Article Snippet: Anti-Wnt-3a antibody (#2391), the anti-β-catenin antibody (#610154), and the monoclonal anti-actin antibody (clone AC-40) (#A3853) were purchased from Cell Signaling Technology (Boston, MA), BD Transduction Laboratories, and Sigma, respectively.

    Techniques: Expressing

    A model of C4ST-1-mediated regulation of Wnt-3a diffusion from Wnt-producing. Step 1 , Wnt acts in an autocrine manner in Wnt-producing cells. Step 2 , secreted Wnt molecules are caught by CS chains expressing on the cell surface or in extracellular matrix.

    Journal: The Journal of Biological Chemistry

    Article Title: Down-regulation of Chondroitin 4-O-Sulfotransferase-1 by Wnt Signaling Triggers Diffusion of Wnt-3a *

    doi: 10.1074/jbc.M110.155093

    Figure Lengend Snippet: A model of C4ST-1-mediated regulation of Wnt-3a diffusion from Wnt-producing. Step 1 , Wnt acts in an autocrine manner in Wnt-producing cells. Step 2 , secreted Wnt molecules are caught by CS chains expressing on the cell surface or in extracellular matrix.

    Article Snippet: Anti-Wnt-3a antibody (#2391), the anti-β-catenin antibody (#610154), and the monoclonal anti-actin antibody (clone AC-40) (#A3853) were purchased from Cell Signaling Technology (Boston, MA), BD Transduction Laboratories, and Sigma, respectively.

    Techniques: Diffusion-based Assay, Expressing

    Wnt-3a can suppress the expression level of C4ST-1 in a non-cell autonomous manner. HeLa cells ( A ) and human embryonic fibroblasts ( B ) were cultured with L or L-Wnt-3a cells for the indicated periods, and C4ST-1 gene expression was measured by real-time

    Journal: The Journal of Biological Chemistry

    Article Title: Down-regulation of Chondroitin 4-O-Sulfotransferase-1 by Wnt Signaling Triggers Diffusion of Wnt-3a *

    doi: 10.1074/jbc.M110.155093

    Figure Lengend Snippet: Wnt-3a can suppress the expression level of C4ST-1 in a non-cell autonomous manner. HeLa cells ( A ) and human embryonic fibroblasts ( B ) were cultured with L or L-Wnt-3a cells for the indicated periods, and C4ST-1 gene expression was measured by real-time

    Article Snippet: Anti-Wnt-3a antibody (#2391), the anti-β-catenin antibody (#610154), and the monoclonal anti-actin antibody (clone AC-40) (#A3853) were purchased from Cell Signaling Technology (Boston, MA), BD Transduction Laboratories, and Sigma, respectively.

    Techniques: Expressing, Cell Culture

    Forced expression of C4ST-1 in L-Wnt-3a cells hardly affected GalNAc4S-6ST and Wnt-3a mRNA levels or Wnt-3a N -glycosylation. A , the mRNA levels of C4ST-1 , GalNAc4S-6ST , and Wnt-3a in parental L cells, L-Wnt-3a cells, and L-Wnt-3a-C4ST-1 clones were analyzed

    Journal: The Journal of Biological Chemistry

    Article Title: Down-regulation of Chondroitin 4-O-Sulfotransferase-1 by Wnt Signaling Triggers Diffusion of Wnt-3a *

    doi: 10.1074/jbc.M110.155093

    Figure Lengend Snippet: Forced expression of C4ST-1 in L-Wnt-3a cells hardly affected GalNAc4S-6ST and Wnt-3a mRNA levels or Wnt-3a N -glycosylation. A , the mRNA levels of C4ST-1 , GalNAc4S-6ST , and Wnt-3a in parental L cells, L-Wnt-3a cells, and L-Wnt-3a-C4ST-1 clones were analyzed

    Article Snippet: Anti-Wnt-3a antibody (#2391), the anti-β-catenin antibody (#610154), and the monoclonal anti-actin antibody (clone AC-40) (#A3853) were purchased from Cell Signaling Technology (Boston, MA), BD Transduction Laboratories, and Sigma, respectively.

    Techniques: Expressing, Clone Assay

    Effects of mechanical vibration stimulation on in vitro osteogenesis-related gene expression for primary rabbit osteoblasts seeded in pTi via qRT-PCR analyses, including ALP, OCN, Runx2, BMP2, OPG, Wnt3a, Lrp6 and β-catenin. Values are all expressed as mean ± S.D. ( n = 4), and the relative expression level of each gene was normalized to β-Actin. * Significant difference from the Control group with P

    Journal: Scientific Reports

    Article Title: Effect of low-level mechanical vibration on osteogenesis and osseointegration of porous titanium implants in the repair of long bone defects

    doi: 10.1038/srep17134

    Figure Lengend Snippet: Effects of mechanical vibration stimulation on in vitro osteogenesis-related gene expression for primary rabbit osteoblasts seeded in pTi via qRT-PCR analyses, including ALP, OCN, Runx2, BMP2, OPG, Wnt3a, Lrp6 and β-catenin. Values are all expressed as mean ± S.D. ( n = 4), and the relative expression level of each gene was normalized to β-Actin. * Significant difference from the Control group with P

    Article Snippet: Furthermore, our results demonstrate that WBV enhanced gene expression in canonical Wnt signaling, as evidenced by increased Wnt3a, Lrp6 and β-catenin mRNA levels.

    Techniques: In Vitro, Expressing, Quantitative RT-PCR, ALP Assay

    Effects of WBV stimulation for 6 weeks and 12 weeks on in vivo osteogenesis-related gene expression in rabbit femora via qRT-PCR analyses, including ALP, OCN, Runx2, BMP2, Sost, OPG, RANKL, Wnt3a, Lrp6 and β-catenin. Values are all expressed as mean ± S.D. ( n = 3 ~ 6) and the relative expression level of each gene was normalized to β-Actin. * Significant difference from the Control group with P

    Journal: Scientific Reports

    Article Title: Effect of low-level mechanical vibration on osteogenesis and osseointegration of porous titanium implants in the repair of long bone defects

    doi: 10.1038/srep17134

    Figure Lengend Snippet: Effects of WBV stimulation for 6 weeks and 12 weeks on in vivo osteogenesis-related gene expression in rabbit femora via qRT-PCR analyses, including ALP, OCN, Runx2, BMP2, Sost, OPG, RANKL, Wnt3a, Lrp6 and β-catenin. Values are all expressed as mean ± S.D. ( n = 3 ~ 6) and the relative expression level of each gene was normalized to β-Actin. * Significant difference from the Control group with P

    Article Snippet: Furthermore, our results demonstrate that WBV enhanced gene expression in canonical Wnt signaling, as evidenced by increased Wnt3a, Lrp6 and β-catenin mRNA levels.

    Techniques: In Vivo, Expressing, Quantitative RT-PCR, ALP Assay

    Effects of mechanical vibration stimulation on in vitro osteogenesis-related protein expression for primary rabbit osteoblasts seeded in pTi via western blotting analyses, including OCN, Runx2, Wnt3a, Lrp6 and β-catenin. Values are all expressed as mean ± S.D. ( n = 3 ~ 4). The relative expression level of each protein was normalized to β-Tubulin. * Significant difference from the Control group with P

    Journal: Scientific Reports

    Article Title: Effect of low-level mechanical vibration on osteogenesis and osseointegration of porous titanium implants in the repair of long bone defects

    doi: 10.1038/srep17134

    Figure Lengend Snippet: Effects of mechanical vibration stimulation on in vitro osteogenesis-related protein expression for primary rabbit osteoblasts seeded in pTi via western blotting analyses, including OCN, Runx2, Wnt3a, Lrp6 and β-catenin. Values are all expressed as mean ± S.D. ( n = 3 ~ 4). The relative expression level of each protein was normalized to β-Tubulin. * Significant difference from the Control group with P

    Article Snippet: Furthermore, our results demonstrate that WBV enhanced gene expression in canonical Wnt signaling, as evidenced by increased Wnt3a, Lrp6 and β-catenin mRNA levels.

    Techniques: In Vitro, Expressing, Western Blot