rabbit anti wnt1  (Danaher Inc)


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    Danaher Inc rabbit anti wnt1
    Effect of <t>Wnt1-positive</t> expression on proliferative and migratory C6 GSCs in different groups. (A) The positive expression of Wnt1 on C6 GSCs was observed by immunofluorescence staining. Scale bar, 100 µm. (B) The statistical data of the number of Wnt1-positive cells are shown in each group. *P<0.05 and ***P<0.001. GSCs, glioma stem cells; ns, no significance.
    Rabbit Anti Wnt1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti wnt1/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti wnt1 - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "Inhibitor of Wnt receptor 1 suppresses the effects of Wnt1, Wnt3a and β‑catenin on the proliferation and migration of C6 GSCs induced by low‑dose radiation"

    Article Title: Inhibitor of Wnt receptor 1 suppresses the effects of Wnt1, Wnt3a and β‑catenin on the proliferation and migration of C6 GSCs induced by low‑dose radiation

    Journal: Oncology Reports

    doi: 10.3892/or.2023.8681

    Effect of Wnt1-positive expression on proliferative and migratory C6 GSCs in different groups. (A) The positive expression of Wnt1 on C6 GSCs was observed by immunofluorescence staining. Scale bar, 100 µm. (B) The statistical data of the number of Wnt1-positive cells are shown in each group. *P<0.05 and ***P<0.001. GSCs, glioma stem cells; ns, no significance.
    Figure Legend Snippet: Effect of Wnt1-positive expression on proliferative and migratory C6 GSCs in different groups. (A) The positive expression of Wnt1 on C6 GSCs was observed by immunofluorescence staining. Scale bar, 100 µm. (B) The statistical data of the number of Wnt1-positive cells are shown in each group. *P<0.05 and ***P<0.001. GSCs, glioma stem cells; ns, no significance.

    Techniques Used: Expressing, Immunofluorescence, Staining

    Changes in Wnt1, Wnt3a and β-catenin protein levels in proliferative and migratory C6 glioma stem cells in different groups. (A) By Western blot analysis, the Wnt1, Wnt3a and β-catenin proteins were detected in the different groups. β-actin was used as the internal control. (B-D) The gray value quantification of (B) Wnt1, (C) Wnt3a and (D) β-catenin protein, normalized to β-actin. *P<0.05, **P<0.01 and ***P<0.001. ns, no significance.
    Figure Legend Snippet: Changes in Wnt1, Wnt3a and β-catenin protein levels in proliferative and migratory C6 glioma stem cells in different groups. (A) By Western blot analysis, the Wnt1, Wnt3a and β-catenin proteins were detected in the different groups. β-actin was used as the internal control. (B-D) The gray value quantification of (B) Wnt1, (C) Wnt3a and (D) β-catenin protein, normalized to β-actin. *P<0.05, **P<0.01 and ***P<0.001. ns, no significance.

    Techniques Used: Western Blot

    antibodies against rabbit polyclonal anti human wnt1  (Abcam)

     
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    Abcam antibodies against rabbit polyclonal anti human wnt1
    miR-424 activates Wnt/β-catenin signaling pathway by directly targeting APC. (A) Wester blots and (B) protein levels of APC, <t>Wnt1,</t> β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 mimics + pcDNA-empty or miR-424 mimics + pcDNA-APC. (C) Western blots and (D) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 inhibitor + si-scramble or miR-424 inhibitor + si-APC. Data are presented as means ± standard deviation of three individual experiments. * P<0.05 and ** P<0.01 vs. control. ## P<0.01 vs. miR-424 mimics + pcDNA-vector or miR-424 inhibitor + si-scramble. APC, adenomatous polyposis coli; miR, microRNA; si, small interfering.
    Antibodies Against Rabbit Polyclonal Anti Human Wnt1, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against rabbit polyclonal anti human wnt1/product/Abcam
    Average 86 stars, based on 1 article reviews
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    antibodies against rabbit polyclonal anti human wnt1 - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "miR‑424‑5p is downregulated in the placentas of patients with preeclampsia and affects trophoblast migration and invasion"

    Article Title: miR‑424‑5p is downregulated in the placentas of patients with preeclampsia and affects trophoblast migration and invasion

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2023.11993

    miR-424 activates Wnt/β-catenin signaling pathway by directly targeting APC. (A) Wester blots and (B) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 mimics + pcDNA-empty or miR-424 mimics + pcDNA-APC. (C) Western blots and (D) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 inhibitor + si-scramble or miR-424 inhibitor + si-APC. Data are presented as means ± standard deviation of three individual experiments. * P<0.05 and ** P<0.01 vs. control. ## P<0.01 vs. miR-424 mimics + pcDNA-vector or miR-424 inhibitor + si-scramble. APC, adenomatous polyposis coli; miR, microRNA; si, small interfering.
    Figure Legend Snippet: miR-424 activates Wnt/β-catenin signaling pathway by directly targeting APC. (A) Wester blots and (B) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 mimics + pcDNA-empty or miR-424 mimics + pcDNA-APC. (C) Western blots and (D) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 inhibitor + si-scramble or miR-424 inhibitor + si-APC. Data are presented as means ± standard deviation of three individual experiments. * P<0.05 and ** P<0.01 vs. control. ## P<0.01 vs. miR-424 mimics + pcDNA-vector or miR-424 inhibitor + si-scramble. APC, adenomatous polyposis coli; miR, microRNA; si, small interfering.

    Techniques Used: Transfection, Western Blot, Standard Deviation, Plasmid Preparation


    Structured Review

    Abcam rabbit polyclonal anti wnt1 antibody
    HepG2 cells were mock infected, or infected with Ad-HCV core or Ad-EGFP for 48 h, and were harvested for preparation of total RNA and protein. ( A ) Infection efficiency of the adenovirus observed under the fluorescent microscope (×10). ( B ) Western blot analysis for HCV core protein expression. ( C ) SLqRT-PCR analysis for miR-152 expression. After normalization to endogenous RNU6B expression, the expression of miR-152 in each group of HepG2 cells was expressed as fold changes in comparison to the levels observed in mock-infected HepG2 cells. ( D ) Real time RT-PCR analysis for <t>Wnt1</t> mRNA expression. ( E ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (HepG2: mock-infected HepG2 cells; Hcv: HepG2 cells infected with the Ad-HCV core; Co: HepG2 cells infected with the Ad-EGFP control; Advirus-Hcv: Adenovirus expressing the HCV core protein, Ad-HCV core; Advirus-Ctrl: Adenovirus expressing the EGFP control protein, Ad-EGFP).
    Rabbit Polyclonal Anti Wnt1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti wnt1 antibody/product/Abcam
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti wnt1 antibody - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells"

    Article Title: HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081730

    HepG2 cells were mock infected, or infected with Ad-HCV core or Ad-EGFP for 48 h, and were harvested for preparation of total RNA and protein. ( A ) Infection efficiency of the adenovirus observed under the fluorescent microscope (×10). ( B ) Western blot analysis for HCV core protein expression. ( C ) SLqRT-PCR analysis for miR-152 expression. After normalization to endogenous RNU6B expression, the expression of miR-152 in each group of HepG2 cells was expressed as fold changes in comparison to the levels observed in mock-infected HepG2 cells. ( D ) Real time RT-PCR analysis for Wnt1 mRNA expression. ( E ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (HepG2: mock-infected HepG2 cells; Hcv: HepG2 cells infected with the Ad-HCV core; Co: HepG2 cells infected with the Ad-EGFP control; Advirus-Hcv: Adenovirus expressing the HCV core protein, Ad-HCV core; Advirus-Ctrl: Adenovirus expressing the EGFP control protein, Ad-EGFP).
    Figure Legend Snippet: HepG2 cells were mock infected, or infected with Ad-HCV core or Ad-EGFP for 48 h, and were harvested for preparation of total RNA and protein. ( A ) Infection efficiency of the adenovirus observed under the fluorescent microscope (×10). ( B ) Western blot analysis for HCV core protein expression. ( C ) SLqRT-PCR analysis for miR-152 expression. After normalization to endogenous RNU6B expression, the expression of miR-152 in each group of HepG2 cells was expressed as fold changes in comparison to the levels observed in mock-infected HepG2 cells. ( D ) Real time RT-PCR analysis for Wnt1 mRNA expression. ( E ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (HepG2: mock-infected HepG2 cells; Hcv: HepG2 cells infected with the Ad-HCV core; Co: HepG2 cells infected with the Ad-EGFP control; Advirus-Hcv: Adenovirus expressing the HCV core protein, Ad-HCV core; Advirus-Ctrl: Adenovirus expressing the EGFP control protein, Ad-EGFP).

    Techniques Used: Infection, Microscopy, Western Blot, Expressing, Quantitative RT-PCR

    The two WT plasmids, pGL3-WNT1-1-wt-3′UTR and pGL3-WNT1-2-wt-3′UTR, including 90 nt spanning each seed match at the 3′UTR, as well as the two mutant controls, pGL3-WNT1-1-mut-3′UTR and pGL3-WNT1-2-mut-3′UTR, were generated based on the firefly luciferase expressing vector pGL3-promoter and confirmed by sequencing. HepG2 Cells were co-transfected with 250 ng of pGL3-WNT1-WT or pGL3-WNT1-Mut constructs with 23.5 nM of miR-152 mimics, miR-152 inhibitor, or their respective negative control. Each sample was co-transfected with 25 ng of pRL-TK plasmid expressing renilla luciferase to monitor the transfection efficiency. A luciferase activity assay was performed 48 hours after transfection with the dual luciferase reporter assay system (Promega). The relative luciferase activity was normalized with renilla luciferase activity. ( A ) WT and Mut 3′-UTRs of WNT1, indicating the interaction sites between miR-152 and 3′-UTR of WNT1. ( B ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-1-WT or WNT1-1-Mut 3′-UTR and miR-152 mimics or its negative control (NC). ( C ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 mimics or miR-152 mimics NC. ( D ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 inhibitor or miR-152 inhibitor NC. Data are shown as means and standard deviations from at least three independent experiments. ** P<0.01.
    Figure Legend Snippet: The two WT plasmids, pGL3-WNT1-1-wt-3′UTR and pGL3-WNT1-2-wt-3′UTR, including 90 nt spanning each seed match at the 3′UTR, as well as the two mutant controls, pGL3-WNT1-1-mut-3′UTR and pGL3-WNT1-2-mut-3′UTR, were generated based on the firefly luciferase expressing vector pGL3-promoter and confirmed by sequencing. HepG2 Cells were co-transfected with 250 ng of pGL3-WNT1-WT or pGL3-WNT1-Mut constructs with 23.5 nM of miR-152 mimics, miR-152 inhibitor, or their respective negative control. Each sample was co-transfected with 25 ng of pRL-TK plasmid expressing renilla luciferase to monitor the transfection efficiency. A luciferase activity assay was performed 48 hours after transfection with the dual luciferase reporter assay system (Promega). The relative luciferase activity was normalized with renilla luciferase activity. ( A ) WT and Mut 3′-UTRs of WNT1, indicating the interaction sites between miR-152 and 3′-UTR of WNT1. ( B ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-1-WT or WNT1-1-Mut 3′-UTR and miR-152 mimics or its negative control (NC). ( C ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 mimics or miR-152 mimics NC. ( D ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 inhibitor or miR-152 inhibitor NC. Data are shown as means and standard deviations from at least three independent experiments. ** P<0.01.

    Techniques Used: Mutagenesis, Generated, Luciferase, Expressing, Plasmid Preparation, Sequencing, Transfection, Construct, Negative Control, Activity Assay, Reporter Assay

    HepG2 cells were transfected with double-stranded miR-152 mimics, single-stranded miR-152 inhibitor, or their relative negative control RNA at a final concentration of 5 nM with a HiPerFect Transfection Reagent kit as indicated for 48 h, and were collected for preparation of total RNA and protein. ( A ) Real time RT-PCR analysis for Wnt1 mRNA expression. ( B ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (inh NC: scrambled oligonucleotides as the negative control for miR-152 inhibitor; inh-152: miR-152 inhibitor; miR NC: scrambled oligonucleotides as the negative control for miR-152 mimics; miR-152: miR-152 mimics; Hcv+miR-152: Adenovirus expressing the HCV core protein+miR-152 mimics).
    Figure Legend Snippet: HepG2 cells were transfected with double-stranded miR-152 mimics, single-stranded miR-152 inhibitor, or their relative negative control RNA at a final concentration of 5 nM with a HiPerFect Transfection Reagent kit as indicated for 48 h, and were collected for preparation of total RNA and protein. ( A ) Real time RT-PCR analysis for Wnt1 mRNA expression. ( B ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (inh NC: scrambled oligonucleotides as the negative control for miR-152 inhibitor; inh-152: miR-152 inhibitor; miR NC: scrambled oligonucleotides as the negative control for miR-152 mimics; miR-152: miR-152 mimics; Hcv+miR-152: Adenovirus expressing the HCV core protein+miR-152 mimics).

    Techniques Used: Transfection, Negative Control, Concentration Assay, Quantitative RT-PCR, Expressing, Western Blot


    Structured Review

    Abcam rabbit anti wnt1
    <t>Wnt1</t> protects primary mesencephalic dopaminergic (DA) neurons against cell death induced by serum deprivation (SD), 6-hydroxydopamine (6-OHDA) and MPP + . Enriched neuronal cultures derived from the mesencephalon of E14 rat embryos maintained for 9-12 days in vitro (DIV) were shifted to a medium without serum and growth factors (A, 12-72 h) or to increasing concentrations (5-50 μM) of 6-OHDA or MPP + (B) in the absence or the presence of a treatment with Wnt1 (100 ng/ml). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : DA neuron survival assessed by counting TH + neurons over the DAPI + nuclei, and expressed as percent (%) of PBS-treated control. C : [ 3 H]dopamine incorporation was assessed 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM), the values expressed as % of control. D: Caspase activity was determined by measuring DEVD-AFC hydrolysis. Enzymatic determinations were performed in lysates from cell cultures deprived of serum or challenged with 6-OHDA or MPP + for 6 h. *p < 0.05 when compared to control (PBS); ° p < 0.05 when compared to sister cultures exposed the cytotoxic insult (within each experimental group). E-J : Representative images showing TH + neurons (in red, E) and DAPI nuclear counterstaining (blue) 24 h after PBS, after 48 h of SD (F), or 24 h after 6-OHDA (G), in absence or the presence of Wnt1 pre-treatment (H,I,J). MPP + sharply decreases TH neurite length, an effect efficiently counteracted by Wnt1.
    Rabbit Anti Wnt1, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti wnt1/product/Abcam
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti wnt1 - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection"

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-6-49

    Wnt1 protects primary mesencephalic dopaminergic (DA) neurons against cell death induced by serum deprivation (SD), 6-hydroxydopamine (6-OHDA) and MPP + . Enriched neuronal cultures derived from the mesencephalon of E14 rat embryos maintained for 9-12 days in vitro (DIV) were shifted to a medium without serum and growth factors (A, 12-72 h) or to increasing concentrations (5-50 μM) of 6-OHDA or MPP + (B) in the absence or the presence of a treatment with Wnt1 (100 ng/ml). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : DA neuron survival assessed by counting TH + neurons over the DAPI + nuclei, and expressed as percent (%) of PBS-treated control. C : [ 3 H]dopamine incorporation was assessed 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM), the values expressed as % of control. D: Caspase activity was determined by measuring DEVD-AFC hydrolysis. Enzymatic determinations were performed in lysates from cell cultures deprived of serum or challenged with 6-OHDA or MPP + for 6 h. *p < 0.05 when compared to control (PBS); ° p < 0.05 when compared to sister cultures exposed the cytotoxic insult (within each experimental group). E-J : Representative images showing TH + neurons (in red, E) and DAPI nuclear counterstaining (blue) 24 h after PBS, after 48 h of SD (F), or 24 h after 6-OHDA (G), in absence or the presence of Wnt1 pre-treatment (H,I,J). MPP + sharply decreases TH neurite length, an effect efficiently counteracted by Wnt1.
    Figure Legend Snippet: Wnt1 protects primary mesencephalic dopaminergic (DA) neurons against cell death induced by serum deprivation (SD), 6-hydroxydopamine (6-OHDA) and MPP + . Enriched neuronal cultures derived from the mesencephalon of E14 rat embryos maintained for 9-12 days in vitro (DIV) were shifted to a medium without serum and growth factors (A, 12-72 h) or to increasing concentrations (5-50 μM) of 6-OHDA or MPP + (B) in the absence or the presence of a treatment with Wnt1 (100 ng/ml). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : DA neuron survival assessed by counting TH + neurons over the DAPI + nuclei, and expressed as percent (%) of PBS-treated control. C : [ 3 H]dopamine incorporation was assessed 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM), the values expressed as % of control. D: Caspase activity was determined by measuring DEVD-AFC hydrolysis. Enzymatic determinations were performed in lysates from cell cultures deprived of serum or challenged with 6-OHDA or MPP + for 6 h. *p < 0.05 when compared to control (PBS); ° p < 0.05 when compared to sister cultures exposed the cytotoxic insult (within each experimental group). E-J : Representative images showing TH + neurons (in red, E) and DAPI nuclear counterstaining (blue) 24 h after PBS, after 48 h of SD (F), or 24 h after 6-OHDA (G), in absence or the presence of Wnt1 pre-treatment (H,I,J). MPP + sharply decreases TH neurite length, an effect efficiently counteracted by Wnt1.

    Techniques Used: Derivative Assay, In Vitro, Activity Assay

    β-catenin depletion abolishes Wnt1- induced TH + neuroprotection . Enriched neuronal cultures were transiently transfected with β-catenin small interference RNA (β-catenin siRNA ) (sc-29210) or control siRNA (β-catenin Ct , see text for details), before being exposed to either Wnt1 or PBS, and DA neuron survival assessed by counting TH + neurons, by assessing [ 3 H]dopamine incorporation and Caspase3 activity. Differences analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B: Effect of SD, 6-OHDA and MPP + treatments in β-catenin mRNA (A) and protein levels (B) showing a significant decrease of β-catenin . Note that preventive application of Wnt1 increases β-catenin both at a mRNA (A) and protein levels (B). *p < 0.05 vs PBS. Depletion of β-catenin via the introduction of β-catenin siRNA shows an almost 40-60% reduction in β-catenin mRNA by Real time PCR (A) and western blotting (B). *p < 0.05 vs control siRNA C-D : Survival of TH + neurons by cell counting (C), [ 3 H]dopamine incorporation (D). Note that in β-catenin siRNA pre-treated cultures, the application of Wnt1 failed to protect TH + neurons against 6-OHDA or MPP + , whereas in cultures pre-treated with a control siRNA, Wnt1 treatment increased TH + neuron survival (C) and [ 3 H]dopamine incorporation (D). E-L : Representative immunocytochemical images show the ability of Wnt1 to efficiently counteracts TH neuron death and neurite loss, an effect abolished by β-catenin silencing *p < 0.05 vs cultures without cytotoxic insult; ** p < 0.05 compared to siRNA Ct + cytotoxic insult (within each each experimental group); ° p < 0.05 compared to Wnt1 treated cultures in the presence of siRNA Ct .
    Figure Legend Snippet: β-catenin depletion abolishes Wnt1- induced TH + neuroprotection . Enriched neuronal cultures were transiently transfected with β-catenin small interference RNA (β-catenin siRNA ) (sc-29210) or control siRNA (β-catenin Ct , see text for details), before being exposed to either Wnt1 or PBS, and DA neuron survival assessed by counting TH + neurons, by assessing [ 3 H]dopamine incorporation and Caspase3 activity. Differences analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B: Effect of SD, 6-OHDA and MPP + treatments in β-catenin mRNA (A) and protein levels (B) showing a significant decrease of β-catenin . Note that preventive application of Wnt1 increases β-catenin both at a mRNA (A) and protein levels (B). *p < 0.05 vs PBS. Depletion of β-catenin via the introduction of β-catenin siRNA shows an almost 40-60% reduction in β-catenin mRNA by Real time PCR (A) and western blotting (B). *p < 0.05 vs control siRNA C-D : Survival of TH + neurons by cell counting (C), [ 3 H]dopamine incorporation (D). Note that in β-catenin siRNA pre-treated cultures, the application of Wnt1 failed to protect TH + neurons against 6-OHDA or MPP + , whereas in cultures pre-treated with a control siRNA, Wnt1 treatment increased TH + neuron survival (C) and [ 3 H]dopamine incorporation (D). E-L : Representative immunocytochemical images show the ability of Wnt1 to efficiently counteracts TH neuron death and neurite loss, an effect abolished by β-catenin silencing *p < 0.05 vs cultures without cytotoxic insult; ** p < 0.05 compared to siRNA Ct + cytotoxic insult (within each each experimental group); ° p < 0.05 compared to Wnt1 treated cultures in the presence of siRNA Ct .

    Techniques Used: Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting

    Knocking down Fzd-1 counteracts Wnt1- induced TH + neuroprotection . Enriched neuronal cultures were processed for Real time PCR using specific primers for Fzd receptors. The effect of knocking down Fzd-1 in Wnt1 neuroprotection was studied with Fzd-1 sense (Fzd-1Ct) or antisense (Fzd-1AS) oligonucleotides. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A : Differential expression and regulation of Fzd transcripts by SD, 6-OHDA and MPP + . B: Western blot (wb) analysis showing down-regulation of Fzd-1 levels in neuronal cultures exposed to the cytotoxic stimuli and the significant reversal induced by Wnt1 . *p < 0.05 vs cultures without cytotoxic insult. Pre-treatment with Fzd-1 AS induced an almost 40-60% decrease of Fzd-1. C-H : Representative confocal images of dual staining with Fzd-1 (green) and TH (red) showing colocalization (orange to yellow) in PBS (C-D) controls. Note the marked loss of Fzd-1 in TH neurons exposed to 6-OHDA (E) or MPP + (F), an effect efficiently counteracted by Wnt1 pre-treatment (G). I-J : Survival of TH + neurons by cell counting (C), [ 3 H]dopamine incorporation (D). K-L : Effect of Fzd-1 AS or Fzd-1 Ct , in β-catenin protein and Caspase3-like activity. Fzd-1 AS pre-treatment prevents Wnt1- induced increased β-catenin protein levels (K) and reverses Wnt1-induced Caspase3-inhibition (L) in 6-OHDA and MPP + -treated cultures. *p < 0.05 vs PBS. *p < 0.05 vs control siRNA. Note that Wnt1 efficiently reversed the dramatic decrease of neurite length caused by 6-OHDA or MPP + in Fzd-1 Ct -treated (M, N), as opposed to Fzd-1 knocked down cultures (O, P). *p < 0.05 vs cultures without insult (within each each experimental group).
    Figure Legend Snippet: Knocking down Fzd-1 counteracts Wnt1- induced TH + neuroprotection . Enriched neuronal cultures were processed for Real time PCR using specific primers for Fzd receptors. The effect of knocking down Fzd-1 in Wnt1 neuroprotection was studied with Fzd-1 sense (Fzd-1Ct) or antisense (Fzd-1AS) oligonucleotides. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A : Differential expression and regulation of Fzd transcripts by SD, 6-OHDA and MPP + . B: Western blot (wb) analysis showing down-regulation of Fzd-1 levels in neuronal cultures exposed to the cytotoxic stimuli and the significant reversal induced by Wnt1 . *p < 0.05 vs cultures without cytotoxic insult. Pre-treatment with Fzd-1 AS induced an almost 40-60% decrease of Fzd-1. C-H : Representative confocal images of dual staining with Fzd-1 (green) and TH (red) showing colocalization (orange to yellow) in PBS (C-D) controls. Note the marked loss of Fzd-1 in TH neurons exposed to 6-OHDA (E) or MPP + (F), an effect efficiently counteracted by Wnt1 pre-treatment (G). I-J : Survival of TH + neurons by cell counting (C), [ 3 H]dopamine incorporation (D). K-L : Effect of Fzd-1 AS or Fzd-1 Ct , in β-catenin protein and Caspase3-like activity. Fzd-1 AS pre-treatment prevents Wnt1- induced increased β-catenin protein levels (K) and reverses Wnt1-induced Caspase3-inhibition (L) in 6-OHDA and MPP + -treated cultures. *p < 0.05 vs PBS. *p < 0.05 vs control siRNA. Note that Wnt1 efficiently reversed the dramatic decrease of neurite length caused by 6-OHDA or MPP + in Fzd-1 Ct -treated (M, N), as opposed to Fzd-1 knocked down cultures (O, P). *p < 0.05 vs cultures without insult (within each each experimental group).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining, Cell Counting, Activity Assay, Inhibition

    Effect of Wnt1-induced TH + neuroprotection on active GSK-3β and Caspase3-like activity . Purified neuronal cultures were exposed to SD, 6-OHDA or MPP + insults as described, and DA neuroprotection examined as above. Active GSK-3β (GSK-3β phospho-Tyr216) protein levels were determined by western blotting (wb). Part of the cultures were pre-treated with Wnt1 (100 ng/ml) or the the specific GSK-3β antagonist, AR-14418 (5 μM), alone or in combination, while other cell cultures were exposed to the specific antagonist of Wnt/β-catenin pathway, Dkk1 (100 ng/ml), 1 hr before Wnt1 (100 ng/ml) treatment. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : Active GSK-3β (GSK-3β phospho-Tyr216) protein levels as determined by wb. A sharp up-regulation is observed after SD, 6-OHDA or MPP + insults, an effect reversed by Wnt1 and AR pre-treatment. B : Survival of TH + neurons exposed to the described insults in the absence or the presence of the different treatments. * p < 0.05 when compared to control cultures without cytotoxic insults. Note that both Wnt1 and AR increase TH + neuron survival, and the combined Wnt1+AR treatment further increase TH + neuron survival. By contrast, exposure of Dkk1-pretreated cultures to SD, 6-OHDA or MPP + reduced Wnt1 protective effect on cell survival. C : Caspase-3 activity shows a sharp inhibition of DEVD-like activity in neurons pre-treated with Wnt1 or AR and exposed to the different insults, whereas previous exposure to Dkk1, counteracts Wnt1-induced Casapse3 inhibition. * p < 0.05 when compared to control cultures without cytotoxic insults.
    Figure Legend Snippet: Effect of Wnt1-induced TH + neuroprotection on active GSK-3β and Caspase3-like activity . Purified neuronal cultures were exposed to SD, 6-OHDA or MPP + insults as described, and DA neuroprotection examined as above. Active GSK-3β (GSK-3β phospho-Tyr216) protein levels were determined by western blotting (wb). Part of the cultures were pre-treated with Wnt1 (100 ng/ml) or the the specific GSK-3β antagonist, AR-14418 (5 μM), alone or in combination, while other cell cultures were exposed to the specific antagonist of Wnt/β-catenin pathway, Dkk1 (100 ng/ml), 1 hr before Wnt1 (100 ng/ml) treatment. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : Active GSK-3β (GSK-3β phospho-Tyr216) protein levels as determined by wb. A sharp up-regulation is observed after SD, 6-OHDA or MPP + insults, an effect reversed by Wnt1 and AR pre-treatment. B : Survival of TH + neurons exposed to the described insults in the absence or the presence of the different treatments. * p < 0.05 when compared to control cultures without cytotoxic insults. Note that both Wnt1 and AR increase TH + neuron survival, and the combined Wnt1+AR treatment further increase TH + neuron survival. By contrast, exposure of Dkk1-pretreated cultures to SD, 6-OHDA or MPP + reduced Wnt1 protective effect on cell survival. C : Caspase-3 activity shows a sharp inhibition of DEVD-like activity in neurons pre-treated with Wnt1 or AR and exposed to the different insults, whereas previous exposure to Dkk1, counteracts Wnt1-induced Casapse3 inhibition. * p < 0.05 when compared to control cultures without cytotoxic insults.

    Techniques Used: Activity Assay, Purification, Western Blot, Inhibition

    Modulation of astroglial Wnt1/β-catenin signaling directs towards TH neuron survival/death . Astrocyte-neuron co-cultures where shifted to serum deprived medium (SD, A), or received 6-OHDA or MPP + (25 μM), with or without Dkk1 (100 ng/ml), a Wnt1-Ab , or AR-14418 (5 μM) as described. A-B : TH + neuron counts (A), and [ 3 H]dopamine uptake (B) 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM) with or without Dkk1, Wnt1-Ab, or AR. * p < 0.05 compared to controls. C: Western blot analysis showing β-catenin levels in neurons exposed to astrocyte insert upon cytotoxic challenge with or without Wnt1-Ab or Dkk1. In this experimental condition, the glial inserts were added on the top of the purified neurons at 9 DIV. D : immunoblotting with Wnt1-Ab (50) using protein extracts from embryonic VM and the NIH/3T3 cell line. 50 ng of recombinant Wnt1 was used as a positive control. E : Dual staining with TH (red) and DAPI depicting TH + neurons in a control (PBS) astrocyte-neuron co-culture. D-P : Confocal images showing dual staining with TH (green) and GFAP (red) in a typical astrocyte-neuron control co-culture at 9 DIV. Note the lenght and branching of TH + processes. Astrocyte coculture induced a significant protection against SD (E), 6-OHDA (H), and the reversal induced by Dkk1 antagonism of Wnt/β-catenin signaling (F,I). By contrast, pharmacological activation of Wnt/β-catenin signaling with AR magnified TH neuroprotection (G,J,K). Astrocyte coculture increases Fzd-1 signal in the long and branched TH + neurites and growth cones (L-N and insert).
    Figure Legend Snippet: Modulation of astroglial Wnt1/β-catenin signaling directs towards TH neuron survival/death . Astrocyte-neuron co-cultures where shifted to serum deprived medium (SD, A), or received 6-OHDA or MPP + (25 μM), with or without Dkk1 (100 ng/ml), a Wnt1-Ab , or AR-14418 (5 μM) as described. A-B : TH + neuron counts (A), and [ 3 H]dopamine uptake (B) 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM) with or without Dkk1, Wnt1-Ab, or AR. * p < 0.05 compared to controls. C: Western blot analysis showing β-catenin levels in neurons exposed to astrocyte insert upon cytotoxic challenge with or without Wnt1-Ab or Dkk1. In this experimental condition, the glial inserts were added on the top of the purified neurons at 9 DIV. D : immunoblotting with Wnt1-Ab (50) using protein extracts from embryonic VM and the NIH/3T3 cell line. 50 ng of recombinant Wnt1 was used as a positive control. E : Dual staining with TH (red) and DAPI depicting TH + neurons in a control (PBS) astrocyte-neuron co-culture. D-P : Confocal images showing dual staining with TH (green) and GFAP (red) in a typical astrocyte-neuron control co-culture at 9 DIV. Note the lenght and branching of TH + processes. Astrocyte coculture induced a significant protection against SD (E), 6-OHDA (H), and the reversal induced by Dkk1 antagonism of Wnt/β-catenin signaling (F,I). By contrast, pharmacological activation of Wnt/β-catenin signaling with AR magnified TH neuroprotection (G,J,K). Astrocyte coculture increases Fzd-1 signal in the long and branched TH + neurites and growth cones (L-N and insert).

    Techniques Used: Western Blot, Purification, Recombinant, Positive Control, Staining, Co-Culture Assay, Activation Assay

    Antagonizing Wnt signaling or Silencing Wnt1 in VM astrocytes fail to protect TH + neurons . Wnt antagonism studies were performed with the extracellular-rich domain (CRD) of Fzd-1 (Fzd-1-CRD, 1 μ/ml) and the CRD of Fzd-2 (Fzd-2-CRD,1 μ/ml). Depletion of Wnt1 in VM astrocytes was achieved by introducing a small interference RNA targeting Wnt1 , Astro siWnt1 (46) in indirect astrocyte-neuron co-cultures (42). A-C: Indirect astrocyte-neuron co-culture efficiently mitigated SD, 6-OHDA- and MPP + -induced decreased TH neuron survival (A), DA uptake (B), and Caspase3 activation (C), whereas these protective effects are reversed by Fzd-1-CRD pre-treatment, but not Fzd-2-CRD. * p < 0.05 when compared to control cultures without cytotoxic insults; ° p < 0.05 compared to neurotoxic exposure within each experimental group. D-F : Glial inserts transiently transfected with siRNA wnt1 or siRNA control were added on the top of the purified neurons at 9 DIV and exposed to cytotoxic stimuli. RT-qPCR analysis (D) and immunocytochemistry (E-F) show an almost 40-60% Wnt1 depletion by 72 h. G-H : In neurons co-cultured with Astro siWnt1 , irrespective of the cytotoxic stimulus, a sharp reduction of TH + neuronal count (G) and increased Caspase3-like activity (H) were observed, compared to neuron co-cultured with Astro Ct (p < 0.05). In β-catenin siRNA- and Fzd-1 AS -knocked down neurons, astrocyte inserts failed to promote neuroprotection. I-S : Confocal images of TH staining (green) in the indirect cocultures showing the protective effect of astrocyte inserts + siRNA Ct (L,O,R) as compared with siRNA Wnt1 (M,P,S). The effect of indirect astrocyte coculture is emphasized in the PBS control cultures (I, N).
    Figure Legend Snippet: Antagonizing Wnt signaling or Silencing Wnt1 in VM astrocytes fail to protect TH + neurons . Wnt antagonism studies were performed with the extracellular-rich domain (CRD) of Fzd-1 (Fzd-1-CRD, 1 μ/ml) and the CRD of Fzd-2 (Fzd-2-CRD,1 μ/ml). Depletion of Wnt1 in VM astrocytes was achieved by introducing a small interference RNA targeting Wnt1 , Astro siWnt1 (46) in indirect astrocyte-neuron co-cultures (42). A-C: Indirect astrocyte-neuron co-culture efficiently mitigated SD, 6-OHDA- and MPP + -induced decreased TH neuron survival (A), DA uptake (B), and Caspase3 activation (C), whereas these protective effects are reversed by Fzd-1-CRD pre-treatment, but not Fzd-2-CRD. * p < 0.05 when compared to control cultures without cytotoxic insults; ° p < 0.05 compared to neurotoxic exposure within each experimental group. D-F : Glial inserts transiently transfected with siRNA wnt1 or siRNA control were added on the top of the purified neurons at 9 DIV and exposed to cytotoxic stimuli. RT-qPCR analysis (D) and immunocytochemistry (E-F) show an almost 40-60% Wnt1 depletion by 72 h. G-H : In neurons co-cultured with Astro siWnt1 , irrespective of the cytotoxic stimulus, a sharp reduction of TH + neuronal count (G) and increased Caspase3-like activity (H) were observed, compared to neuron co-cultured with Astro Ct (p < 0.05). In β-catenin siRNA- and Fzd-1 AS -knocked down neurons, astrocyte inserts failed to promote neuroprotection. I-S : Confocal images of TH staining (green) in the indirect cocultures showing the protective effect of astrocyte inserts + siRNA Ct (L,O,R) as compared with siRNA Wnt1 (M,P,S). The effect of indirect astrocyte coculture is emphasized in the PBS control cultures (I, N).

    Techniques Used: Co-Culture Assay, Activation Assay, Transfection, Purification, Quantitative RT-PCR, Immunocytochemistry, Cell Culture, Activity Assay, Staining

    Effect of intracerebral infusion of a Fzd receptor antagonist within the intact SNpc in the number of TH + Nissl + and Fluorojade-C (FJC) cell bodies . The effect of blocking Wnt/β-catenin signaling was assessed using the negative modulator of Wnt1 signaling, Dkk1 (1 μg/μl) or saline, unilaterally infused into the left intact SNpc, as described. Mice were sacrificed 1-7 days post-infusion, and the brains processed for TH + Nissl + cell counts (M) and Fluorojade C staining (FJC, N) in consecutive midbrain sections. A-L: C onfocal images showing TH + (green) neurons counterstained with DAPI (blu) of coronal midbrain sections at the level of the SNpc 1 day after unilateral saline (A-C) or 1 (D-F), 3 (G-I) and 7 (J-L) days after unilateral Dkk1 infusion. In Dkk1-infused mice, a decrease of TH + immunofluorescent signal was observed starting 1 d (D) in the ipsilateral (E), but not contralateral (F), non-infused SN, with a peak TH + loss at 3 days (see H compared to I), and a stabilization observed by 7 days (F). M . Total number of TH + and Nissl + counted throught the entire rostro-caudal axis of the SNpc. Treatment groups were averaged (n = 4/time-point, means ± S.E.M.) * p < 0.05 vs contralateral side, within each respective group. B . Total number of Fluorojade C (FJC) stained cells in SNpc ipsilateral and contralateral to the infusion was calculated for each side, averaged for each animal (n = 4/time-point) and normalized to the number of TH + neurons in SNpc per section. *p < 0.05 vs saline injected side.
    Figure Legend Snippet: Effect of intracerebral infusion of a Fzd receptor antagonist within the intact SNpc in the number of TH + Nissl + and Fluorojade-C (FJC) cell bodies . The effect of blocking Wnt/β-catenin signaling was assessed using the negative modulator of Wnt1 signaling, Dkk1 (1 μg/μl) or saline, unilaterally infused into the left intact SNpc, as described. Mice were sacrificed 1-7 days post-infusion, and the brains processed for TH + Nissl + cell counts (M) and Fluorojade C staining (FJC, N) in consecutive midbrain sections. A-L: C onfocal images showing TH + (green) neurons counterstained with DAPI (blu) of coronal midbrain sections at the level of the SNpc 1 day after unilateral saline (A-C) or 1 (D-F), 3 (G-I) and 7 (J-L) days after unilateral Dkk1 infusion. In Dkk1-infused mice, a decrease of TH + immunofluorescent signal was observed starting 1 d (D) in the ipsilateral (E), but not contralateral (F), non-infused SN, with a peak TH + loss at 3 days (see H compared to I), and a stabilization observed by 7 days (F). M . Total number of TH + and Nissl + counted throught the entire rostro-caudal axis of the SNpc. Treatment groups were averaged (n = 4/time-point, means ± S.E.M.) * p < 0.05 vs contralateral side, within each respective group. B . Total number of Fluorojade C (FJC) stained cells in SNpc ipsilateral and contralateral to the infusion was calculated for each side, averaged for each animal (n = 4/time-point) and normalized to the number of TH + neurons in SNpc per section. *p < 0.05 vs saline injected side.

    Techniques Used: Blocking Assay, Staining, Injection

    Effect of pharmacological activation of Wnt/β-catenin signaling in TH + neuroprotection against intracerebral Dkk1 or systemic MPTP treatment . To mimick the activation of Wnt1/β-catenin signaling, we selected the specific GSK-3β inhibitor, AR (10 mg/kg twice a day) starting 72 h before unilateral Dkk1 infusion within the SN, or before the systemic (i.p.) treatment with the parkinsonian neurotoxin, MPTP (15 mg kg -1 , 4 times a day at 2 h intervals), and mice sacrificed at the peak degeneration phase (3 days post-treatment). A-B : Representative confocal images showing dual localization of TH + neurons (green) and GFAP + astrocytes (red) in Saline (A), MPTP (B) and AR/MPTP (C) 4d post-MPTP, showing the sharp decrease of TH neurons associated to the marked astrocytosis and the remarkable protective effect of AR (C). C : The total number of TH + and Nissl + neurons was counted throught the entire rostro-caudal axis of the SNpc as above. Treatment groups were averaged (n = 4/time-point, means ± S.E.M.) * p < 0.05 vs unifused side (for Dkk1), within each respective group. Dkk1 and MPTP significantly reduced TH + and Nissl + neurons 4 d post-treatment. MPTP systemic treatment reduces TH + neuron numbers in both left and both sides. Note the remarkable counteraction afforded by AR in increasing TH + neurons to unlesioned saline-treated control. *p < 0.05 vs -MPTP.
    Figure Legend Snippet: Effect of pharmacological activation of Wnt/β-catenin signaling in TH + neuroprotection against intracerebral Dkk1 or systemic MPTP treatment . To mimick the activation of Wnt1/β-catenin signaling, we selected the specific GSK-3β inhibitor, AR (10 mg/kg twice a day) starting 72 h before unilateral Dkk1 infusion within the SN, or before the systemic (i.p.) treatment with the parkinsonian neurotoxin, MPTP (15 mg kg -1 , 4 times a day at 2 h intervals), and mice sacrificed at the peak degeneration phase (3 days post-treatment). A-B : Representative confocal images showing dual localization of TH + neurons (green) and GFAP + astrocytes (red) in Saline (A), MPTP (B) and AR/MPTP (C) 4d post-MPTP, showing the sharp decrease of TH neurons associated to the marked astrocytosis and the remarkable protective effect of AR (C). C : The total number of TH + and Nissl + neurons was counted throught the entire rostro-caudal axis of the SNpc as above. Treatment groups were averaged (n = 4/time-point, means ± S.E.M.) * p < 0.05 vs unifused side (for Dkk1), within each respective group. Dkk1 and MPTP significantly reduced TH + and Nissl + neurons 4 d post-treatment. MPTP systemic treatment reduces TH + neuron numbers in both left and both sides. Note the remarkable counteraction afforded by AR in increasing TH + neurons to unlesioned saline-treated control. *p < 0.05 vs -MPTP.

    Techniques Used: Activation Assay

    Schematic illustration of Wnt1/Fzd-1/β-catenin signaling as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk . Crosstalk between astrocytes and DA neurons represent a cardinal neuroprotective mechanism against inflammation, oxidative stress and growth factor deprivation (10). Here astrocyte-DA neuron crosstalk via Wnt1 is emphasized. Astrocyte-derived Wnt, via activation of Fzd-1 receptors, may contribute to maintain the integrity of DA neurons by influencing Wnt signaling components, including blockade of GSK-3β-induced phosphorylation (P) and proteosomal degradation of the neuronal pool of β-catenin. Stabilized β-catenin can translocate into the nucleus and associate with a family of transcription factors and regulate the expression of Wnt target genes involved in DA neuron survival. β-catenin may also function as a pivotal defense molecule against oxidative stress (79), and can act as a coactivator for several nuclear receptors involved in the maintenance/protection of DA neurons (81). Crosstalk with up-stream survival pathways converging to β-catenin stabilization can also be envisaged (26, 27). Neurotoxic injury or increased oxidative load as a result of aging may antagonize Wnt/β-catenin signaling in DA neurons by up-regulating active GSK-3β, leading to β-catenin degradation and increased DA neuron vulnerability, which may underlie a progressive DA neuron deficit. Neuronal injury also triggers reactive astrocyte expression of a panel of growth and neurotrophic factors, anti-oxidant and neuroprotective mechanisms, among which astrocyte Wnt1 via Fzd-1 receptors may function as a vital component of DA neurons self-protective machinery shifting the balance towards the programming of cell survival/neurorescue.
    Figure Legend Snippet: Schematic illustration of Wnt1/Fzd-1/β-catenin signaling as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk . Crosstalk between astrocytes and DA neurons represent a cardinal neuroprotective mechanism against inflammation, oxidative stress and growth factor deprivation (10). Here astrocyte-DA neuron crosstalk via Wnt1 is emphasized. Astrocyte-derived Wnt, via activation of Fzd-1 receptors, may contribute to maintain the integrity of DA neurons by influencing Wnt signaling components, including blockade of GSK-3β-induced phosphorylation (P) and proteosomal degradation of the neuronal pool of β-catenin. Stabilized β-catenin can translocate into the nucleus and associate with a family of transcription factors and regulate the expression of Wnt target genes involved in DA neuron survival. β-catenin may also function as a pivotal defense molecule against oxidative stress (79), and can act as a coactivator for several nuclear receptors involved in the maintenance/protection of DA neurons (81). Crosstalk with up-stream survival pathways converging to β-catenin stabilization can also be envisaged (26, 27). Neurotoxic injury or increased oxidative load as a result of aging may antagonize Wnt/β-catenin signaling in DA neurons by up-regulating active GSK-3β, leading to β-catenin degradation and increased DA neuron vulnerability, which may underlie a progressive DA neuron deficit. Neuronal injury also triggers reactive astrocyte expression of a panel of growth and neurotrophic factors, anti-oxidant and neuroprotective mechanisms, among which astrocyte Wnt1 via Fzd-1 receptors may function as a vital component of DA neurons self-protective machinery shifting the balance towards the programming of cell survival/neurorescue.

    Techniques Used: Derivative Assay, Activation Assay, Expressing


    Structured Review

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    6-OHDA treatment downregulated the protein level of <t>Wnt1</t> in U251 cells. (a) U251 cells were treated with vehicle or 6-OHDA (50 μ M) for 3, 6, 9, 12, and 24 h, and the protein level of Wnt1 was detected by Western blot with β -actin as an internal control. (b) The relative band intensities of Wnt1 were measured by Quantity One software and normalized to the expression of β -actin in U251 cells. All of the data are presented as the mean ± SD from three independent experiments. Differences among various time points were examined by using ANOVA followed by Tukey-Kramer tests for post hoc multiple comparisons. F value = 67.10, df between groups = 5, df within groups = 12, and df total = 17.
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    Images

    1) Product Images from "Wnt1 Promotes EAAT2 Expression and Mediates the Protective Effects of Astrocytes on Dopaminergic Cells in Parkinson's Disease"

    Article Title: Wnt1 Promotes EAAT2 Expression and Mediates the Protective Effects of Astrocytes on Dopaminergic Cells in Parkinson's Disease

    Journal: Neural Plasticity

    doi: 10.1155/2019/1247276

    6-OHDA treatment downregulated the protein level of Wnt1 in U251 cells. (a) U251 cells were treated with vehicle or 6-OHDA (50 μ M) for 3, 6, 9, 12, and 24 h, and the protein level of Wnt1 was detected by Western blot with β -actin as an internal control. (b) The relative band intensities of Wnt1 were measured by Quantity One software and normalized to the expression of β -actin in U251 cells. All of the data are presented as the mean ± SD from three independent experiments. Differences among various time points were examined by using ANOVA followed by Tukey-Kramer tests for post hoc multiple comparisons. F value = 67.10, df between groups = 5, df within groups = 12, and df total = 17.
    Figure Legend Snippet: 6-OHDA treatment downregulated the protein level of Wnt1 in U251 cells. (a) U251 cells were treated with vehicle or 6-OHDA (50 μ M) for 3, 6, 9, 12, and 24 h, and the protein level of Wnt1 was detected by Western blot with β -actin as an internal control. (b) The relative band intensities of Wnt1 were measured by Quantity One software and normalized to the expression of β -actin in U251 cells. All of the data are presented as the mean ± SD from three independent experiments. Differences among various time points were examined by using ANOVA followed by Tukey-Kramer tests for post hoc multiple comparisons. F value = 67.10, df between groups = 5, df within groups = 12, and df total = 17.

    Techniques Used: Western Blot, Software, Expressing

    Evaluation of the Wnt1 overexpression vector in U251 cells by Western blot and PCR. Forty-eight hours after transfection with the Wnt1 expression vector or empty vector, U251 cells were harvested, and the mRNA and protein levels of Wnt1 were determined by real-time PCR and Western blot. The mRNA (a) and protein (b, c) levels of Wnt1 were significantly higher in U251 cells overexpressing Wnt1 than in control cells, whereas the empty vector did not influence Wnt1 expression. ∗∗∗∗ P < 0.0001. For Western blot assay, differences among groups were examined by using ANOVA followed by Tukey-Kramer tests for post hoc multiple comparisons (c). F value = 161.42, df between groups = 2, df within groups = 6, and df total = 8.
    Figure Legend Snippet: Evaluation of the Wnt1 overexpression vector in U251 cells by Western blot and PCR. Forty-eight hours after transfection with the Wnt1 expression vector or empty vector, U251 cells were harvested, and the mRNA and protein levels of Wnt1 were determined by real-time PCR and Western blot. The mRNA (a) and protein (b, c) levels of Wnt1 were significantly higher in U251 cells overexpressing Wnt1 than in control cells, whereas the empty vector did not influence Wnt1 expression. ∗∗∗∗ P < 0.0001. For Western blot assay, differences among groups were examined by using ANOVA followed by Tukey-Kramer tests for post hoc multiple comparisons (c). F value = 161.42, df between groups = 2, df within groups = 6, and df total = 8.

    Techniques Used: Over Expression, Plasmid Preparation, Western Blot, Transfection, Expressing, Real-time Polymerase Chain Reaction

    Coculturing with U251-Wnt1 cells attenuated 6-OHDA-induced injury in SH-SY5Y cells. SH-SY5Y cells were indirectly cocultured with U251-Wnt1 cells or U251-EV cells, with SH-SY5Y cells cultured alone as the control, and then treated with 50 μ M 6-OHDA for 24 h; cell viability was assessed using the MTT assay. The data are expressed as percentages relative to the control group with 0 μ M 6-OHDA and are presented as the mean ± SD from four independent experiments. ∗ P < 0.05 compared to the control with 0 μ M 6-OHDA; # P < 0.05 compared to the control group at the corresponding 6-OHDA concentration.
    Figure Legend Snippet: Coculturing with U251-Wnt1 cells attenuated 6-OHDA-induced injury in SH-SY5Y cells. SH-SY5Y cells were indirectly cocultured with U251-Wnt1 cells or U251-EV cells, with SH-SY5Y cells cultured alone as the control, and then treated with 50 μ M 6-OHDA for 24 h; cell viability was assessed using the MTT assay. The data are expressed as percentages relative to the control group with 0 μ M 6-OHDA and are presented as the mean ± SD from four independent experiments. ∗ P < 0.05 compared to the control with 0 μ M 6-OHDA; # P < 0.05 compared to the control group at the corresponding 6-OHDA concentration.

    Techniques Used: Cell Culture, MTT Assay, Concentration Assay

    Effect of coculturing with U251-Wnt1 cells on 6-OHDA-induced damage in SH-SY5Y cells measured by flow cytometry. SH-SY5Y cells were indirectly cocultured with U251 cells, U251-Wnt1 cells, or U251-EV cells and then treated with or without 50 μ M 6-OHDA or 100 ng/ml DKK-1 for 24 h, and a combination of annexin V-FITC and PI staining was performed followed by flow cytometry. (a) Representative set of flow cytometric two-parameter dot plots in the corresponding groups. The lower right quadrant, which shows annexin V-FITC-positive and PI-negative cells, represents early apoptotic cells. The upper right quadrant, which shows both annexin V-FITC- and PI-positive cells, represents cells in the end stages of apoptosis and necrosis. (b) The percentage of cells under apoptosis and necrosis. Data are presented as the mean ± SD from 3 independent experiments. Differences among groups were examined by using ANOVA followed by Tukey-Kramer tests for post hoc multiple comparisons. F value = 28.59, df between groups = 5, df within groups = 12, and df total = 17.
    Figure Legend Snippet: Effect of coculturing with U251-Wnt1 cells on 6-OHDA-induced damage in SH-SY5Y cells measured by flow cytometry. SH-SY5Y cells were indirectly cocultured with U251 cells, U251-Wnt1 cells, or U251-EV cells and then treated with or without 50 μ M 6-OHDA or 100 ng/ml DKK-1 for 24 h, and a combination of annexin V-FITC and PI staining was performed followed by flow cytometry. (a) Representative set of flow cytometric two-parameter dot plots in the corresponding groups. The lower right quadrant, which shows annexin V-FITC-positive and PI-negative cells, represents early apoptotic cells. The upper right quadrant, which shows both annexin V-FITC- and PI-positive cells, represents cells in the end stages of apoptosis and necrosis. (b) The percentage of cells under apoptosis and necrosis. Data are presented as the mean ± SD from 3 independent experiments. Differences among groups were examined by using ANOVA followed by Tukey-Kramer tests for post hoc multiple comparisons. F value = 28.59, df between groups = 5, df within groups = 12, and df total = 17.

    Techniques Used: Flow Cytometry, Staining

    Wnt1 overexpression decreased the glutamate level in culture medium. SH-SY5Y cells were indirectly cocultured with U251 cells, U251-Wnt1 cells, or U251-EV cells and then treated with or without 50 μ M 6-OHDA or 100 ng/ml DKK-1 for 24 h, and the glutamate levels in culture media were detected by ELISA. The data are expressed as percentages relative to the control group. All of the data are presented as the mean ± SD from 7 independent experiments. Differences among groups were examined by using ANOVA followed by Tukey-Kramer tests for post hoc multiple comparisons. F value = 15.87, df between groups = 5, df within groups = 36, and df total = 41.
    Figure Legend Snippet: Wnt1 overexpression decreased the glutamate level in culture medium. SH-SY5Y cells were indirectly cocultured with U251 cells, U251-Wnt1 cells, or U251-EV cells and then treated with or without 50 μ M 6-OHDA or 100 ng/ml DKK-1 for 24 h, and the glutamate levels in culture media were detected by ELISA. The data are expressed as percentages relative to the control group. All of the data are presented as the mean ± SD from 7 independent experiments. Differences among groups were examined by using ANOVA followed by Tukey-Kramer tests for post hoc multiple comparisons. F value = 15.87, df between groups = 5, df within groups = 36, and df total = 41.

    Techniques Used: Over Expression, Enzyme-linked Immunosorbent Assay

    Wnt1 overexpression attenuated the inhibitory effect of 6-OHDA on EAAT2 expression in U251 cells. U251 cells, U251-Wnt1 cells, or U251-EV cells were treated with vehicle, 6-OHDA (50 μ M), or/and DKK-1 (100 ng/ml) for 24 h, and the protein levels of EAAT2 (a) were detected by Western blot with β -actin as an internal control. The relative band intensities of EAAT2 (b) were measured by Quantity One software and normalized to the expression of β -actin. Differences among groups were examined using ANOVA followed by using Tukey-Kramer tests for post hoc multiple comparisons. Data are presented as the mean ± SD of 3 experiments. F value = 25.29, df between groups = 4, df within groups = 10, and df total = 14.
    Figure Legend Snippet: Wnt1 overexpression attenuated the inhibitory effect of 6-OHDA on EAAT2 expression in U251 cells. U251 cells, U251-Wnt1 cells, or U251-EV cells were treated with vehicle, 6-OHDA (50 μ M), or/and DKK-1 (100 ng/ml) for 24 h, and the protein levels of EAAT2 (a) were detected by Western blot with β -actin as an internal control. The relative band intensities of EAAT2 (b) were measured by Quantity One software and normalized to the expression of β -actin. Differences among groups were examined using ANOVA followed by using Tukey-Kramer tests for post hoc multiple comparisons. Data are presented as the mean ± SD of 3 experiments. F value = 25.29, df between groups = 4, df within groups = 10, and df total = 14.

    Techniques Used: Over Expression, Expressing, Western Blot, Software

    Wnt1 overexpression activated the Wnt/ β -catenin pathway in U251 cells. U251 cells, U251-Wnt1 cells, or U251-EV cells were treated with vehicle, 6-OHDA (50 μ M), or/and DKK-1 (100 ng/ml) for 24 h, and the protein levels of β -catenin (a) were detected by Western blot with β -actin as an internal control. The relative band intensities of β -catenin (b) were measured by Quantity One software and normalized to the expression of β -actin. Differences among groups were examined using ANOVA followed by using Tukey-Kramer tests for post hoc multiple comparisons. Data are presented as the mean ± SD of 3 experiments. F value = 30.23, df between groups = 4, df within groups = 10, and df total = 14.
    Figure Legend Snippet: Wnt1 overexpression activated the Wnt/ β -catenin pathway in U251 cells. U251 cells, U251-Wnt1 cells, or U251-EV cells were treated with vehicle, 6-OHDA (50 μ M), or/and DKK-1 (100 ng/ml) for 24 h, and the protein levels of β -catenin (a) were detected by Western blot with β -actin as an internal control. The relative band intensities of β -catenin (b) were measured by Quantity One software and normalized to the expression of β -actin. Differences among groups were examined using ANOVA followed by using Tukey-Kramer tests for post hoc multiple comparisons. Data are presented as the mean ± SD of 3 experiments. F value = 30.23, df between groups = 4, df within groups = 10, and df total = 14.

    Techniques Used: Over Expression, Western Blot, Software, Expressing

    Immunofluorescence staining of p65 protein in U251 cells. U251 cells, U251-Wnt1 cells, or U251-EV cells were treated with vehicle, 6-OHDA (50 μ M), or/and DKK-1 (100 ng/ml) for 24 h, and the expression and distribution of p65 were examined using an antibody against p65 (red). Nuclei were counterstained with DAPI (blue). All images were captured using a confocal laser scanning microscope.
    Figure Legend Snippet: Immunofluorescence staining of p65 protein in U251 cells. U251 cells, U251-Wnt1 cells, or U251-EV cells were treated with vehicle, 6-OHDA (50 μ M), or/and DKK-1 (100 ng/ml) for 24 h, and the expression and distribution of p65 were examined using an antibody against p65 (red). Nuclei were counterstained with DAPI (blue). All images were captured using a confocal laser scanning microscope.

    Techniques Used: Immunofluorescence, Staining, Expressing, Laser-Scanning Microscopy


    Structured Review

    Abcam rabbit anti wnt1
    (A) In situ hybridization for Dkk1 on coronal sections at E9.5 revealed that Dkk1 mRNA is highly expressed in the diencephalon and in a salt-and-pepper pattern in the ventral midbrain, with higher expression rostrally and lower caudally. Scale bars = 100 µm. Abbreviations: m- midbrain, d - diencephalon. (B) Immunohistochemistry for <t>Wnt1</t> on an adjacent section to the Dkk1 -probed section shown in (A) revealed that Wnt1 protein and Dkk1 mRNA are expressed in the VM at the same time and in a complementary anterior-posterior (not shown) and ventro-lateral manner (B). Scale bar = 50 µm. (C) At E10.5, Dkk1 is expressed in the medial part of the floor plate in the ventricular, intermediate and marginal zones. Scale bars = 100 µm upper panels, 50 µm bottom panels (high magnification). (D) Imunohistochemistry for Lmx1a revealed that, at E10.5, Dkk1 + cells are found in the medial part of the Lmx1a expression domain. Scale bars = 100 µm.
    Rabbit Anti Wnt1, supplied by Abcam, 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 "Dkk1 Regulates Ventral Midbrain Dopaminergic Differentiation and Morphogenesis"

    Article Title: Dkk1 Regulates Ventral Midbrain Dopaminergic Differentiation and Morphogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015786

    (A) In situ hybridization for Dkk1 on coronal sections at E9.5 revealed that Dkk1 mRNA is highly expressed in the diencephalon and in a salt-and-pepper pattern in the ventral midbrain, with higher expression rostrally and lower caudally. Scale bars = 100 µm. Abbreviations: m- midbrain, d - diencephalon. (B) Immunohistochemistry for Wnt1 on an adjacent section to the Dkk1 -probed section shown in (A) revealed that Wnt1 protein and Dkk1 mRNA are expressed in the VM at the same time and in a complementary anterior-posterior (not shown) and ventro-lateral manner (B). Scale bar = 50 µm. (C) At E10.5, Dkk1 is expressed in the medial part of the floor plate in the ventricular, intermediate and marginal zones. Scale bars = 100 µm upper panels, 50 µm bottom panels (high magnification). (D) Imunohistochemistry for Lmx1a revealed that, at E10.5, Dkk1 + cells are found in the medial part of the Lmx1a expression domain. Scale bars = 100 µm.
    Figure Legend Snippet: (A) In situ hybridization for Dkk1 on coronal sections at E9.5 revealed that Dkk1 mRNA is highly expressed in the diencephalon and in a salt-and-pepper pattern in the ventral midbrain, with higher expression rostrally and lower caudally. Scale bars = 100 µm. Abbreviations: m- midbrain, d - diencephalon. (B) Immunohistochemistry for Wnt1 on an adjacent section to the Dkk1 -probed section shown in (A) revealed that Wnt1 protein and Dkk1 mRNA are expressed in the VM at the same time and in a complementary anterior-posterior (not shown) and ventro-lateral manner (B). Scale bar = 50 µm. (C) At E10.5, Dkk1 is expressed in the medial part of the floor plate in the ventricular, intermediate and marginal zones. Scale bars = 100 µm upper panels, 50 µm bottom panels (high magnification). (D) Imunohistochemistry for Lmx1a revealed that, at E10.5, Dkk1 + cells are found in the medial part of the Lmx1a expression domain. Scale bars = 100 µm.

    Techniques Used: In Situ Hybridization, Expressing, Immunohistochemistry

    rabbit anti wnt1 antibody  (Danaher Inc)


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    Danaher Inc rabbit anti wnt1 antibody
    Rabbit Anti Wnt1 Antibody, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti wnt1 antibody/product/Danaher Inc
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    rabbit anti wnt induced signaling protein 1  (Danaher Inc)


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    Danaher Inc rabbit anti wnt induced signaling protein 1
    Rabbit Anti Wnt Induced Signaling Protein 1, supplied by Danaher Inc, 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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    Abcam rabbit anti wnt1
    Rabbit Anti Wnt1, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti wnt1/product/Abcam
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    Structured Review

    Abcam rabbit anti wnt1 antibody
    FSTL1 interacts with canonical Wnt ligands. A , FSTL1 interacts with <t>Wnt1.</t> HEK293T cells were transfected with Wnt1 plasmid in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1 to pull down Wnt1 ( left panel ). Reciprocal IP was performed ( right panel ). Western blotting was performed on whole lysates and precipitates as indicated. B , FSTL1 interacts with Wnt2 and Wnt3a. HEK293T cells were transfected with FATL1-HA in the absence or the presence of Wnt2-V5 or Wnt3a-V5 plasmid. Cell lysates were immunoprecipitated with IgG or anti-V5 antibody to pull down FSTL1 ( left panel ). Reciprocal IP was also performed ( right panel s ). C , FSTL1 interacts with Wnt8a, Wnt8b, Wnt9a, and Wnt10b. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of V5-tagged Wnt8a, Wnt8b, Wnt9a, and Wnt10b plasmids. Cell lysates were immunoprecipitated with anti-V5, followed by Western blotting with anti-FSTL1 or anti-V5 antibody on the precipitates and lysates as indicated. D , interaction of FSTL1 and Wnt2 occurs in the extracellular space. HEK293T cells were transfected with Wnt2-V5 in the absence or the presence of FSTL1-HA plasmid. Cell culture medium was immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and medium as indicated. E and F , interaction of FSTL1 and Wnt3a ( E ) or Wnt10b ( F ) occurs in the extracellular space. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of Wnt3a-V5 ( E ) or Wnt10b-V5 ( F ) plasmid. Cell culture medium was immunoprecipitated with anti-V5 antibody, followed by Western blotting with anti-V5 or anti-FSTL1 on the precipitates and medium as indicated. G , ALK3 does not interact with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 in the absence or the presence of FSTL1-HA or ALK3-HA plasmid. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and lysates as indicated. H , FSTL1 interacts with Wnt1 and Wnt3a in the kidney. Lysates from mouse kidneys with UUO for 7 days were immunoprecipitated with a combination of anti-Wnt1 and anti-Wnt3a antibodies or normal IgG, and the precipitates and lysates were subjected to Western blotting as indicated. I , FSTL1 directly interacts with Wnt3a. Recombinant Wnt3a protein (1.5 μg) was incubated with and without recombinant FSTL1 protein (3 μg) in 300 μl Tris-buffered saline/0.1% Triton X-100 overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated with anti-FSTL1 antibodies followed by Protein G agarose. Eluted proteins and inputs were analyzed by Western blotting as indicated. J , domain organization of full-length (FL) and mutant FSTL1 proteins. K , interactions of EC–VWC and EC domains of FSTL1 with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of FL, EC–VWC, or EC plasmid. Cell lysates were immunoprecipitated with anti-FSTL1. Western blotting was performed on whole lysates and precipitates as indicated. L , VWC but not FS domain of FSTL1 interacts with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of 3× FLAG-FS or 3× FLAG-VWC plasmid. Cell lysates were immunoprecipitated with anti-FLAG. Western blotting was performed on whole lysates and precipitates as indicated. EC, the extracellular calcium–binding domain; FS, the follistatin-like domain; FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IgG, immunoglobulin G; IP, immunoprecipitation; UUO, unilateral ureteral obstruction; VWC, a domain with homology to von Willebrand factor type C domain.
    Rabbit Anti Wnt1 Antibody, supplied by Abcam, 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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    Images

    1) Product Images from "Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo"

    Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.102010

    FSTL1 interacts with canonical Wnt ligands. A , FSTL1 interacts with Wnt1. HEK293T cells were transfected with Wnt1 plasmid in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1 to pull down Wnt1 ( left panel ). Reciprocal IP was performed ( right panel ). Western blotting was performed on whole lysates and precipitates as indicated. B , FSTL1 interacts with Wnt2 and Wnt3a. HEK293T cells were transfected with FATL1-HA in the absence or the presence of Wnt2-V5 or Wnt3a-V5 plasmid. Cell lysates were immunoprecipitated with IgG or anti-V5 antibody to pull down FSTL1 ( left panel ). Reciprocal IP was also performed ( right panel s ). C , FSTL1 interacts with Wnt8a, Wnt8b, Wnt9a, and Wnt10b. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of V5-tagged Wnt8a, Wnt8b, Wnt9a, and Wnt10b plasmids. Cell lysates were immunoprecipitated with anti-V5, followed by Western blotting with anti-FSTL1 or anti-V5 antibody on the precipitates and lysates as indicated. D , interaction of FSTL1 and Wnt2 occurs in the extracellular space. HEK293T cells were transfected with Wnt2-V5 in the absence or the presence of FSTL1-HA plasmid. Cell culture medium was immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and medium as indicated. E and F , interaction of FSTL1 and Wnt3a ( E ) or Wnt10b ( F ) occurs in the extracellular space. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of Wnt3a-V5 ( E ) or Wnt10b-V5 ( F ) plasmid. Cell culture medium was immunoprecipitated with anti-V5 antibody, followed by Western blotting with anti-V5 or anti-FSTL1 on the precipitates and medium as indicated. G , ALK3 does not interact with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 in the absence or the presence of FSTL1-HA or ALK3-HA plasmid. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and lysates as indicated. H , FSTL1 interacts with Wnt1 and Wnt3a in the kidney. Lysates from mouse kidneys with UUO for 7 days were immunoprecipitated with a combination of anti-Wnt1 and anti-Wnt3a antibodies or normal IgG, and the precipitates and lysates were subjected to Western blotting as indicated. I , FSTL1 directly interacts with Wnt3a. Recombinant Wnt3a protein (1.5 μg) was incubated with and without recombinant FSTL1 protein (3 μg) in 300 μl Tris-buffered saline/0.1% Triton X-100 overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated with anti-FSTL1 antibodies followed by Protein G agarose. Eluted proteins and inputs were analyzed by Western blotting as indicated. J , domain organization of full-length (FL) and mutant FSTL1 proteins. K , interactions of EC–VWC and EC domains of FSTL1 with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of FL, EC–VWC, or EC plasmid. Cell lysates were immunoprecipitated with anti-FSTL1. Western blotting was performed on whole lysates and precipitates as indicated. L , VWC but not FS domain of FSTL1 interacts with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of 3× FLAG-FS or 3× FLAG-VWC plasmid. Cell lysates were immunoprecipitated with anti-FLAG. Western blotting was performed on whole lysates and precipitates as indicated. EC, the extracellular calcium–binding domain; FS, the follistatin-like domain; FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IgG, immunoglobulin G; IP, immunoprecipitation; UUO, unilateral ureteral obstruction; VWC, a domain with homology to von Willebrand factor type C domain.
    Figure Legend Snippet: FSTL1 interacts with canonical Wnt ligands. A , FSTL1 interacts with Wnt1. HEK293T cells were transfected with Wnt1 plasmid in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1 to pull down Wnt1 ( left panel ). Reciprocal IP was performed ( right panel ). Western blotting was performed on whole lysates and precipitates as indicated. B , FSTL1 interacts with Wnt2 and Wnt3a. HEK293T cells were transfected with FATL1-HA in the absence or the presence of Wnt2-V5 or Wnt3a-V5 plasmid. Cell lysates were immunoprecipitated with IgG or anti-V5 antibody to pull down FSTL1 ( left panel ). Reciprocal IP was also performed ( right panel s ). C , FSTL1 interacts with Wnt8a, Wnt8b, Wnt9a, and Wnt10b. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of V5-tagged Wnt8a, Wnt8b, Wnt9a, and Wnt10b plasmids. Cell lysates were immunoprecipitated with anti-V5, followed by Western blotting with anti-FSTL1 or anti-V5 antibody on the precipitates and lysates as indicated. D , interaction of FSTL1 and Wnt2 occurs in the extracellular space. HEK293T cells were transfected with Wnt2-V5 in the absence or the presence of FSTL1-HA plasmid. Cell culture medium was immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and medium as indicated. E and F , interaction of FSTL1 and Wnt3a ( E ) or Wnt10b ( F ) occurs in the extracellular space. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of Wnt3a-V5 ( E ) or Wnt10b-V5 ( F ) plasmid. Cell culture medium was immunoprecipitated with anti-V5 antibody, followed by Western blotting with anti-V5 or anti-FSTL1 on the precipitates and medium as indicated. G , ALK3 does not interact with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 in the absence or the presence of FSTL1-HA or ALK3-HA plasmid. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and lysates as indicated. H , FSTL1 interacts with Wnt1 and Wnt3a in the kidney. Lysates from mouse kidneys with UUO for 7 days were immunoprecipitated with a combination of anti-Wnt1 and anti-Wnt3a antibodies or normal IgG, and the precipitates and lysates were subjected to Western blotting as indicated. I , FSTL1 directly interacts with Wnt3a. Recombinant Wnt3a protein (1.5 μg) was incubated with and without recombinant FSTL1 protein (3 μg) in 300 μl Tris-buffered saline/0.1% Triton X-100 overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated with anti-FSTL1 antibodies followed by Protein G agarose. Eluted proteins and inputs were analyzed by Western blotting as indicated. J , domain organization of full-length (FL) and mutant FSTL1 proteins. K , interactions of EC–VWC and EC domains of FSTL1 with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of FL, EC–VWC, or EC plasmid. Cell lysates were immunoprecipitated with anti-FSTL1. Western blotting was performed on whole lysates and precipitates as indicated. L , VWC but not FS domain of FSTL1 interacts with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of 3× FLAG-FS or 3× FLAG-VWC plasmid. Cell lysates were immunoprecipitated with anti-FLAG. Western blotting was performed on whole lysates and precipitates as indicated. EC, the extracellular calcium–binding domain; FS, the follistatin-like domain; FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IgG, immunoglobulin G; IP, immunoprecipitation; UUO, unilateral ureteral obstruction; VWC, a domain with homology to von Willebrand factor type C domain.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Cell Culture, Recombinant, Incubation, Mutagenesis, Binding Assay

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    Danaher Inc rabbit anti wnt1
    Effect of <t>Wnt1-positive</t> expression on proliferative and migratory C6 GSCs in different groups. (A) The positive expression of Wnt1 on C6 GSCs was observed by immunofluorescence staining. Scale bar, 100 µm. (B) The statistical data of the number of Wnt1-positive cells are shown in each group. *P<0.05 and ***P<0.001. GSCs, glioma stem cells; ns, no significance.
    Rabbit Anti Wnt1, supplied by Danaher Inc, 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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    Abcam antibodies against rabbit polyclonal anti human wnt1
    miR-424 activates Wnt/β-catenin signaling pathway by directly targeting APC. (A) Wester blots and (B) protein levels of APC, <t>Wnt1,</t> β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 mimics + pcDNA-empty or miR-424 mimics + pcDNA-APC. (C) Western blots and (D) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 inhibitor + si-scramble or miR-424 inhibitor + si-APC. Data are presented as means ± standard deviation of three individual experiments. * P<0.05 and ** P<0.01 vs. control. ## P<0.01 vs. miR-424 mimics + pcDNA-vector or miR-424 inhibitor + si-scramble. APC, adenomatous polyposis coli; miR, microRNA; si, small interfering.
    Antibodies Against Rabbit Polyclonal Anti Human Wnt1, supplied by Abcam, 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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    Abcam rabbit polyclonal anti wnt1 antibody
    HepG2 cells were mock infected, or infected with Ad-HCV core or Ad-EGFP for 48 h, and were harvested for preparation of total RNA and protein. ( A ) Infection efficiency of the adenovirus observed under the fluorescent microscope (×10). ( B ) Western blot analysis for HCV core protein expression. ( C ) SLqRT-PCR analysis for miR-152 expression. After normalization to endogenous RNU6B expression, the expression of miR-152 in each group of HepG2 cells was expressed as fold changes in comparison to the levels observed in mock-infected HepG2 cells. ( D ) Real time RT-PCR analysis for <t>Wnt1</t> mRNA expression. ( E ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (HepG2: mock-infected HepG2 cells; Hcv: HepG2 cells infected with the Ad-HCV core; Co: HepG2 cells infected with the Ad-EGFP control; Advirus-Hcv: Adenovirus expressing the HCV core protein, Ad-HCV core; Advirus-Ctrl: Adenovirus expressing the EGFP control protein, Ad-EGFP).
    Rabbit Polyclonal Anti Wnt1 Antibody, supplied by Abcam, 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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    Abcam rabbit anti wnt1
    <t>Wnt1</t> protects primary mesencephalic dopaminergic (DA) neurons against cell death induced by serum deprivation (SD), 6-hydroxydopamine (6-OHDA) and MPP + . Enriched neuronal cultures derived from the mesencephalon of E14 rat embryos maintained for 9-12 days in vitro (DIV) were shifted to a medium without serum and growth factors (A, 12-72 h) or to increasing concentrations (5-50 μM) of 6-OHDA or MPP + (B) in the absence or the presence of a treatment with Wnt1 (100 ng/ml). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : DA neuron survival assessed by counting TH + neurons over the DAPI + nuclei, and expressed as percent (%) of PBS-treated control. C : [ 3 H]dopamine incorporation was assessed 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM), the values expressed as % of control. D: Caspase activity was determined by measuring DEVD-AFC hydrolysis. Enzymatic determinations were performed in lysates from cell cultures deprived of serum or challenged with 6-OHDA or MPP + for 6 h. *p < 0.05 when compared to control (PBS); ° p < 0.05 when compared to sister cultures exposed the cytotoxic insult (within each experimental group). E-J : Representative images showing TH + neurons (in red, E) and DAPI nuclear counterstaining (blue) 24 h after PBS, after 48 h of SD (F), or 24 h after 6-OHDA (G), in absence or the presence of Wnt1 pre-treatment (H,I,J). MPP + sharply decreases TH neurite length, an effect efficiently counteracted by Wnt1.
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    Danaher Inc rabbit anti wnt1 antibody
    <t>Wnt1</t> protects primary mesencephalic dopaminergic (DA) neurons against cell death induced by serum deprivation (SD), 6-hydroxydopamine (6-OHDA) and MPP + . Enriched neuronal cultures derived from the mesencephalon of E14 rat embryos maintained for 9-12 days in vitro (DIV) were shifted to a medium without serum and growth factors (A, 12-72 h) or to increasing concentrations (5-50 μM) of 6-OHDA or MPP + (B) in the absence or the presence of a treatment with Wnt1 (100 ng/ml). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : DA neuron survival assessed by counting TH + neurons over the DAPI + nuclei, and expressed as percent (%) of PBS-treated control. C : [ 3 H]dopamine incorporation was assessed 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM), the values expressed as % of control. D: Caspase activity was determined by measuring DEVD-AFC hydrolysis. Enzymatic determinations were performed in lysates from cell cultures deprived of serum or challenged with 6-OHDA or MPP + for 6 h. *p < 0.05 when compared to control (PBS); ° p < 0.05 when compared to sister cultures exposed the cytotoxic insult (within each experimental group). E-J : Representative images showing TH + neurons (in red, E) and DAPI nuclear counterstaining (blue) 24 h after PBS, after 48 h of SD (F), or 24 h after 6-OHDA (G), in absence or the presence of Wnt1 pre-treatment (H,I,J). MPP + sharply decreases TH neurite length, an effect efficiently counteracted by Wnt1.
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    Danaher Inc rabbit anti wnt induced signaling protein 1
    <t>Wnt1</t> protects primary mesencephalic dopaminergic (DA) neurons against cell death induced by serum deprivation (SD), 6-hydroxydopamine (6-OHDA) and MPP + . Enriched neuronal cultures derived from the mesencephalon of E14 rat embryos maintained for 9-12 days in vitro (DIV) were shifted to a medium without serum and growth factors (A, 12-72 h) or to increasing concentrations (5-50 μM) of 6-OHDA or MPP + (B) in the absence or the presence of a treatment with Wnt1 (100 ng/ml). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : DA neuron survival assessed by counting TH + neurons over the DAPI + nuclei, and expressed as percent (%) of PBS-treated control. C : [ 3 H]dopamine incorporation was assessed 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM), the values expressed as % of control. D: Caspase activity was determined by measuring DEVD-AFC hydrolysis. Enzymatic determinations were performed in lysates from cell cultures deprived of serum or challenged with 6-OHDA or MPP + for 6 h. *p < 0.05 when compared to control (PBS); ° p < 0.05 when compared to sister cultures exposed the cytotoxic insult (within each experimental group). E-J : Representative images showing TH + neurons (in red, E) and DAPI nuclear counterstaining (blue) 24 h after PBS, after 48 h of SD (F), or 24 h after 6-OHDA (G), in absence or the presence of Wnt1 pre-treatment (H,I,J). MPP + sharply decreases TH neurite length, an effect efficiently counteracted by Wnt1.
    Rabbit Anti Wnt Induced Signaling Protein 1, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti wnt induced signaling protein 1/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    86
    Abcam rabbit anti wnt1 antibody
    FSTL1 interacts with canonical Wnt ligands. A , FSTL1 interacts with <t>Wnt1.</t> HEK293T cells were transfected with Wnt1 plasmid in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1 to pull down Wnt1 ( left panel ). Reciprocal IP was performed ( right panel ). Western blotting was performed on whole lysates and precipitates as indicated. B , FSTL1 interacts with Wnt2 and Wnt3a. HEK293T cells were transfected with FATL1-HA in the absence or the presence of Wnt2-V5 or Wnt3a-V5 plasmid. Cell lysates were immunoprecipitated with IgG or anti-V5 antibody to pull down FSTL1 ( left panel ). Reciprocal IP was also performed ( right panel s ). C , FSTL1 interacts with Wnt8a, Wnt8b, Wnt9a, and Wnt10b. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of V5-tagged Wnt8a, Wnt8b, Wnt9a, and Wnt10b plasmids. Cell lysates were immunoprecipitated with anti-V5, followed by Western blotting with anti-FSTL1 or anti-V5 antibody on the precipitates and lysates as indicated. D , interaction of FSTL1 and Wnt2 occurs in the extracellular space. HEK293T cells were transfected with Wnt2-V5 in the absence or the presence of FSTL1-HA plasmid. Cell culture medium was immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and medium as indicated. E and F , interaction of FSTL1 and Wnt3a ( E ) or Wnt10b ( F ) occurs in the extracellular space. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of Wnt3a-V5 ( E ) or Wnt10b-V5 ( F ) plasmid. Cell culture medium was immunoprecipitated with anti-V5 antibody, followed by Western blotting with anti-V5 or anti-FSTL1 on the precipitates and medium as indicated. G , ALK3 does not interact with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 in the absence or the presence of FSTL1-HA or ALK3-HA plasmid. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and lysates as indicated. H , FSTL1 interacts with Wnt1 and Wnt3a in the kidney. Lysates from mouse kidneys with UUO for 7 days were immunoprecipitated with a combination of anti-Wnt1 and anti-Wnt3a antibodies or normal IgG, and the precipitates and lysates were subjected to Western blotting as indicated. I , FSTL1 directly interacts with Wnt3a. Recombinant Wnt3a protein (1.5 μg) was incubated with and without recombinant FSTL1 protein (3 μg) in 300 μl Tris-buffered saline/0.1% Triton X-100 overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated with anti-FSTL1 antibodies followed by Protein G agarose. Eluted proteins and inputs were analyzed by Western blotting as indicated. J , domain organization of full-length (FL) and mutant FSTL1 proteins. K , interactions of EC–VWC and EC domains of FSTL1 with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of FL, EC–VWC, or EC plasmid. Cell lysates were immunoprecipitated with anti-FSTL1. Western blotting was performed on whole lysates and precipitates as indicated. L , VWC but not FS domain of FSTL1 interacts with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of 3× FLAG-FS or 3× FLAG-VWC plasmid. Cell lysates were immunoprecipitated with anti-FLAG. Western blotting was performed on whole lysates and precipitates as indicated. EC, the extracellular calcium–binding domain; FS, the follistatin-like domain; FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IgG, immunoglobulin G; IP, immunoprecipitation; UUO, unilateral ureteral obstruction; VWC, a domain with homology to von Willebrand factor type C domain.
    Rabbit Anti Wnt1 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti wnt1 antibody/product/Abcam
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti wnt1 antibody - by Bioz Stars, 2024-06
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    Image Search Results


    Effect of Wnt1-positive expression on proliferative and migratory C6 GSCs in different groups. (A) The positive expression of Wnt1 on C6 GSCs was observed by immunofluorescence staining. Scale bar, 100 µm. (B) The statistical data of the number of Wnt1-positive cells are shown in each group. *P<0.05 and ***P<0.001. GSCs, glioma stem cells; ns, no significance.

    Journal: Oncology Reports

    Article Title: Inhibitor of Wnt receptor 1 suppresses the effects of Wnt1, Wnt3a and β‑catenin on the proliferation and migration of C6 GSCs induced by low‑dose radiation

    doi: 10.3892/or.2023.8681

    Figure Lengend Snippet: Effect of Wnt1-positive expression on proliferative and migratory C6 GSCs in different groups. (A) The positive expression of Wnt1 on C6 GSCs was observed by immunofluorescence staining. Scale bar, 100 µm. (B) The statistical data of the number of Wnt1-positive cells are shown in each group. *P<0.05 and ***P<0.001. GSCs, glioma stem cells; ns, no significance.

    Article Snippet: The fixed cells were incubated at 4°C overnight with primary antibodies, including rabbit anti-γ-H 2 AX (1:2,000; cat. no. ab11174; Abcam), rabbit anti-Wnt1 (1:3,000; cat. no. ab15151; Abcam), rabbit anti-Wnt3a (1:2,000; cat. no. ab28472; Abcam) and rabbit anti-β-catenin (1:5,000; cat. no. C2206; MiliporeSigma).

    Techniques: Expressing, Immunofluorescence, Staining

    Changes in Wnt1, Wnt3a and β-catenin protein levels in proliferative and migratory C6 glioma stem cells in different groups. (A) By Western blot analysis, the Wnt1, Wnt3a and β-catenin proteins were detected in the different groups. β-actin was used as the internal control. (B-D) The gray value quantification of (B) Wnt1, (C) Wnt3a and (D) β-catenin protein, normalized to β-actin. *P<0.05, **P<0.01 and ***P<0.001. ns, no significance.

    Journal: Oncology Reports

    Article Title: Inhibitor of Wnt receptor 1 suppresses the effects of Wnt1, Wnt3a and β‑catenin on the proliferation and migration of C6 GSCs induced by low‑dose radiation

    doi: 10.3892/or.2023.8681

    Figure Lengend Snippet: Changes in Wnt1, Wnt3a and β-catenin protein levels in proliferative and migratory C6 glioma stem cells in different groups. (A) By Western blot analysis, the Wnt1, Wnt3a and β-catenin proteins were detected in the different groups. β-actin was used as the internal control. (B-D) The gray value quantification of (B) Wnt1, (C) Wnt3a and (D) β-catenin protein, normalized to β-actin. *P<0.05, **P<0.01 and ***P<0.001. ns, no significance.

    Article Snippet: The fixed cells were incubated at 4°C overnight with primary antibodies, including rabbit anti-γ-H 2 AX (1:2,000; cat. no. ab11174; Abcam), rabbit anti-Wnt1 (1:3,000; cat. no. ab15151; Abcam), rabbit anti-Wnt3a (1:2,000; cat. no. ab28472; Abcam) and rabbit anti-β-catenin (1:5,000; cat. no. C2206; MiliporeSigma).

    Techniques: Western Blot

    miR-424 activates Wnt/β-catenin signaling pathway by directly targeting APC. (A) Wester blots and (B) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 mimics + pcDNA-empty or miR-424 mimics + pcDNA-APC. (C) Western blots and (D) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 inhibitor + si-scramble or miR-424 inhibitor + si-APC. Data are presented as means ± standard deviation of three individual experiments. * P<0.05 and ** P<0.01 vs. control. ## P<0.01 vs. miR-424 mimics + pcDNA-vector or miR-424 inhibitor + si-scramble. APC, adenomatous polyposis coli; miR, microRNA; si, small interfering.

    Journal: Experimental and Therapeutic Medicine

    Article Title: miR‑424‑5p is downregulated in the placentas of patients with preeclampsia and affects trophoblast migration and invasion

    doi: 10.3892/etm.2023.11993

    Figure Lengend Snippet: miR-424 activates Wnt/β-catenin signaling pathway by directly targeting APC. (A) Wester blots and (B) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 mimics + pcDNA-empty or miR-424 mimics + pcDNA-APC. (C) Western blots and (D) protein levels of APC, Wnt1, β-catenin, MMP-9 and MMP2 in HTR-8/SVneo cells transfected with miR-424 inhibitor + si-scramble or miR-424 inhibitor + si-APC. Data are presented as means ± standard deviation of three individual experiments. * P<0.05 and ** P<0.01 vs. control. ## P<0.01 vs. miR-424 mimics + pcDNA-vector or miR-424 inhibitor + si-scramble. APC, adenomatous polyposis coli; miR, microRNA; si, small interfering.

    Article Snippet: The PVDF membrane was blocked with 1% BSA (Sigma-Aldrich; Merck KGaA) for 2 h at room temperature, followed by incubation overnight at 4˚C with primary antibodies including rabbit monoclonal anti-human APC (1:1,000; cat. no. 2504; Cell Signaling Technology, Inc.), rabbit monoclonal anti-human β-catenin (1:1,000; cat. no. 8480; Cell Signaling Technology), rabbit monoclonal anti-human MMP-9 (1:1,000; cat. no. 13667; Cell Signaling Technology), rabbit monoclonal anti-human MMP2 (1:1,000; cat. no. 40994; Cell Signaling Technology), the antibodies against rabbit polyclonal anti-human Wnt1 (1:1,000; cat. no. ab15251; Abcam) and rabbit polyclonal anti-human β-actin (1:2,000; cat. no. ab8227; Abcam).

    Techniques: Transfection, Western Blot, Standard Deviation, Plasmid Preparation

    HepG2 cells were mock infected, or infected with Ad-HCV core or Ad-EGFP for 48 h, and were harvested for preparation of total RNA and protein. ( A ) Infection efficiency of the adenovirus observed under the fluorescent microscope (×10). ( B ) Western blot analysis for HCV core protein expression. ( C ) SLqRT-PCR analysis for miR-152 expression. After normalization to endogenous RNU6B expression, the expression of miR-152 in each group of HepG2 cells was expressed as fold changes in comparison to the levels observed in mock-infected HepG2 cells. ( D ) Real time RT-PCR analysis for Wnt1 mRNA expression. ( E ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (HepG2: mock-infected HepG2 cells; Hcv: HepG2 cells infected with the Ad-HCV core; Co: HepG2 cells infected with the Ad-EGFP control; Advirus-Hcv: Adenovirus expressing the HCV core protein, Ad-HCV core; Advirus-Ctrl: Adenovirus expressing the EGFP control protein, Ad-EGFP).

    Journal: PLoS ONE

    Article Title: HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells

    doi: 10.1371/journal.pone.0081730

    Figure Lengend Snippet: HepG2 cells were mock infected, or infected with Ad-HCV core or Ad-EGFP for 48 h, and were harvested for preparation of total RNA and protein. ( A ) Infection efficiency of the adenovirus observed under the fluorescent microscope (×10). ( B ) Western blot analysis for HCV core protein expression. ( C ) SLqRT-PCR analysis for miR-152 expression. After normalization to endogenous RNU6B expression, the expression of miR-152 in each group of HepG2 cells was expressed as fold changes in comparison to the levels observed in mock-infected HepG2 cells. ( D ) Real time RT-PCR analysis for Wnt1 mRNA expression. ( E ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (HepG2: mock-infected HepG2 cells; Hcv: HepG2 cells infected with the Ad-HCV core; Co: HepG2 cells infected with the Ad-EGFP control; Advirus-Hcv: Adenovirus expressing the HCV core protein, Ad-HCV core; Advirus-Ctrl: Adenovirus expressing the EGFP control protein, Ad-EGFP).

    Article Snippet: Membranes were blocked with 5% BSA in TBS and 0.1% Tween-20, and incubated with primary rabbit polyclonal anti-Wnt1 antibody (Abcam, Cambridge, UK) or mouse monoclonal anti-Flag (a flag tag was added into the Ad-HCV core vector) antibody (Cell signaling Technology, Inc.) and horseradish peroxidase–linked anti-mouse conjugates (DAKO) according to the standard Western blot protocol. α-Tubulin was included as a loading control.

    Techniques: Infection, Microscopy, Western Blot, Expressing, Quantitative RT-PCR

    The two WT plasmids, pGL3-WNT1-1-wt-3′UTR and pGL3-WNT1-2-wt-3′UTR, including 90 nt spanning each seed match at the 3′UTR, as well as the two mutant controls, pGL3-WNT1-1-mut-3′UTR and pGL3-WNT1-2-mut-3′UTR, were generated based on the firefly luciferase expressing vector pGL3-promoter and confirmed by sequencing. HepG2 Cells were co-transfected with 250 ng of pGL3-WNT1-WT or pGL3-WNT1-Mut constructs with 23.5 nM of miR-152 mimics, miR-152 inhibitor, or their respective negative control. Each sample was co-transfected with 25 ng of pRL-TK plasmid expressing renilla luciferase to monitor the transfection efficiency. A luciferase activity assay was performed 48 hours after transfection with the dual luciferase reporter assay system (Promega). The relative luciferase activity was normalized with renilla luciferase activity. ( A ) WT and Mut 3′-UTRs of WNT1, indicating the interaction sites between miR-152 and 3′-UTR of WNT1. ( B ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-1-WT or WNT1-1-Mut 3′-UTR and miR-152 mimics or its negative control (NC). ( C ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 mimics or miR-152 mimics NC. ( D ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 inhibitor or miR-152 inhibitor NC. Data are shown as means and standard deviations from at least three independent experiments. ** P<0.01.

    Journal: PLoS ONE

    Article Title: HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells

    doi: 10.1371/journal.pone.0081730

    Figure Lengend Snippet: The two WT plasmids, pGL3-WNT1-1-wt-3′UTR and pGL3-WNT1-2-wt-3′UTR, including 90 nt spanning each seed match at the 3′UTR, as well as the two mutant controls, pGL3-WNT1-1-mut-3′UTR and pGL3-WNT1-2-mut-3′UTR, were generated based on the firefly luciferase expressing vector pGL3-promoter and confirmed by sequencing. HepG2 Cells were co-transfected with 250 ng of pGL3-WNT1-WT or pGL3-WNT1-Mut constructs with 23.5 nM of miR-152 mimics, miR-152 inhibitor, or their respective negative control. Each sample was co-transfected with 25 ng of pRL-TK plasmid expressing renilla luciferase to monitor the transfection efficiency. A luciferase activity assay was performed 48 hours after transfection with the dual luciferase reporter assay system (Promega). The relative luciferase activity was normalized with renilla luciferase activity. ( A ) WT and Mut 3′-UTRs of WNT1, indicating the interaction sites between miR-152 and 3′-UTR of WNT1. ( B ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-1-WT or WNT1-1-Mut 3′-UTR and miR-152 mimics or its negative control (NC). ( C ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 mimics or miR-152 mimics NC. ( D ) Dual luciferase assay of HepG2 cells co-transfected with the firefly luciferase constructs containing the WNT1-2-WT or WNT1-2-Mut 3′-UTR and miR-152 inhibitor or miR-152 inhibitor NC. Data are shown as means and standard deviations from at least three independent experiments. ** P<0.01.

    Article Snippet: Membranes were blocked with 5% BSA in TBS and 0.1% Tween-20, and incubated with primary rabbit polyclonal anti-Wnt1 antibody (Abcam, Cambridge, UK) or mouse monoclonal anti-Flag (a flag tag was added into the Ad-HCV core vector) antibody (Cell signaling Technology, Inc.) and horseradish peroxidase–linked anti-mouse conjugates (DAKO) according to the standard Western blot protocol. α-Tubulin was included as a loading control.

    Techniques: Mutagenesis, Generated, Luciferase, Expressing, Plasmid Preparation, Sequencing, Transfection, Construct, Negative Control, Activity Assay, Reporter Assay

    HepG2 cells were transfected with double-stranded miR-152 mimics, single-stranded miR-152 inhibitor, or their relative negative control RNA at a final concentration of 5 nM with a HiPerFect Transfection Reagent kit as indicated for 48 h, and were collected for preparation of total RNA and protein. ( A ) Real time RT-PCR analysis for Wnt1 mRNA expression. ( B ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (inh NC: scrambled oligonucleotides as the negative control for miR-152 inhibitor; inh-152: miR-152 inhibitor; miR NC: scrambled oligonucleotides as the negative control for miR-152 mimics; miR-152: miR-152 mimics; Hcv+miR-152: Adenovirus expressing the HCV core protein+miR-152 mimics).

    Journal: PLoS ONE

    Article Title: HCV Core Protein-Induced Down-Regulation of microRNA-152 Promoted Aberrant Proliferation by Regulating Wnt1 in HepG2 Cells

    doi: 10.1371/journal.pone.0081730

    Figure Lengend Snippet: HepG2 cells were transfected with double-stranded miR-152 mimics, single-stranded miR-152 inhibitor, or their relative negative control RNA at a final concentration of 5 nM with a HiPerFect Transfection Reagent kit as indicated for 48 h, and were collected for preparation of total RNA and protein. ( A ) Real time RT-PCR analysis for Wnt1 mRNA expression. ( B ) Western blot analysis for Wnt1 protein expression. Data are shown as means and standard deviations from triplicate experiments. * P<0.05. (inh NC: scrambled oligonucleotides as the negative control for miR-152 inhibitor; inh-152: miR-152 inhibitor; miR NC: scrambled oligonucleotides as the negative control for miR-152 mimics; miR-152: miR-152 mimics; Hcv+miR-152: Adenovirus expressing the HCV core protein+miR-152 mimics).

    Article Snippet: Membranes were blocked with 5% BSA in TBS and 0.1% Tween-20, and incubated with primary rabbit polyclonal anti-Wnt1 antibody (Abcam, Cambridge, UK) or mouse monoclonal anti-Flag (a flag tag was added into the Ad-HCV core vector) antibody (Cell signaling Technology, Inc.) and horseradish peroxidase–linked anti-mouse conjugates (DAKO) according to the standard Western blot protocol. α-Tubulin was included as a loading control.

    Techniques: Transfection, Negative Control, Concentration Assay, Quantitative RT-PCR, Expressing, Western Blot

    Wnt1 protects primary mesencephalic dopaminergic (DA) neurons against cell death induced by serum deprivation (SD), 6-hydroxydopamine (6-OHDA) and MPP + . Enriched neuronal cultures derived from the mesencephalon of E14 rat embryos maintained for 9-12 days in vitro (DIV) were shifted to a medium without serum and growth factors (A, 12-72 h) or to increasing concentrations (5-50 μM) of 6-OHDA or MPP + (B) in the absence or the presence of a treatment with Wnt1 (100 ng/ml). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : DA neuron survival assessed by counting TH + neurons over the DAPI + nuclei, and expressed as percent (%) of PBS-treated control. C : [ 3 H]dopamine incorporation was assessed 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM), the values expressed as % of control. D: Caspase activity was determined by measuring DEVD-AFC hydrolysis. Enzymatic determinations were performed in lysates from cell cultures deprived of serum or challenged with 6-OHDA or MPP + for 6 h. *p < 0.05 when compared to control (PBS); ° p < 0.05 when compared to sister cultures exposed the cytotoxic insult (within each experimental group). E-J : Representative images showing TH + neurons (in red, E) and DAPI nuclear counterstaining (blue) 24 h after PBS, after 48 h of SD (F), or 24 h after 6-OHDA (G), in absence or the presence of Wnt1 pre-treatment (H,I,J). MPP + sharply decreases TH neurite length, an effect efficiently counteracted by Wnt1.

    Journal: Molecular Neurodegeneration

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    doi: 10.1186/1750-1326-6-49

    Figure Lengend Snippet: Wnt1 protects primary mesencephalic dopaminergic (DA) neurons against cell death induced by serum deprivation (SD), 6-hydroxydopamine (6-OHDA) and MPP + . Enriched neuronal cultures derived from the mesencephalon of E14 rat embryos maintained for 9-12 days in vitro (DIV) were shifted to a medium without serum and growth factors (A, 12-72 h) or to increasing concentrations (5-50 μM) of 6-OHDA or MPP + (B) in the absence or the presence of a treatment with Wnt1 (100 ng/ml). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : DA neuron survival assessed by counting TH + neurons over the DAPI + nuclei, and expressed as percent (%) of PBS-treated control. C : [ 3 H]dopamine incorporation was assessed 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM), the values expressed as % of control. D: Caspase activity was determined by measuring DEVD-AFC hydrolysis. Enzymatic determinations were performed in lysates from cell cultures deprived of serum or challenged with 6-OHDA or MPP + for 6 h. *p < 0.05 when compared to control (PBS); ° p < 0.05 when compared to sister cultures exposed the cytotoxic insult (within each experimental group). E-J : Representative images showing TH + neurons (in red, E) and DAPI nuclear counterstaining (blue) 24 h after PBS, after 48 h of SD (F), or 24 h after 6-OHDA (G), in absence or the presence of Wnt1 pre-treatment (H,I,J). MPP + sharply decreases TH neurite length, an effect efficiently counteracted by Wnt1.

    Article Snippet: After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: rabbit anti-TH (Chemicon); rat anti-DAT (Millipore), rabbit anti-Wnt1 (Abcam), mouse anti-β-catenin (Transduction Labs), mouse anti-GSK-3β (Transduction Labs), mouse anti-GSK-3β phospho-Tyr216 (BD Biosciences), goat anti-Fzd-1 (Santa Cruz Biotechnology, Inc), β-actin (Cell Signaling).

    Techniques: Derivative Assay, In Vitro, Activity Assay

    β-catenin depletion abolishes Wnt1- induced TH + neuroprotection . Enriched neuronal cultures were transiently transfected with β-catenin small interference RNA (β-catenin siRNA ) (sc-29210) or control siRNA (β-catenin Ct , see text for details), before being exposed to either Wnt1 or PBS, and DA neuron survival assessed by counting TH + neurons, by assessing [ 3 H]dopamine incorporation and Caspase3 activity. Differences analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B: Effect of SD, 6-OHDA and MPP + treatments in β-catenin mRNA (A) and protein levels (B) showing a significant decrease of β-catenin . Note that preventive application of Wnt1 increases β-catenin both at a mRNA (A) and protein levels (B). *p < 0.05 vs PBS. Depletion of β-catenin via the introduction of β-catenin siRNA shows an almost 40-60% reduction in β-catenin mRNA by Real time PCR (A) and western blotting (B). *p < 0.05 vs control siRNA C-D : Survival of TH + neurons by cell counting (C), [ 3 H]dopamine incorporation (D). Note that in β-catenin siRNA pre-treated cultures, the application of Wnt1 failed to protect TH + neurons against 6-OHDA or MPP + , whereas in cultures pre-treated with a control siRNA, Wnt1 treatment increased TH + neuron survival (C) and [ 3 H]dopamine incorporation (D). E-L : Representative immunocytochemical images show the ability of Wnt1 to efficiently counteracts TH neuron death and neurite loss, an effect abolished by β-catenin silencing *p < 0.05 vs cultures without cytotoxic insult; ** p < 0.05 compared to siRNA Ct + cytotoxic insult (within each each experimental group); ° p < 0.05 compared to Wnt1 treated cultures in the presence of siRNA Ct .

    Journal: Molecular Neurodegeneration

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    doi: 10.1186/1750-1326-6-49

    Figure Lengend Snippet: β-catenin depletion abolishes Wnt1- induced TH + neuroprotection . Enriched neuronal cultures were transiently transfected with β-catenin small interference RNA (β-catenin siRNA ) (sc-29210) or control siRNA (β-catenin Ct , see text for details), before being exposed to either Wnt1 or PBS, and DA neuron survival assessed by counting TH + neurons, by assessing [ 3 H]dopamine incorporation and Caspase3 activity. Differences analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B: Effect of SD, 6-OHDA and MPP + treatments in β-catenin mRNA (A) and protein levels (B) showing a significant decrease of β-catenin . Note that preventive application of Wnt1 increases β-catenin both at a mRNA (A) and protein levels (B). *p < 0.05 vs PBS. Depletion of β-catenin via the introduction of β-catenin siRNA shows an almost 40-60% reduction in β-catenin mRNA by Real time PCR (A) and western blotting (B). *p < 0.05 vs control siRNA C-D : Survival of TH + neurons by cell counting (C), [ 3 H]dopamine incorporation (D). Note that in β-catenin siRNA pre-treated cultures, the application of Wnt1 failed to protect TH + neurons against 6-OHDA or MPP + , whereas in cultures pre-treated with a control siRNA, Wnt1 treatment increased TH + neuron survival (C) and [ 3 H]dopamine incorporation (D). E-L : Representative immunocytochemical images show the ability of Wnt1 to efficiently counteracts TH neuron death and neurite loss, an effect abolished by β-catenin silencing *p < 0.05 vs cultures without cytotoxic insult; ** p < 0.05 compared to siRNA Ct + cytotoxic insult (within each each experimental group); ° p < 0.05 compared to Wnt1 treated cultures in the presence of siRNA Ct .

    Article Snippet: After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: rabbit anti-TH (Chemicon); rat anti-DAT (Millipore), rabbit anti-Wnt1 (Abcam), mouse anti-β-catenin (Transduction Labs), mouse anti-GSK-3β (Transduction Labs), mouse anti-GSK-3β phospho-Tyr216 (BD Biosciences), goat anti-Fzd-1 (Santa Cruz Biotechnology, Inc), β-actin (Cell Signaling).

    Techniques: Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting

    Knocking down Fzd-1 counteracts Wnt1- induced TH + neuroprotection . Enriched neuronal cultures were processed for Real time PCR using specific primers for Fzd receptors. The effect of knocking down Fzd-1 in Wnt1 neuroprotection was studied with Fzd-1 sense (Fzd-1Ct) or antisense (Fzd-1AS) oligonucleotides. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A : Differential expression and regulation of Fzd transcripts by SD, 6-OHDA and MPP + . B: Western blot (wb) analysis showing down-regulation of Fzd-1 levels in neuronal cultures exposed to the cytotoxic stimuli and the significant reversal induced by Wnt1 . *p < 0.05 vs cultures without cytotoxic insult. Pre-treatment with Fzd-1 AS induced an almost 40-60% decrease of Fzd-1. C-H : Representative confocal images of dual staining with Fzd-1 (green) and TH (red) showing colocalization (orange to yellow) in PBS (C-D) controls. Note the marked loss of Fzd-1 in TH neurons exposed to 6-OHDA (E) or MPP + (F), an effect efficiently counteracted by Wnt1 pre-treatment (G). I-J : Survival of TH + neurons by cell counting (C), [ 3 H]dopamine incorporation (D). K-L : Effect of Fzd-1 AS or Fzd-1 Ct , in β-catenin protein and Caspase3-like activity. Fzd-1 AS pre-treatment prevents Wnt1- induced increased β-catenin protein levels (K) and reverses Wnt1-induced Caspase3-inhibition (L) in 6-OHDA and MPP + -treated cultures. *p < 0.05 vs PBS. *p < 0.05 vs control siRNA. Note that Wnt1 efficiently reversed the dramatic decrease of neurite length caused by 6-OHDA or MPP + in Fzd-1 Ct -treated (M, N), as opposed to Fzd-1 knocked down cultures (O, P). *p < 0.05 vs cultures without insult (within each each experimental group).

    Journal: Molecular Neurodegeneration

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    doi: 10.1186/1750-1326-6-49

    Figure Lengend Snippet: Knocking down Fzd-1 counteracts Wnt1- induced TH + neuroprotection . Enriched neuronal cultures were processed for Real time PCR using specific primers for Fzd receptors. The effect of knocking down Fzd-1 in Wnt1 neuroprotection was studied with Fzd-1 sense (Fzd-1Ct) or antisense (Fzd-1AS) oligonucleotides. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A : Differential expression and regulation of Fzd transcripts by SD, 6-OHDA and MPP + . B: Western blot (wb) analysis showing down-regulation of Fzd-1 levels in neuronal cultures exposed to the cytotoxic stimuli and the significant reversal induced by Wnt1 . *p < 0.05 vs cultures without cytotoxic insult. Pre-treatment with Fzd-1 AS induced an almost 40-60% decrease of Fzd-1. C-H : Representative confocal images of dual staining with Fzd-1 (green) and TH (red) showing colocalization (orange to yellow) in PBS (C-D) controls. Note the marked loss of Fzd-1 in TH neurons exposed to 6-OHDA (E) or MPP + (F), an effect efficiently counteracted by Wnt1 pre-treatment (G). I-J : Survival of TH + neurons by cell counting (C), [ 3 H]dopamine incorporation (D). K-L : Effect of Fzd-1 AS or Fzd-1 Ct , in β-catenin protein and Caspase3-like activity. Fzd-1 AS pre-treatment prevents Wnt1- induced increased β-catenin protein levels (K) and reverses Wnt1-induced Caspase3-inhibition (L) in 6-OHDA and MPP + -treated cultures. *p < 0.05 vs PBS. *p < 0.05 vs control siRNA. Note that Wnt1 efficiently reversed the dramatic decrease of neurite length caused by 6-OHDA or MPP + in Fzd-1 Ct -treated (M, N), as opposed to Fzd-1 knocked down cultures (O, P). *p < 0.05 vs cultures without insult (within each each experimental group).

    Article Snippet: After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: rabbit anti-TH (Chemicon); rat anti-DAT (Millipore), rabbit anti-Wnt1 (Abcam), mouse anti-β-catenin (Transduction Labs), mouse anti-GSK-3β (Transduction Labs), mouse anti-GSK-3β phospho-Tyr216 (BD Biosciences), goat anti-Fzd-1 (Santa Cruz Biotechnology, Inc), β-actin (Cell Signaling).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining, Cell Counting, Activity Assay, Inhibition

    Effect of Wnt1-induced TH + neuroprotection on active GSK-3β and Caspase3-like activity . Purified neuronal cultures were exposed to SD, 6-OHDA or MPP + insults as described, and DA neuroprotection examined as above. Active GSK-3β (GSK-3β phospho-Tyr216) protein levels were determined by western blotting (wb). Part of the cultures were pre-treated with Wnt1 (100 ng/ml) or the the specific GSK-3β antagonist, AR-14418 (5 μM), alone or in combination, while other cell cultures were exposed to the specific antagonist of Wnt/β-catenin pathway, Dkk1 (100 ng/ml), 1 hr before Wnt1 (100 ng/ml) treatment. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : Active GSK-3β (GSK-3β phospho-Tyr216) protein levels as determined by wb. A sharp up-regulation is observed after SD, 6-OHDA or MPP + insults, an effect reversed by Wnt1 and AR pre-treatment. B : Survival of TH + neurons exposed to the described insults in the absence or the presence of the different treatments. * p < 0.05 when compared to control cultures without cytotoxic insults. Note that both Wnt1 and AR increase TH + neuron survival, and the combined Wnt1+AR treatment further increase TH + neuron survival. By contrast, exposure of Dkk1-pretreated cultures to SD, 6-OHDA or MPP + reduced Wnt1 protective effect on cell survival. C : Caspase-3 activity shows a sharp inhibition of DEVD-like activity in neurons pre-treated with Wnt1 or AR and exposed to the different insults, whereas previous exposure to Dkk1, counteracts Wnt1-induced Casapse3 inhibition. * p < 0.05 when compared to control cultures without cytotoxic insults.

    Journal: Molecular Neurodegeneration

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    doi: 10.1186/1750-1326-6-49

    Figure Lengend Snippet: Effect of Wnt1-induced TH + neuroprotection on active GSK-3β and Caspase3-like activity . Purified neuronal cultures were exposed to SD, 6-OHDA or MPP + insults as described, and DA neuroprotection examined as above. Active GSK-3β (GSK-3β phospho-Tyr216) protein levels were determined by western blotting (wb). Part of the cultures were pre-treated with Wnt1 (100 ng/ml) or the the specific GSK-3β antagonist, AR-14418 (5 μM), alone or in combination, while other cell cultures were exposed to the specific antagonist of Wnt/β-catenin pathway, Dkk1 (100 ng/ml), 1 hr before Wnt1 (100 ng/ml) treatment. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B : Active GSK-3β (GSK-3β phospho-Tyr216) protein levels as determined by wb. A sharp up-regulation is observed after SD, 6-OHDA or MPP + insults, an effect reversed by Wnt1 and AR pre-treatment. B : Survival of TH + neurons exposed to the described insults in the absence or the presence of the different treatments. * p < 0.05 when compared to control cultures without cytotoxic insults. Note that both Wnt1 and AR increase TH + neuron survival, and the combined Wnt1+AR treatment further increase TH + neuron survival. By contrast, exposure of Dkk1-pretreated cultures to SD, 6-OHDA or MPP + reduced Wnt1 protective effect on cell survival. C : Caspase-3 activity shows a sharp inhibition of DEVD-like activity in neurons pre-treated with Wnt1 or AR and exposed to the different insults, whereas previous exposure to Dkk1, counteracts Wnt1-induced Casapse3 inhibition. * p < 0.05 when compared to control cultures without cytotoxic insults.

    Article Snippet: After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: rabbit anti-TH (Chemicon); rat anti-DAT (Millipore), rabbit anti-Wnt1 (Abcam), mouse anti-β-catenin (Transduction Labs), mouse anti-GSK-3β (Transduction Labs), mouse anti-GSK-3β phospho-Tyr216 (BD Biosciences), goat anti-Fzd-1 (Santa Cruz Biotechnology, Inc), β-actin (Cell Signaling).

    Techniques: Activity Assay, Purification, Western Blot, Inhibition

    Modulation of astroglial Wnt1/β-catenin signaling directs towards TH neuron survival/death . Astrocyte-neuron co-cultures where shifted to serum deprived medium (SD, A), or received 6-OHDA or MPP + (25 μM), with or without Dkk1 (100 ng/ml), a Wnt1-Ab , or AR-14418 (5 μM) as described. A-B : TH + neuron counts (A), and [ 3 H]dopamine uptake (B) 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM) with or without Dkk1, Wnt1-Ab, or AR. * p < 0.05 compared to controls. C: Western blot analysis showing β-catenin levels in neurons exposed to astrocyte insert upon cytotoxic challenge with or without Wnt1-Ab or Dkk1. In this experimental condition, the glial inserts were added on the top of the purified neurons at 9 DIV. D : immunoblotting with Wnt1-Ab (50) using protein extracts from embryonic VM and the NIH/3T3 cell line. 50 ng of recombinant Wnt1 was used as a positive control. E : Dual staining with TH (red) and DAPI depicting TH + neurons in a control (PBS) astrocyte-neuron co-culture. D-P : Confocal images showing dual staining with TH (green) and GFAP (red) in a typical astrocyte-neuron control co-culture at 9 DIV. Note the lenght and branching of TH + processes. Astrocyte coculture induced a significant protection against SD (E), 6-OHDA (H), and the reversal induced by Dkk1 antagonism of Wnt/β-catenin signaling (F,I). By contrast, pharmacological activation of Wnt/β-catenin signaling with AR magnified TH neuroprotection (G,J,K). Astrocyte coculture increases Fzd-1 signal in the long and branched TH + neurites and growth cones (L-N and insert).

    Journal: Molecular Neurodegeneration

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    doi: 10.1186/1750-1326-6-49

    Figure Lengend Snippet: Modulation of astroglial Wnt1/β-catenin signaling directs towards TH neuron survival/death . Astrocyte-neuron co-cultures where shifted to serum deprived medium (SD, A), or received 6-OHDA or MPP + (25 μM), with or without Dkk1 (100 ng/ml), a Wnt1-Ab , or AR-14418 (5 μM) as described. A-B : TH + neuron counts (A), and [ 3 H]dopamine uptake (B) 48 h after SD or 24 h after 6-OHDA or MPP + (25 μM) with or without Dkk1, Wnt1-Ab, or AR. * p < 0.05 compared to controls. C: Western blot analysis showing β-catenin levels in neurons exposed to astrocyte insert upon cytotoxic challenge with or without Wnt1-Ab or Dkk1. In this experimental condition, the glial inserts were added on the top of the purified neurons at 9 DIV. D : immunoblotting with Wnt1-Ab (50) using protein extracts from embryonic VM and the NIH/3T3 cell line. 50 ng of recombinant Wnt1 was used as a positive control. E : Dual staining with TH (red) and DAPI depicting TH + neurons in a control (PBS) astrocyte-neuron co-culture. D-P : Confocal images showing dual staining with TH (green) and GFAP (red) in a typical astrocyte-neuron control co-culture at 9 DIV. Note the lenght and branching of TH + processes. Astrocyte coculture induced a significant protection against SD (E), 6-OHDA (H), and the reversal induced by Dkk1 antagonism of Wnt/β-catenin signaling (F,I). By contrast, pharmacological activation of Wnt/β-catenin signaling with AR magnified TH neuroprotection (G,J,K). Astrocyte coculture increases Fzd-1 signal in the long and branched TH + neurites and growth cones (L-N and insert).

    Article Snippet: After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: rabbit anti-TH (Chemicon); rat anti-DAT (Millipore), rabbit anti-Wnt1 (Abcam), mouse anti-β-catenin (Transduction Labs), mouse anti-GSK-3β (Transduction Labs), mouse anti-GSK-3β phospho-Tyr216 (BD Biosciences), goat anti-Fzd-1 (Santa Cruz Biotechnology, Inc), β-actin (Cell Signaling).

    Techniques: Western Blot, Purification, Recombinant, Positive Control, Staining, Co-Culture Assay, Activation Assay

    Antagonizing Wnt signaling or Silencing Wnt1 in VM astrocytes fail to protect TH + neurons . Wnt antagonism studies were performed with the extracellular-rich domain (CRD) of Fzd-1 (Fzd-1-CRD, 1 μ/ml) and the CRD of Fzd-2 (Fzd-2-CRD,1 μ/ml). Depletion of Wnt1 in VM astrocytes was achieved by introducing a small interference RNA targeting Wnt1 , Astro siWnt1 (46) in indirect astrocyte-neuron co-cultures (42). A-C: Indirect astrocyte-neuron co-culture efficiently mitigated SD, 6-OHDA- and MPP + -induced decreased TH neuron survival (A), DA uptake (B), and Caspase3 activation (C), whereas these protective effects are reversed by Fzd-1-CRD pre-treatment, but not Fzd-2-CRD. * p < 0.05 when compared to control cultures without cytotoxic insults; ° p < 0.05 compared to neurotoxic exposure within each experimental group. D-F : Glial inserts transiently transfected with siRNA wnt1 or siRNA control were added on the top of the purified neurons at 9 DIV and exposed to cytotoxic stimuli. RT-qPCR analysis (D) and immunocytochemistry (E-F) show an almost 40-60% Wnt1 depletion by 72 h. G-H : In neurons co-cultured with Astro siWnt1 , irrespective of the cytotoxic stimulus, a sharp reduction of TH + neuronal count (G) and increased Caspase3-like activity (H) were observed, compared to neuron co-cultured with Astro Ct (p < 0.05). In β-catenin siRNA- and Fzd-1 AS -knocked down neurons, astrocyte inserts failed to promote neuroprotection. I-S : Confocal images of TH staining (green) in the indirect cocultures showing the protective effect of astrocyte inserts + siRNA Ct (L,O,R) as compared with siRNA Wnt1 (M,P,S). The effect of indirect astrocyte coculture is emphasized in the PBS control cultures (I, N).

    Journal: Molecular Neurodegeneration

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    doi: 10.1186/1750-1326-6-49

    Figure Lengend Snippet: Antagonizing Wnt signaling or Silencing Wnt1 in VM astrocytes fail to protect TH + neurons . Wnt antagonism studies were performed with the extracellular-rich domain (CRD) of Fzd-1 (Fzd-1-CRD, 1 μ/ml) and the CRD of Fzd-2 (Fzd-2-CRD,1 μ/ml). Depletion of Wnt1 in VM astrocytes was achieved by introducing a small interference RNA targeting Wnt1 , Astro siWnt1 (46) in indirect astrocyte-neuron co-cultures (42). A-C: Indirect astrocyte-neuron co-culture efficiently mitigated SD, 6-OHDA- and MPP + -induced decreased TH neuron survival (A), DA uptake (B), and Caspase3 activation (C), whereas these protective effects are reversed by Fzd-1-CRD pre-treatment, but not Fzd-2-CRD. * p < 0.05 when compared to control cultures without cytotoxic insults; ° p < 0.05 compared to neurotoxic exposure within each experimental group. D-F : Glial inserts transiently transfected with siRNA wnt1 or siRNA control were added on the top of the purified neurons at 9 DIV and exposed to cytotoxic stimuli. RT-qPCR analysis (D) and immunocytochemistry (E-F) show an almost 40-60% Wnt1 depletion by 72 h. G-H : In neurons co-cultured with Astro siWnt1 , irrespective of the cytotoxic stimulus, a sharp reduction of TH + neuronal count (G) and increased Caspase3-like activity (H) were observed, compared to neuron co-cultured with Astro Ct (p < 0.05). In β-catenin siRNA- and Fzd-1 AS -knocked down neurons, astrocyte inserts failed to promote neuroprotection. I-S : Confocal images of TH staining (green) in the indirect cocultures showing the protective effect of astrocyte inserts + siRNA Ct (L,O,R) as compared with siRNA Wnt1 (M,P,S). The effect of indirect astrocyte coculture is emphasized in the PBS control cultures (I, N).

    Article Snippet: After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: rabbit anti-TH (Chemicon); rat anti-DAT (Millipore), rabbit anti-Wnt1 (Abcam), mouse anti-β-catenin (Transduction Labs), mouse anti-GSK-3β (Transduction Labs), mouse anti-GSK-3β phospho-Tyr216 (BD Biosciences), goat anti-Fzd-1 (Santa Cruz Biotechnology, Inc), β-actin (Cell Signaling).

    Techniques: Co-Culture Assay, Activation Assay, Transfection, Purification, Quantitative RT-PCR, Immunocytochemistry, Cell Culture, Activity Assay, Staining

    Effect of intracerebral infusion of a Fzd receptor antagonist within the intact SNpc in the number of TH + Nissl + and Fluorojade-C (FJC) cell bodies . The effect of blocking Wnt/β-catenin signaling was assessed using the negative modulator of Wnt1 signaling, Dkk1 (1 μg/μl) or saline, unilaterally infused into the left intact SNpc, as described. Mice were sacrificed 1-7 days post-infusion, and the brains processed for TH + Nissl + cell counts (M) and Fluorojade C staining (FJC, N) in consecutive midbrain sections. A-L: C onfocal images showing TH + (green) neurons counterstained with DAPI (blu) of coronal midbrain sections at the level of the SNpc 1 day after unilateral saline (A-C) or 1 (D-F), 3 (G-I) and 7 (J-L) days after unilateral Dkk1 infusion. In Dkk1-infused mice, a decrease of TH + immunofluorescent signal was observed starting 1 d (D) in the ipsilateral (E), but not contralateral (F), non-infused SN, with a peak TH + loss at 3 days (see H compared to I), and a stabilization observed by 7 days (F). M . Total number of TH + and Nissl + counted throught the entire rostro-caudal axis of the SNpc. Treatment groups were averaged (n = 4/time-point, means ± S.E.M.) * p < 0.05 vs contralateral side, within each respective group. B . Total number of Fluorojade C (FJC) stained cells in SNpc ipsilateral and contralateral to the infusion was calculated for each side, averaged for each animal (n = 4/time-point) and normalized to the number of TH + neurons in SNpc per section. *p < 0.05 vs saline injected side.

    Journal: Molecular Neurodegeneration

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    doi: 10.1186/1750-1326-6-49

    Figure Lengend Snippet: Effect of intracerebral infusion of a Fzd receptor antagonist within the intact SNpc in the number of TH + Nissl + and Fluorojade-C (FJC) cell bodies . The effect of blocking Wnt/β-catenin signaling was assessed using the negative modulator of Wnt1 signaling, Dkk1 (1 μg/μl) or saline, unilaterally infused into the left intact SNpc, as described. Mice were sacrificed 1-7 days post-infusion, and the brains processed for TH + Nissl + cell counts (M) and Fluorojade C staining (FJC, N) in consecutive midbrain sections. A-L: C onfocal images showing TH + (green) neurons counterstained with DAPI (blu) of coronal midbrain sections at the level of the SNpc 1 day after unilateral saline (A-C) or 1 (D-F), 3 (G-I) and 7 (J-L) days after unilateral Dkk1 infusion. In Dkk1-infused mice, a decrease of TH + immunofluorescent signal was observed starting 1 d (D) in the ipsilateral (E), but not contralateral (F), non-infused SN, with a peak TH + loss at 3 days (see H compared to I), and a stabilization observed by 7 days (F). M . Total number of TH + and Nissl + counted throught the entire rostro-caudal axis of the SNpc. Treatment groups were averaged (n = 4/time-point, means ± S.E.M.) * p < 0.05 vs contralateral side, within each respective group. B . Total number of Fluorojade C (FJC) stained cells in SNpc ipsilateral and contralateral to the infusion was calculated for each side, averaged for each animal (n = 4/time-point) and normalized to the number of TH + neurons in SNpc per section. *p < 0.05 vs saline injected side.

    Article Snippet: After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: rabbit anti-TH (Chemicon); rat anti-DAT (Millipore), rabbit anti-Wnt1 (Abcam), mouse anti-β-catenin (Transduction Labs), mouse anti-GSK-3β (Transduction Labs), mouse anti-GSK-3β phospho-Tyr216 (BD Biosciences), goat anti-Fzd-1 (Santa Cruz Biotechnology, Inc), β-actin (Cell Signaling).

    Techniques: Blocking Assay, Staining, Injection

    Effect of pharmacological activation of Wnt/β-catenin signaling in TH + neuroprotection against intracerebral Dkk1 or systemic MPTP treatment . To mimick the activation of Wnt1/β-catenin signaling, we selected the specific GSK-3β inhibitor, AR (10 mg/kg twice a day) starting 72 h before unilateral Dkk1 infusion within the SN, or before the systemic (i.p.) treatment with the parkinsonian neurotoxin, MPTP (15 mg kg -1 , 4 times a day at 2 h intervals), and mice sacrificed at the peak degeneration phase (3 days post-treatment). A-B : Representative confocal images showing dual localization of TH + neurons (green) and GFAP + astrocytes (red) in Saline (A), MPTP (B) and AR/MPTP (C) 4d post-MPTP, showing the sharp decrease of TH neurons associated to the marked astrocytosis and the remarkable protective effect of AR (C). C : The total number of TH + and Nissl + neurons was counted throught the entire rostro-caudal axis of the SNpc as above. Treatment groups were averaged (n = 4/time-point, means ± S.E.M.) * p < 0.05 vs unifused side (for Dkk1), within each respective group. Dkk1 and MPTP significantly reduced TH + and Nissl + neurons 4 d post-treatment. MPTP systemic treatment reduces TH + neuron numbers in both left and both sides. Note the remarkable counteraction afforded by AR in increasing TH + neurons to unlesioned saline-treated control. *p < 0.05 vs -MPTP.

    Journal: Molecular Neurodegeneration

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    doi: 10.1186/1750-1326-6-49

    Figure Lengend Snippet: Effect of pharmacological activation of Wnt/β-catenin signaling in TH + neuroprotection against intracerebral Dkk1 or systemic MPTP treatment . To mimick the activation of Wnt1/β-catenin signaling, we selected the specific GSK-3β inhibitor, AR (10 mg/kg twice a day) starting 72 h before unilateral Dkk1 infusion within the SN, or before the systemic (i.p.) treatment with the parkinsonian neurotoxin, MPTP (15 mg kg -1 , 4 times a day at 2 h intervals), and mice sacrificed at the peak degeneration phase (3 days post-treatment). A-B : Representative confocal images showing dual localization of TH + neurons (green) and GFAP + astrocytes (red) in Saline (A), MPTP (B) and AR/MPTP (C) 4d post-MPTP, showing the sharp decrease of TH neurons associated to the marked astrocytosis and the remarkable protective effect of AR (C). C : The total number of TH + and Nissl + neurons was counted throught the entire rostro-caudal axis of the SNpc as above. Treatment groups were averaged (n = 4/time-point, means ± S.E.M.) * p < 0.05 vs unifused side (for Dkk1), within each respective group. Dkk1 and MPTP significantly reduced TH + and Nissl + neurons 4 d post-treatment. MPTP systemic treatment reduces TH + neuron numbers in both left and both sides. Note the remarkable counteraction afforded by AR in increasing TH + neurons to unlesioned saline-treated control. *p < 0.05 vs -MPTP.

    Article Snippet: After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: rabbit anti-TH (Chemicon); rat anti-DAT (Millipore), rabbit anti-Wnt1 (Abcam), mouse anti-β-catenin (Transduction Labs), mouse anti-GSK-3β (Transduction Labs), mouse anti-GSK-3β phospho-Tyr216 (BD Biosciences), goat anti-Fzd-1 (Santa Cruz Biotechnology, Inc), β-actin (Cell Signaling).

    Techniques: Activation Assay

    Schematic illustration of Wnt1/Fzd-1/β-catenin signaling as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk . Crosstalk between astrocytes and DA neurons represent a cardinal neuroprotective mechanism against inflammation, oxidative stress and growth factor deprivation (10). Here astrocyte-DA neuron crosstalk via Wnt1 is emphasized. Astrocyte-derived Wnt, via activation of Fzd-1 receptors, may contribute to maintain the integrity of DA neurons by influencing Wnt signaling components, including blockade of GSK-3β-induced phosphorylation (P) and proteosomal degradation of the neuronal pool of β-catenin. Stabilized β-catenin can translocate into the nucleus and associate with a family of transcription factors and regulate the expression of Wnt target genes involved in DA neuron survival. β-catenin may also function as a pivotal defense molecule against oxidative stress (79), and can act as a coactivator for several nuclear receptors involved in the maintenance/protection of DA neurons (81). Crosstalk with up-stream survival pathways converging to β-catenin stabilization can also be envisaged (26, 27). Neurotoxic injury or increased oxidative load as a result of aging may antagonize Wnt/β-catenin signaling in DA neurons by up-regulating active GSK-3β, leading to β-catenin degradation and increased DA neuron vulnerability, which may underlie a progressive DA neuron deficit. Neuronal injury also triggers reactive astrocyte expression of a panel of growth and neurotrophic factors, anti-oxidant and neuroprotective mechanisms, among which astrocyte Wnt1 via Fzd-1 receptors may function as a vital component of DA neurons self-protective machinery shifting the balance towards the programming of cell survival/neurorescue.

    Journal: Molecular Neurodegeneration

    Article Title: A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

    doi: 10.1186/1750-1326-6-49

    Figure Lengend Snippet: Schematic illustration of Wnt1/Fzd-1/β-catenin signaling as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk . Crosstalk between astrocytes and DA neurons represent a cardinal neuroprotective mechanism against inflammation, oxidative stress and growth factor deprivation (10). Here astrocyte-DA neuron crosstalk via Wnt1 is emphasized. Astrocyte-derived Wnt, via activation of Fzd-1 receptors, may contribute to maintain the integrity of DA neurons by influencing Wnt signaling components, including blockade of GSK-3β-induced phosphorylation (P) and proteosomal degradation of the neuronal pool of β-catenin. Stabilized β-catenin can translocate into the nucleus and associate with a family of transcription factors and regulate the expression of Wnt target genes involved in DA neuron survival. β-catenin may also function as a pivotal defense molecule against oxidative stress (79), and can act as a coactivator for several nuclear receptors involved in the maintenance/protection of DA neurons (81). Crosstalk with up-stream survival pathways converging to β-catenin stabilization can also be envisaged (26, 27). Neurotoxic injury or increased oxidative load as a result of aging may antagonize Wnt/β-catenin signaling in DA neurons by up-regulating active GSK-3β, leading to β-catenin degradation and increased DA neuron vulnerability, which may underlie a progressive DA neuron deficit. Neuronal injury also triggers reactive astrocyte expression of a panel of growth and neurotrophic factors, anti-oxidant and neuroprotective mechanisms, among which astrocyte Wnt1 via Fzd-1 receptors may function as a vital component of DA neurons self-protective machinery shifting the balance towards the programming of cell survival/neurorescue.

    Article Snippet: After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: rabbit anti-TH (Chemicon); rat anti-DAT (Millipore), rabbit anti-Wnt1 (Abcam), mouse anti-β-catenin (Transduction Labs), mouse anti-GSK-3β (Transduction Labs), mouse anti-GSK-3β phospho-Tyr216 (BD Biosciences), goat anti-Fzd-1 (Santa Cruz Biotechnology, Inc), β-actin (Cell Signaling).

    Techniques: Derivative Assay, Activation Assay, Expressing

    FSTL1 interacts with canonical Wnt ligands. A , FSTL1 interacts with Wnt1. HEK293T cells were transfected with Wnt1 plasmid in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1 to pull down Wnt1 ( left panel ). Reciprocal IP was performed ( right panel ). Western blotting was performed on whole lysates and precipitates as indicated. B , FSTL1 interacts with Wnt2 and Wnt3a. HEK293T cells were transfected with FATL1-HA in the absence or the presence of Wnt2-V5 or Wnt3a-V5 plasmid. Cell lysates were immunoprecipitated with IgG or anti-V5 antibody to pull down FSTL1 ( left panel ). Reciprocal IP was also performed ( right panel s ). C , FSTL1 interacts with Wnt8a, Wnt8b, Wnt9a, and Wnt10b. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of V5-tagged Wnt8a, Wnt8b, Wnt9a, and Wnt10b plasmids. Cell lysates were immunoprecipitated with anti-V5, followed by Western blotting with anti-FSTL1 or anti-V5 antibody on the precipitates and lysates as indicated. D , interaction of FSTL1 and Wnt2 occurs in the extracellular space. HEK293T cells were transfected with Wnt2-V5 in the absence or the presence of FSTL1-HA plasmid. Cell culture medium was immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and medium as indicated. E and F , interaction of FSTL1 and Wnt3a ( E ) or Wnt10b ( F ) occurs in the extracellular space. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of Wnt3a-V5 ( E ) or Wnt10b-V5 ( F ) plasmid. Cell culture medium was immunoprecipitated with anti-V5 antibody, followed by Western blotting with anti-V5 or anti-FSTL1 on the precipitates and medium as indicated. G , ALK3 does not interact with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 in the absence or the presence of FSTL1-HA or ALK3-HA plasmid. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and lysates as indicated. H , FSTL1 interacts with Wnt1 and Wnt3a in the kidney. Lysates from mouse kidneys with UUO for 7 days were immunoprecipitated with a combination of anti-Wnt1 and anti-Wnt3a antibodies or normal IgG, and the precipitates and lysates were subjected to Western blotting as indicated. I , FSTL1 directly interacts with Wnt3a. Recombinant Wnt3a protein (1.5 μg) was incubated with and without recombinant FSTL1 protein (3 μg) in 300 μl Tris-buffered saline/0.1% Triton X-100 overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated with anti-FSTL1 antibodies followed by Protein G agarose. Eluted proteins and inputs were analyzed by Western blotting as indicated. J , domain organization of full-length (FL) and mutant FSTL1 proteins. K , interactions of EC–VWC and EC domains of FSTL1 with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of FL, EC–VWC, or EC plasmid. Cell lysates were immunoprecipitated with anti-FSTL1. Western blotting was performed on whole lysates and precipitates as indicated. L , VWC but not FS domain of FSTL1 interacts with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of 3× FLAG-FS or 3× FLAG-VWC plasmid. Cell lysates were immunoprecipitated with anti-FLAG. Western blotting was performed on whole lysates and precipitates as indicated. EC, the extracellular calcium–binding domain; FS, the follistatin-like domain; FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IgG, immunoglobulin G; IP, immunoprecipitation; UUO, unilateral ureteral obstruction; VWC, a domain with homology to von Willebrand factor type C domain.

    Journal: The Journal of Biological Chemistry

    Article Title: Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/β-catenin signaling in obstructed kidneys in vivo

    doi: 10.1016/j.jbc.2022.102010

    Figure Lengend Snippet: FSTL1 interacts with canonical Wnt ligands. A , FSTL1 interacts with Wnt1. HEK293T cells were transfected with Wnt1 plasmid in the absence or the presence of FSTL1-HA plasmid. Cell lysates were immunoprecipitated with anti-FSTL1 to pull down Wnt1 ( left panel ). Reciprocal IP was performed ( right panel ). Western blotting was performed on whole lysates and precipitates as indicated. B , FSTL1 interacts with Wnt2 and Wnt3a. HEK293T cells were transfected with FATL1-HA in the absence or the presence of Wnt2-V5 or Wnt3a-V5 plasmid. Cell lysates were immunoprecipitated with IgG or anti-V5 antibody to pull down FSTL1 ( left panel ). Reciprocal IP was also performed ( right panel s ). C , FSTL1 interacts with Wnt8a, Wnt8b, Wnt9a, and Wnt10b. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of V5-tagged Wnt8a, Wnt8b, Wnt9a, and Wnt10b plasmids. Cell lysates were immunoprecipitated with anti-V5, followed by Western blotting with anti-FSTL1 or anti-V5 antibody on the precipitates and lysates as indicated. D , interaction of FSTL1 and Wnt2 occurs in the extracellular space. HEK293T cells were transfected with Wnt2-V5 in the absence or the presence of FSTL1-HA plasmid. Cell culture medium was immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and medium as indicated. E and F , interaction of FSTL1 and Wnt3a ( E ) or Wnt10b ( F ) occurs in the extracellular space. HEK293T cells were transfected with FSTL1-HA in the absence or the presence of Wnt3a-V5 ( E ) or Wnt10b-V5 ( F ) plasmid. Cell culture medium was immunoprecipitated with anti-V5 antibody, followed by Western blotting with anti-V5 or anti-FSTL1 on the precipitates and medium as indicated. G , ALK3 does not interact with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 in the absence or the presence of FSTL1-HA or ALK3-HA plasmid. Cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting with anti-V5 or anti-HA antibody on the precipitates and lysates as indicated. H , FSTL1 interacts with Wnt1 and Wnt3a in the kidney. Lysates from mouse kidneys with UUO for 7 days were immunoprecipitated with a combination of anti-Wnt1 and anti-Wnt3a antibodies or normal IgG, and the precipitates and lysates were subjected to Western blotting as indicated. I , FSTL1 directly interacts with Wnt3a. Recombinant Wnt3a protein (1.5 μg) was incubated with and without recombinant FSTL1 protein (3 μg) in 300 μl Tris-buffered saline/0.1% Triton X-100 overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated with anti-FSTL1 antibodies followed by Protein G agarose. Eluted proteins and inputs were analyzed by Western blotting as indicated. J , domain organization of full-length (FL) and mutant FSTL1 proteins. K , interactions of EC–VWC and EC domains of FSTL1 with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of FL, EC–VWC, or EC plasmid. Cell lysates were immunoprecipitated with anti-FSTL1. Western blotting was performed on whole lysates and precipitates as indicated. L , VWC but not FS domain of FSTL1 interacts with Wnt3a. HEK293T cells were transfected with Wnt3a-V5 plasmid in the absence or the presence of 3× FLAG-FS or 3× FLAG-VWC plasmid. Cell lysates were immunoprecipitated with anti-FLAG. Western blotting was performed on whole lysates and precipitates as indicated. EC, the extracellular calcium–binding domain; FS, the follistatin-like domain; FSTL1, follistatin-like 1; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IgG, immunoglobulin G; IP, immunoprecipitation; UUO, unilateral ureteral obstruction; VWC, a domain with homology to von Willebrand factor type C domain.

    Article Snippet: Preabsorbed lysates were incubated with rabbit anti-Wnt1 antibody (catalog no.: ab15251; Abcam) and rabbit anti-WNT3a antibody (catlog no.: 2721; Cell Signaling Technology) or normal rabbit immunoglobulin G at 4 °C overnight.

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Cell Culture, Recombinant, Incubation, Mutagenesis, Binding Assay