murine sfrp1  (Addgene inc)

Journal: Journal of Bone and Mineral Research

Article Title: Osteomodulin deficiency in mice causes a specific reduction of transversal cortical bone size

doi: 10.1093/jbmr/zjae072

Figure Lengend Snippet: Omd is a WNT1-induced gene. A) Scatter plot of microarray expression values. Average signal intensities of transcripts in ST2 cells 5 days after transfection with a Wnt1 expression plasmid or an empty pLNCX vector are shown with the 3 most differentially expressed genes labeled and marked by circles. B) Confirmation of microarray results by qRT-PCR. Expression of Omd , Aspn , and Postn in ST2 cells 5 days after transfection with a wild-type Wnt1 expression plasmid or a plasmid encoding the osteogenesis imperfecta-causing G177C variant of Wnt1 relative to the expression in ST2 cells transfected with the empty pLNCX vector (dashed line). C) Expression of Apcdd1, Omd , Aspn , and Postn in ST2 cells stimulated for the indicated duration with either recombinant WNT1/SFRP1, SFRP1, WNT3a, or WNT5a compared with PBS treated controls (indicated by dashed lines). Data are presented as box plots with dots for individual datapoints. Data were analyzed by Student t-test (B) or one-way ANOVA with Tukey’s post-hoc test (C). * P < .05, * * P < .01, * * * P < .001, * * * * P < .0001.

Article Snippet: ST2 cells were cultured to 80%–90% confluency and stimulated with the following recombinant murine WNT ligands administered in FCS- and antibiotic-free α-MEM for 0.5, 2, or 6 hr as indicated prior to RNA isolation: 100 ng/ml recombinant murine WNT1/SFRP1 complex (reWNT1/SFRP1, Bio-Techne, United States), 50 ng/ml recombinant murine SFRP1 (reSFRP1, Bio-Techne, United States) as an equimolar control, 100 ng/ml recombinant murine WNT3a (reWNT3a, PeproTech, United States), and 100 ng/ml recombinant murine WNT5a (reWNT5a, R&D Systems, United States).

Techniques: Microarray, Expressing, Transfection, Plasmid Preparation, Labeling, Quantitative RT-PCR, Variant Assay, Recombinant

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibition results in altered dendritic spine development. (A) Representative dendritic segments of cortical neurons expressing empty vector (EV), Wif1, Sfrp1, Fzd8CRD or Dvl1ΔPDZ. (B) Quantification of dendritic protrusion density. (C) Percent of all dendritic protrusions classified as either spines or filopodia. Quantification of average protrusion length (D) and average spine head width (E) for each treatment. *** p < 0.001, ** p < 0.01, * p < 0.05. n = number of neurons: EV n = 31, Wif1 n = 34 Sfrp1 n = 23, Fzd8CRD n = 25, Dvl1ΔPDZ n = 25. Scale bar: 5 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Inhibition, Expressing, Plasmid Preparation

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Expression of Wnt inhibitors or Wnt2 shifts the distribution of dendritic protrusion lengths. (A) Relative frequency distributions of dendritic protrusion length comparing Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ expressing neurons to EV control. (B) Relative frequency distribution of protrusion length comparing Wnt2 expressing neurons to EV control. n = number of neurons: EV(A) n = 31, Wif1 n = 34, Sfrp1 n = 23, Fzd8CRD n = 25, Dvl1ΔPDZ n = 25; EV(B) n = 29, Wnt2 n = 25. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Expressing, Control

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibition results in decreased dendrite elaboration. (A) Representative cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD or Dvl1ΔPDZ. (B) Quantification of the total dendrite length per neuron for each treatment. (C) Quantification of the number of dendritic endpoints per neuron for each treatment. (D–G) Sholl analysis of dendritic complexity comparing neurons treated with each Wnt inhibitor to control neurons. *** p < 0.001, ** p < 0.01, * p < 0.05. n = number of neurons: EV n = 56, Wif1 n = 49, Sfrp1 n = 39, Fzd8CRD n = 39, Dvl1ΔPDZ n = 39. Scale bar: 50 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Inhibition, Expressing, Control

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibition does not affect primary dendrite number on cortical neurons. (A) Representative cell bodies of cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ either alone, or in combination with BDNF. (B) Quantification of the number of primary dendrites per neuron for each treatment. *** p < 0.001. n = number of neurons: EV n = 31, BDNF n = 31, Wif1 n = 34, BDNF + Wif1 n = 29, Sfrp1 n = 22, BDNF + Sfrp1 n = 21, Fzd8CRD n = 25, BDNF + Fzd8CRD n = 25, Dvl1ΔPDZ n = 25, BDNF + Dvl1ΔPDZ n = 30. Scale bar: 5 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Inhibition, Expressing

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Wnt inhibitors interfere with BDNF-induced dendritic spine formation. (A) Representative dendritic segments of cortical neurons expressing EV, BDNF with EV, BDNF with Wif1, BDNF with Sfrp1, BDNF with Fzd8CRD and BDNF with Dvl1ΔPDZ. (B) Quantification of dendritic protrusion density. (C) Fraction of all spines classified as either spines or filopodia. Quantification of average protrusion length (D) and average spine head width (E) for each treatment. *** p < 0.001, ** p < 0.01, * p < 0.5. (* = relative to EV, # = relative to EV + BDNF.) n = number of neurons: EV n = 31, BDNF + EV n = 30, BDNF + Wif1 n = 29, BDNF + Sfrp1 n = 25, BDNF + Fzd8CRD n = 25, BDNF + Dvl1ΔPDZ n = 30. Scale bar: 5 μm.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Expressing

Journal: Molecular and Cellular Neurosciences

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation ☆

doi: 10.1016/j.mcn.2013.04.006

Figure Lengend Snippet: Transfection with Wnt inhibitors does not prevent BDNF-induced c-Fos expression in cortical neurons. (A) GFP fluorescence, c-Fos immunoreactivity and DAPI fluorescence of representative cell bodies of cortical neurons expressing EV, Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ either alone, or in combination with BDNF. (B) Quantification of normalized nuclear c-Fos immunoreactivity per neuron for each treatment. *** p < 0.001. n = 10 neurons for each treatment.

Article Snippet: pCMV-BDNF was constructed by inserting the open reading frame for murine BDNF into the pEGFP-N1 backbone (Clontech, Mountain View, CA). pBR22-Wif1 expresses murine Wif1 and was a generous gift from Dr. Ken Iwatsuki (Ajinomoto Co., Tokyo, Japan). pCS2 +-Sfrp1 (Addgene plasmid 16693, Cambridge, MA) expresses murine Sfrp1 (Randall Moon Lab). pRK5-mFzd8CRD-IgG (Addgene plasmid 16689) expresses a fusion protein consisting of the extracellular domain of the murine Frizzled-8 protein fused with the human immunoglobulin heavy chain ( ). pCS2 +-mDvl1ΔPDZ-HA expresses a C-terminally HA tagged murine Dishevelled-1 with a deletion of amino acids 276–336 ( ). pTRE-tight-BDNF and pTRE-tight-Wnt2 were constructed by inserting the ORFs for murine BDNF and murine Wnt2, respectively, into the pTRE-tight backbone (Clontech). pTRE-tight-EGFP was constructed by inserting the EGFP ORF from pEGFP-N1 (Clontech) into the pTRE-tight backbone. pCMV-rtTA was constructed by inserting the ORF for the reverse-tet-Transactivator protein from pTRIPZ (Thermo Scientific) into the pEGFP-N1 backbone from which the EGFP coding region was deleted.

Techniques: Transfection, Expressing, Fluorescence