Journal: PLoS Genetics

Article Title: The planar cell polarity protein VANG-1/Vangl negatively regulates Wnt/β-catenin signaling through a Dvl dependent mechanism

doi: 10.1371/journal.pgen.1007840

Figure Lengend Snippet: ( A ) TOPFLASH reporter activity in wild type and Vangl1/2 double mutant (DKO) HEK293 cells treated with 100 ng/ml Wnt3a or the corresponding amount of 0.1% BSA in PBS in the absence (left panel) or presence (right panel) of R-Spondin. Rescue experiments were performed with expression of wild type and C-terminally truncated Vangl2 (fused to EGFP). Statistical significance was calculated using a Student’s t-test. *, p<0.05; **, p<0.01. ( B ) Western blot analysis of Vangl1 and Vangl2 expression in control, Vangl1/2 DKO and DKO cells expressing wild type or C-terminally truncated Vangl2 (fused to EGFP). Note that endogenous Vangl2 expression is not visible at this exposure. A longer exposure is provided in .

Article Snippet: Treatments used were: recombinant human Wnt3a (CF 5036-WN-CF RnD Systems), recombinant human R-Spondin-1 (120–38, PeproTech) and LGK 974 (974–02, Stem RD) for inhibition of endogenous Wnt ligands secretion.

Techniques: Activity Assay, Mutagenesis, Expressing, Western Blot, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: IFATS collection: Adipose stromal cell differentiation is reduced by endothelial cell contact and paracrine communication: role of canonical Wnt signaling.

doi: 10.1634/stemcells.2008-0277

Figure Lengend Snippet: Figure 5. Effect of canonical Wnt signaling on adipogenic differentiation of adipose stromal cells (ASC). (A): ASC were treated with either Wnt signaling agonist (1 M; Calbiochem) or recombinant Wnt protein (Wnt3a; 10 ng/ml) for 3 days prior to anal- ysis of activation of canonical Wnt signaling using the TCF7-driven Topflash reporter as- say. Note a 60% increase in Topflash activ- ity with Wnt agonist stimulation. (B): Adi- pogenic differentiation of ASC after 3 days using gene expression analysis of PPAR and LPL by reverse transcription polymerase chain reaction. A significant (, p .03) decrease was noted in adipogenic differenti- ation upon stimulation with Wnt agonist or a Wnt3a recombinant protein. Abbreviations: BM, basal medium; DM, differentiation me- dium; PPAR, peroxisome proliferator-acti- vated receptor.

Article Snippet: Thus, we examined the specific effect of canonical Wnt signaling stimulation by either the Wnt agonist ([(2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl) pyrimidine)]; Calbiochem, San Diego, http://www.emdbiosciences.com) or recombinant Wnt3a protein (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) on both downstream Wnt pathway activation and features of adipogenesis.

Techniques: Recombinant, Activation Assay, Gene Expression, Reverse Transcription, Polymerase Chain Reaction

Journal: Molecular Brain

Article Title: Wnt3a upregulates brain-derived insulin by increasing NeuroD1 via Wnt/β-catenin signaling in the hypothalamus

doi: 10.1186/s13041-016-0207-5

Figure Lengend Snippet: Wnt3a upregulates insulin synthesis and secretion from N39, a hypothalamic neuronal cell line. a N39 cells were treated with vehicle (PBS) or Wnt3a (25 or 100 ng/mL) for 6, 12, and 24 h, and the levels of Ins2 mRNA were measured by qRT-PCR and normalized to the levels of GAPDH mRNA ( n = 23). b N39 cells were treated with vehicle or Wnt3a (100 ng/mL) for 24 h, and the induction of insulin was examined by immunofluorescence analysis with an antibody against proinsulin. Nuclei were stained with Hoechst 33342 dye. Scale bar, 20 μm. c Culture media were collected for 24 h after treatment with vehicle or Wnt3a (100 ng/mL), concentrated in Vivaspin columns, and insulin concentrations were measured by ELISA ( n = 6). Data are means + SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with vehicle control at each time point

Article Snippet: Recombinant mouse Wnt3a (1324-WN, R&D Systems) was dissolved in phosphate-buffered saline (PBS) containing 0.2 % BSA.

Techniques: Quantitative RT-PCR, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecular Brain

Article Title: Wnt3a upregulates brain-derived insulin by increasing NeuroD1 via Wnt/β-catenin signaling in the hypothalamus

doi: 10.1186/s13041-016-0207-5

Figure Lengend Snippet: Wnt3a induces the expression of Ins2 by activating Wnt/β-catenin signaling. a N39 cells were treated with vehicle or Wnt3a (25 or 100 ng/mL) for 12 and 24 h. Active (Non-phospho) β-catenin and total β-catenin were detected using immunoblot assay. b and c The intensity of bands shown in ( a ) was quantified by using the ImageJ software with normalization to GAPDH ( n = 6). d N39 cells were treated with vehicle or Wnt3a (100 ng/mL) for 24 h, and active β-catenin was examined by immunofluorescence analysis; Hoechst 33342 dye was used for nuclear staining. Scale bar, 20 μm. e N39 cells were treated with 1 μM BIO, a GSK3 inhibitor, for 12 h, and the level of Ins2 mRNA was measured by qRT-PCR ( n = 9). f N39 cells were treated with vehicle (DMSO) or 1 μM BIO for 1, 3, 6, and 12 h to observe accumulation of active β-catenin and total β-catenin ( n = 6). g and h Quantification of immunoblot data in ( f ) using the ImageJ software was performed with normalization to GAPDH. Data are means + SEM. ** p < 0.01, *** p < 0.001 compared with vehicle treatment

Article Snippet: Recombinant mouse Wnt3a (1324-WN, R&D Systems) was dissolved in phosphate-buffered saline (PBS) containing 0.2 % BSA.

Techniques: Expressing, Western Blot, Software, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Molecular Brain

Article Title: Wnt3a upregulates brain-derived insulin by increasing NeuroD1 via Wnt/β-catenin signaling in the hypothalamus

doi: 10.1186/s13041-016-0207-5

Figure Lengend Snippet: Wnt3a increases the expression of NeuroD1 by activating Wnt/β-catenin signaling in N39 cells. a NeuroD1 mRNA levels were quantified using qRT-PCR after vehicle or Wnt3a (25 or 100 ng/mL) treatment for 6, 12, and 24 h ( n = 23). b NeuroD1 protein levels were measured by immunoblot assay after vehicle or Wnt3a (20 or 100 ng/mL) treatment for 12 and 24 h. c The intensity of bands shown in ( b ) was quantified by using ImageJ with normalization to GAPDH ( n = 6). d After treatment with vehicle or Wnt3a (100 ng/mL) for 24 h, NeuroD1 was examined using the immunofluorescence assay; the nuclei were stained with Hoechst 33342 dye. Scale bar, 20 μm. e The level of NeuroD1 mRNA was determined by qRT-PCR 12 h after treatment with 1 μM BIO ( n = 9). f Cells were treated with 1 μM BIO, and the level of NeuroD1 was measured by immunoblot assay after treatment for 1, 3, 6, and 12 h. g The intensity of NeuroD1 bands in ( f ) was quantified using the ImageJ software and normalized to GAPDH ( n = 6). Data are means + SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with vehicle treatment

Article Snippet: Recombinant mouse Wnt3a (1324-WN, R&D Systems) was dissolved in phosphate-buffered saline (PBS) containing 0.2 % BSA.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Software

Journal: Molecular Brain

Article Title: Wnt3a upregulates brain-derived insulin by increasing NeuroD1 via Wnt/β-catenin signaling in the hypothalamus

doi: 10.1186/s13041-016-0207-5

Figure Lengend Snippet: Wnt3a-induced Ins2 upregulation is blocked by knockdown of NeuroD1. NeuroD1 was knocked down by infecting N39 cells with lentivirus containing shRNA for 72 h. a N39 cells were infected by lentiviral shRNA against NeuroD1 for 48 h and then treated with Wnt3a (100 ng/mL) for 24 h. The levels of active and total β-catenin and NeuroD1 protein were determined by immunoblot assay. b The intensity of NeuroD1 signal in ( a ) was quantified using the ImageJ software ( n = 4). The levels of NeuroD1 mRNA ( c ) and Ins2 mRNA ( d ) were measured by qRT-PCR in N39 cells that were infected with lentiviral non-targeting or NeuroD1 shRNA for 48 h and then treated with Wnt3a (100 ng/mL) for 24 h ( n = 9). Data are means + SEM. *** p < 0.001 compared with vehicle treatment in shNon-target. ### p < 0.001 compared with Wnt3a treatment in shNon-target. N.S., not significant within shNeuroD1 samples

Article Snippet: Recombinant mouse Wnt3a (1324-WN, R&D Systems) was dissolved in phosphate-buffered saline (PBS) containing 0.2 % BSA.

Techniques: Knockdown, shRNA, Infection, Western Blot, Software, Quantitative RT-PCR

Journal: Molecular Brain

Article Title: Wnt3a upregulates brain-derived insulin by increasing NeuroD1 via Wnt/β-catenin signaling in the hypothalamus

doi: 10.1186/s13041-016-0207-5

Figure Lengend Snippet: Wnt3a upregulates NeuroD1 and Ins2 in the mouse hypothalamus. The basal mRNA levels of Ins2 and Wnt3a were measured by qRT-PCR in hypothalamic tissues from C57BL/6 male mice ( n = 6−9) ( a ). C57BL/6 male mice received icv injections of vehicle or Wnt3a (4 or 20 ng), and hypothalamic tissues were dissected after 24 h. The mRNA levels of Ins2 ( b ) and NeuroD1 ( c ) were measured by qRT-PCR ( n = 6−9). Data are means + SEM. ### p < 0.001 compared with Ins2 mRNA, *** p < 0.001 compared with vehicle administration

Article Snippet: Recombinant mouse Wnt3a (1324-WN, R&D Systems) was dissolved in phosphate-buffered saline (PBS) containing 0.2 % BSA.

Techniques: Quantitative RT-PCR

Journal: Molecular Brain

Article Title: Wnt3a upregulates brain-derived insulin by increasing NeuroD1 via Wnt/β-catenin signaling in the hypothalamus

doi: 10.1186/s13041-016-0207-5

Figure Lengend Snippet: The proposed regulatory mechanism of insulin expression by the Wnt/β-catenin/NeuroD1 pathway in the hypothalamus. Wnt3a might bind to LRP and Frizzed receptor to activate the canonical Wnt pathway. GSK3 phosphorylates β-catenin and induces its degradation; Wnt3a inhibits GSK3, leading to β-catenin accumulation. Accumulated β-catenin translocates into the nucleus and enhances the expression of NeuroD1 with TCF. Up-regulated NeuroD1 also translocates into the nucleus, where it induces the production of insulin

Article Snippet: Recombinant mouse Wnt3a (1324-WN, R&D Systems) was dissolved in phosphate-buffered saline (PBS) containing 0.2 % BSA.

Techniques: Expressing