Journal: Communications Biology

Article Title: PRICKLE3 protects VANGL proteins from CK1-mediated phosphorylation and RNF43-mediated degradation

doi: 10.1038/s42003-025-09422-9

Figure Lengend Snippet: a General schematic of the TetON inducible gene expression system used in this study. In the absence of doxycycline, the reverse tetracycline-controlled transactivator (rtTA) is inactive. Upon doxycycline addition, rtTA binds to the tetO promoter and activates transcription of the gene of interest, leading to protein expression. b Doxycycline-induced overexpression of HA-tagged PRICKLE3 in HEK T-REx 293 PRICKLE3 TetON cells and of PRICKLE1 in the corresponding inducible cell line. c Induction of HA-PRICKLE3 expression by doxycycline in HEK T-REx 293 PRICKLE3 TetON cells. HEK T-REx 293 wildtype (WT) cells served as a control for doxycycline effects. Arrowheads indicate phosphorylation-dependent shifts in the electrophoretic mobility of VANGL proteins. α-TUBULIN was used as a loading control. Representative result from n = 5. d , e Densitometric quantification of Western blot signals. Values were normalized to untreated cells. Statistical analysis was performed using an unpaired t -test; corresponding p -values are shown ( n = 5). f Effect of recombinant WNT stimulation on VANGL phosphorylation. HEK T-REx 293 PRICKLE3 TetON cells were pre-treated overnight with the porcupine inhibitor LGK-974 to block endogenous WNT ligand secretion and subsequently stimulated with 100 ng/ml human recombinant WNT5A or WNT3A for 3 h. Arrowheads indicate phosphorylation-dependent mobility shifts of ROR. α-TUBULIN served as a loading control. Representative result from n = 4. g , h Densitometric quantification of Western blot signals from panel f. Data were normalized to untreated controls. Statistical analysis was performed using an unpaired t -test; corresponding p -values are reported ( n = 4).

Article Snippet: In selected wells, cells were additionally treated with 1 μM LGK-974 (StemRD, #974-02) and/or 100 ng/mL recombinant human WNT3A (R&D Systems, #1324-WN).

Techniques: Gene Expression, Expressing, Over Expression, Control, Phospho-proteomics, Western Blot, Recombinant, Blocking Assay

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

Journal: Cancer Research Communications

Article Title: WNT4 Regulates Cellular Metabolism via Intracellular Activity at the Mitochondria in Breast and Gynecologic Cancers

doi: 10.1158/2767-9764.CRC-23-0275

Figure Lengend Snippet: BioID supports WNT4 localization to the mitochondria. A, Proteins enriched in HT1080 Wnt-BirA versus parental HT1080 cells lacking BirA construct expression. B, Overlap of proteins identified in HT1080 (A) versus HT1080-PKO and MM134 identifies n = 72 “high-confidence” WNT4-associated proteins. C, Gene ontology analysis for cellular compartment for WNT3A- versus WNT4-associated proteins. Dashed line = 1.3 ( P = 0.05). D, Network analysis of WNT3A- versus WNT4-associated proteins via subcell barcode. Enrichments against cell line HCC287 background shown; parallel results observed with other cell line background data, for example, MCF7. E, Proteins with predicted cytosolic or mitochondrial localization (subcell barcode) among “high-confidence” WNT4-associated proteins. Red = predicted mitochondrial localization, pink = mTOR complex in mitochondrial dynamics, biogenesis, and autophagy. F, Biotin treatment and streptavidin pulldown was performed as for MS studies, and candidate WNT4-associated proteins from E detected by immunoblotting. Total protein by Ponceau.

Article Snippet: Blots were probed with Streptavidin-HRP (Cell Signaling Technology #3999; RRID:AB_10830897) or antibodies used according to manufacturer's recommendations: WNT4 (R&D Systems, MAB4751; RRID:AB_2215448); WNT3A (Novus Biologicals, MAB13242); anti-HA-HRP conjugate (Cell Signaling Technology #2999; RRID:AB_1264166); DHRS2 (Sigma HPA053915; RRID:AB_2682307); mTOR (Cell Signaling Technology #2983; RRID:AB_2105622); STAT1 (Sigma HPA000931; RRID:AB_1080100).

Techniques: Construct, Expressing, Western Blot

Journal: European journal of immunology

Article Title: Recombinant Wnt3a and Wnt5a elicit macrophage cytokine production and tolerization to microbial stimulation via Toll-like receptor 4.

doi: 10.1002/eji.201343959

Figure Lengend Snippet: Figure 2. Recombinant Wnt3a but not Wnt5a-induced elevated Axin2 expression in mouse BM-derived macrophages. The mRNA expression of the gene encoding for Axin2 was determined by qRT-PCR following stimulation with recombinant Wnt5a and Wnt3a (300 ng/mL) for 4 h; values were normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT) and depicted as fold-change upon Wnt treatment compared with unstimulated control cells in the same experiment. Bars with medians represent the 25th and 75th percentiles; whiskers indicate the largest and smallest values. Dotted line indicates a fold-change of 1. Data are cumulative from seven independent experiments with one data point per condition per experiment. Wilcoxon matched-pairs rank sum test was performed to compare Wnt-induced relative gene expression to untreated control cultures. **p < 0.01, *p < 0.05.

Article Snippet: BMderived macrophages were stimulated for the times indicated with recombinant Wnt proteins, TLR agonists, or recombinant mouse cytokines (IFN-γ, 10 ng/mL; R&D Systems; IL-4, 10 ng/mL; PeproTech).

Techniques: Recombinant, Expressing, Derivative Assay, Quantitative RT-PCR, Control, Gene Expression

Journal: European journal of immunology

Article Title: Recombinant Wnt3a and Wnt5a elicit macrophage cytokine production and tolerization to microbial stimulation via Toll-like receptor 4.

doi: 10.1002/eji.201343959

Figure Lengend Snippet: Figure 3. Soluble Wnt pathway inhibitors do not impair macrophage cytokine responses induced by recombinant (A) Wnt5a and (B) Wnt3a. Macrophages were stimulated with recombinant Wnt5a and Wnt3a (300 ng/mL) in the presence or absence of sFRP1 (1 μg/mL), sFRP5 (1 μg/mL), or DKK1 (0.1 μg/mL) for 4 h. Cytokine concentrations were determined in cell culture supernatants using ELISA. Data are means ± SD of triplicate wells of one experiment representative of two to four independent experiments each performed in triplicates using at least two different lots of each of the recombinant Wnt proteins.

Article Snippet: BMderived macrophages were stimulated for the times indicated with recombinant Wnt proteins, TLR agonists, or recombinant mouse cytokines (IFN-γ, 10 ng/mL; R&D Systems; IL-4, 10 ng/mL; PeproTech).

Techniques: Recombinant, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Recombinant Wnt3a and Wnt5a elicit macrophage cytokine production and tolerization to microbial stimulation via Toll-like receptor 4.

doi: 10.1002/eji.201343959

Figure Lengend Snippet: Figure 4. Wnt5a- and Wnt3a-induced macrophage inflammatory gene expression and cytokine responses are TLR4 and MyD88 dependent. BM-derived macrophages from WT, TLR4−/−, and MyD88−/−mice were stimulated with recombinant Wnt5a and Wnt3a (300 ng/mL) for 4 h. The TLR agonists, LPS (100 EU/mL; TLR4), and Pam3CSK4 (10 ng/mL; TLR2), as well as unstimulated cells were used as controls. (A) The mRNA expression of the genes encoding IL-12p40, IL-6, TNF, and IL-10 was assessed by qRT-PCR and is depicted as relative gene expression after normalization to hypoxanthine phosphoribosyltransferase 1 (HPRT) mRNA expression. Means ± SEM of three independent experiments (one data point per condition per experiment) are shown. (B) Concentrations of IL-12p40, IL-6, TNF, and IL-10 were determined in the cell culture supernatants by ELISA. Means ± SEM of four independent experiments (one data point per experiment) are shown. Groups treated with the same stimulus in panels A and B were compared using ANOVA one-way analysis of variance with Tukey posttest, α = 0.05, 95% confidence interval. *p < 0.05.

Article Snippet: BMderived macrophages were stimulated for the times indicated with recombinant Wnt proteins, TLR agonists, or recombinant mouse cytokines (IFN-γ, 10 ng/mL; R&D Systems; IL-4, 10 ng/mL; PeproTech).

Techniques: Gene Expression, Derivative Assay, Recombinant, Expressing, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Recombinant Wnt3a and Wnt5a elicit macrophage cytokine production and tolerization to microbial stimulation via Toll-like receptor 4.

doi: 10.1002/eji.201343959

Figure Lengend Snippet: Figure 5. Suppression of TLR-induced cytokine responses after Wnt prestimulation is dependent on TLR4. BM-derived macrophages were prein- cubated with recombinant Wnt5a and Wnt3a for 20 h and subsequently stimulated with (A) the TLR2 agonist Pam3CSK4 (10 ng/mL) or (B) the TLR9 agonist CpG (250 ng/mL). TNF and IL-6 concentrations were determined in cell culture supernatants by ELISA at 6 h post-stimulation. Data show means ± SD of triplicate wells representative of four (Pam3CSK4) and two (CpG) independent experiments each performed in triplicates. (C) BM-derived macrophages from WT and TLR4−/−mice were preincubated (first stimulation) for 20 h with medium alone (med), recombinant Wnt proteins (300 ng/mL), or LPS (1 EU/mL). Subsequently, cells were left untreated (med) or stimulated with Pam3CSK4 (10 ng/mL; second stimulation). TNF and IL-6 concentrations in cell culture supernatants were determined by ELISA 6 h after Pam3CSK4 stimulation. Data show means ± SD of triplicate wells of one experiment representative of results obtained in two (TNF) and three (IL-6) independent experiments using at least two different Wnt lots.

Article Snippet: BMderived macrophages were stimulated for the times indicated with recombinant Wnt proteins, TLR agonists, or recombinant mouse cytokines (IFN-γ, 10 ng/mL; R&D Systems; IL-4, 10 ng/mL; PeproTech).

Techniques: Derivative Assay, Recombinant, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Recombinant Wnt3a and Wnt5a elicit macrophage cytokine production and tolerization to microbial stimulation via Toll-like receptor 4.

doi: 10.1002/eji.201343959

Figure Lengend Snippet: Figure 6. Polymyxin B (PxB) inhibits macrophage cytokine production in response to recombinant Wnt5a and Wnt3a. (A) BM-derived macrophages generated from WT mice were left unstimulated (unst) or were stimulated for 4 h with recombinant Wnt5a and Wnt3a (300 ng/mL) or LPS (100 EU/mL) in the presence or absence of PxB (25 μg/mL). Concentrations of IL-12p40, IL-6, TNF, and IL-10 in the supernatant were determined by ELISA in three to six independent experiments with one data point per condition per experiment and are shown comparing medium- versus PxB-treated cells within individual experiments. Data were analyzed by Wilcoxon matched-pairs ranked sum test. **p < 0.01, *p < 0.05, n.s.: not significant. (B) Percentages of the cytokine concentrations remaining after PxB treatment, calculated from the raw data of three to six independent experiments described above. Means are indicated.

Article Snippet: BMderived macrophages were stimulated for the times indicated with recombinant Wnt proteins, TLR agonists, or recombinant mouse cytokines (IFN-γ, 10 ng/mL; R&D Systems; IL-4, 10 ng/mL; PeproTech).

Techniques: Recombinant, Derivative Assay, Generated, Enzyme-linked Immunosorbent Assay