recombinant wnt11 protein  (R&D Systems)

Journal: Communications Biology

Article Title: Wnt11 mediates fibroblast–smooth muscle cell interaction to promote neurogenic bladder fibrosis in rats

doi: 10.1038/s42003-026-09647-2

Figure Lengend Snippet: a Relative mRNA level of WNT11 in fibrotic bladders following BPNI and SCI ( n = 8). Western blotting of WNT11 expression in BPNI ( b ) and SCI ( c )-induced NB. Representative photomicrographs showed that WNT11 (green) was increased in rats after BPNI ( d ) and SCI ( e ). Scale bar = 400 μm. Protein levels of fibroblasts differentiation-related markers (α-SMA and Col-1) in BFs isolated from rats treated with BPNI ( f ) and SCI ( g ). Representative western blotting showing the phenotypic transformation of SMCs (SMTN and MYH10) in BPNI ( h ) and SCI ( i ) rats. Western blotting analyses showing protein levels of WNT11 and markers of fibroblastic differentiation (α-SMA and Col-1) and SMCs phenotypic transformation (SMTN and MYH10) in BFs ( j ) and SMCs ( k ) treated with TGF-β1 at different doses, respectively. Data are shown as the mean ± SD, and P values were determined by the one-way ANOVA followed by Tukey’s post-hoc test ( a ), * P < 0.05, ** P < 0.01.

Article Snippet: To maximally activate or inhibit the WNT cascade in vivo, rats were treated with recombinant WNT11 protein (#6179-WN-010, R&D Systems) or LGK974 (#HY-17545, MedChemExpress) immediately after the surgical procedure.

Techniques: Western Blot, Expressing, Isolation, Transformation Assay

Journal: Communications Biology

Article Title: Wnt11 mediates fibroblast–smooth muscle cell interaction to promote neurogenic bladder fibrosis in rats

doi: 10.1038/s42003-026-09647-2

Figure Lengend Snippet: a – n Eight-week-old male Sprague–Dawley rats were subjected to sham, BPNI, or SCI operation. Vehicle, WNT11, or LGK974 were administered to rats every day after the surgical procedure for 2 weeks, and these rats were euthanized at post 4 weeks of operation. a Schematic of the experimental protocol and treatment regimen. b Representative bladder images in rats treated with vehicle, WNT11, or LGK974 post BPNI or SCI. Representative images of cystometrograms in rats after BPNI ( c ) or SCI ( d ). Statistical results for the intravesical pressure in rats after BPNI ( e ) or SCI ( f ) ( n = 5). Histological analysis of the Masson’s trichrome staining in bladders after BPNI ( g ) or SCI ( h ). Scale bar = 200 μm. Quantifications of detrusor thickness ( i , j ) and collagen content ( k , l ) by Masson’s trichrome staining were performed to assess the bladder fibrosis ( n = 5). Western blotting showing the protein levels of WNT11 and markers of fibroblastic differentiation (α-SMA and Col-1) and SMCs phenotypic transformation (SMTN and MYH10) in bladders after BPNI ( m ) or SCI ( n ). Western blotting analyses showing protein levels of TGF-β1 and markers of fibroblastic differentiation (α-SMA and Col-1) and SMCs phenotypic transformation (SMTN and MYH10) in BFs ( o ) and SMCs ( p ) treated with WNT11 at different doses. Western blotting analyses showing siWNT11 transfection inhibiting fibroblastic differentiation and SMCs phenotypic transformation in BFs ( q ) and SMCs ( r ) with TGF-β1 treatment, respectively. Data are shown as the mean ± SD, and P values were determined by the two-way ANOVA followed by Tukey’s post-hoc test ( e , f , i – l ), ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: To maximally activate or inhibit the WNT cascade in vivo, rats were treated with recombinant WNT11 protein (#6179-WN-010, R&D Systems) or LGK974 (#HY-17545, MedChemExpress) immediately after the surgical procedure.

Techniques: Staining, Western Blot, Transformation Assay, Transfection

Journal: Communications Biology

Article Title: Wnt11 mediates fibroblast–smooth muscle cell interaction to promote neurogenic bladder fibrosis in rats

doi: 10.1038/s42003-026-09647-2

Figure Lengend Snippet: Representative western blotting and quantitative analyses for FZD10 and Vangl2 in bladders after BPNI ( a ) or SCI ( b ) ( n = 6). Interaction between endogenous WNT11 and Vangl2 in BFs ( c ) and SMCs ( d ). e Confocal microscopy analysis of BFs and SMCs showing the colocalization of WNT11 (red) and Vangl2 (green). Scale bar = 20 μm. Quantification of colocalization was ascertained by ImageJ software in five randomly chosen fields. Numbers in the images correspond to the average Pearson’s correlation coefficient ± SD. Representative Western blotting and summarized data showing the relative protein levels of Vangl2 and markers of fibroblastic differentiation (α-SMA and Col-1) and SMCs phenotypic transformation (SMTN and MYH10) in BFs ( f ) and SMCs ( g ) ( n = 4). Data are shown as the mean ± SD. P values were determined by the two-tailed unpaired Student’s t -test ( a , b ) and the two-way ANOVA followed by Tukey’s post-hoc test ( f , g ), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: To maximally activate or inhibit the WNT cascade in vivo, rats were treated with recombinant WNT11 protein (#6179-WN-010, R&D Systems) or LGK974 (#HY-17545, MedChemExpress) immediately after the surgical procedure.

Techniques: Western Blot, Confocal Microscopy, Software, Transformation Assay, Two Tailed Test

Journal: Communications Biology

Article Title: Wnt11 mediates fibroblast–smooth muscle cell interaction to promote neurogenic bladder fibrosis in rats

doi: 10.1038/s42003-026-09647-2

Figure Lengend Snippet: Representative western blotting and quantitative analyses for the phosphorylation levels of JNK and c-JUN in bladders after BPNI ( a ) or SCI ( b ) ( n = 3). BFs ( c ) and SMCs ( d ) developed higher expression levels of p-JNK and p-c-JUN upon WNT11 treatment as indicated by the western blotting and quantitative analyses ( n = 4). Representative immunofluorescence staining and quantitative analyses showing that WNT11 treatment increased the fluorescence intensity and accumulation of p-c-JUN in the nucleus of BFs ( e ) and SMCs ( f ) ( n = 4). Scale bar=100μm. Protein levels of p-JNK, p-c-JUN, and markers of fibroblastic differentiation (α-SMA and Col-1) and SMCs phenotypic transformation (SMTN and MYH10) in BFs ( g ) and SMCs ( h ) treated with WNT11 with or without SP600125 by western blotting ( n = 3). Data are shown as the mean ± SD. P values were determined by the two-way ANOVA followed by Tukey’s post-hoc test ( a , b , g , h ) and the two-tailed unpaired student’s t -test ( c – f ), * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To maximally activate or inhibit the WNT cascade in vivo, rats were treated with recombinant WNT11 protein (#6179-WN-010, R&D Systems) or LGK974 (#HY-17545, MedChemExpress) immediately after the surgical procedure.

Techniques: Western Blot, Phospho-proteomics, Expressing, Immunofluorescence, Staining, Fluorescence, Transformation Assay, Two Tailed Test

Journal: Communications Biology

Article Title: Wnt11 mediates fibroblast–smooth muscle cell interaction to promote neurogenic bladder fibrosis in rats

doi: 10.1038/s42003-026-09647-2

Figure Lengend Snippet: Relative mRNA levels of DVL1, DVL2, and DVL3 in fibrotic bladders at 4 weeks after BPNI ( a ) and SCI ( b ) ( n = 6). Representative western blotting and quantitative analyses for the protein levels of DVL1, DVL2, and DVL3 in bladders after BPNI ( c ) or SCI ( d ) ( n = 6). e Representative western blotting and quantitative analyses revealing the protein levels of DVL2 and markers of fibroblastic differentiation, and the phosphorylation levels of JNK and c-JUN in BFs ( n = 3). f Protein levels of p-c-JUN, p-JNK, DVL2, SMTN, and MYH10 in SMCs treated with WNT11 and/or siDVL2 ( n = 3). Immunoprecipitation demonstrated that DVL2 bound to Vangl2 in BFs ( g ) and SMCs ( h ). Representative western blotting showing the presence of the target proteins (input) and immunoprecipitation showing the interaction of Flag-DVL2, Flag-DEP, Flag-DIX, and Flag-PDZ with endogenous Vangl2 in HEK293T cells using antibodies to Flag ( i ) and IgG ( j ). Data are shown as the mean ± SD. P values were determined by the two-tailed unpaired Student’s t -test ( a – d ) and the two-way ANOVA followed by Tukey’s post-hoc test ( e , f ), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: To maximally activate or inhibit the WNT cascade in vivo, rats were treated with recombinant WNT11 protein (#6179-WN-010, R&D Systems) or LGK974 (#HY-17545, MedChemExpress) immediately after the surgical procedure.

Techniques: Western Blot, Phospho-proteomics, Immunoprecipitation, Two Tailed Test

Journal: Communications Biology

Article Title: Wnt11 mediates fibroblast–smooth muscle cell interaction to promote neurogenic bladder fibrosis in rats

doi: 10.1038/s42003-026-09647-2

Figure Lengend Snippet: Under the condition of bladder denervation, the WNT11/Vangl2 signal was transmitted into the cytoplasm via DVL2, followed by activating PCP signaling to promote fibroblastic differentiation and SMC phenotypic transformation, which also depended on the interaction of TGF-β1/Smad2/3 signaling at the level of cytomembrane and nucleus, eventually resulting in NB fibrosis.

Article Snippet: To maximally activate or inhibit the WNT cascade in vivo, rats were treated with recombinant WNT11 protein (#6179-WN-010, R&D Systems) or LGK974 (#HY-17545, MedChemExpress) immediately after the surgical procedure.

Techniques: Transformation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Cecal growth factors promote enteric neurosphere formation and hindgut colonization in the avian model

doi: 10.3389/fcell.2025.1681844

Figure Lengend Snippet: Isolation, characterization and differentiation of chicken enteric neurospheres. Schematic figure of avian enteric neurosphere generation (A) . Brightfield images show examples of neurospheres generated from E8 ENSCs that emerged 1, 3, and 7 days in GDNF-containing medium (B–D) . Representative immunofluorescence images of 7-day-old neurospheres generated from E8 chicken intestine (E–G) . Samples were cultured under three different conditions: Exp.1: control medium ( (E) , CTRL), Exp. 2: GDNF containing cell culture media ( (F) , GDNF), and Exp.3: GWEN ( (G) , ceca-derived mesenchymal growth factors: GDNF, WNT11, ET-3, Noggin) supplemented media. Immunofluorescence of cross-sections confirms the presence of neurons expressing TUJ1, and neural crest cells expressing PHOX2B. Compared to CTRL and GDNF-only conditions, GWEN treatment resulted in a robust increase in TUJ1+/PHOX2B+ cell aggregates on the surface of the neurosphere and single Phox2b+ cells in the center of the aggregates. The inset shows a magnified view of a PHOX2B+/TUJ1+ aggregate, indicating enhanced differentiation of neural crest derivatives (E–G) . Scale bar = 100 µm. CTRL, control; NS, neurosphere.

Article Snippet: To assess the effect of ceca-specific mesenchymal factors on enteric neural stem cells, the culture medium was further supplemented with 10 ng/mL glial cell line-derived neurotrophic factor (GDNF; R&D Systems, 212-GD-010), 200 ng/mL WNT11 (Origene, TP761904), 250 ng/mL endothelin-3 (ET-3; R&D Systems, 1,162/100U), and 100 ng/mL Noggin (R&D Systems, 6997-NG-025).

Techniques: Isolation, Generated, Immunofluorescence, Cell Culture, Control, Derivative Assay, Expressing

Journal: Human Molecular Genetics

Article Title: WNT11, a new gene associated with early onset osteoporosis, is required for osteoblastogenesis

doi: 10.1093/hmg/ddab349

Figure Lengend Snippet: Molecular characteristics of the WNT11 variants

Article Snippet: Mutant cell lines were treated with recombinant human WNT11 protein (rhWnt11), Chinese hamster ovary derived (CHO derived) (R&D Systems, Minneapolis, MN) for 24 h. Additional experiments were performed in mutant cells cultured with 100 ng/ml recombinant human WNT5A protein (rhWnt5a), CHO derived, and with recombinant human WNT3A protein (rhWnt3a), CHO derived (R&D Systems, Minneapolis, MN) for 24 h. Furthermore, control U2OS cells and mutant WNT11 cells were treated with recombinant human RSPO2 protein (rhRspo2) (from mouse myeloma cell line), rhWnt3a, rhWnt5a and rhWnt11 (100 ng/ml) for 5 h for immunofluorescence analysis.

Techniques:

Journal: Human Molecular Genetics

Article Title: WNT11, a new gene associated with early onset osteoporosis, is required for osteoblastogenesis

doi: 10.1093/hmg/ddab349

Figure Lengend Snippet: WNT11 heterozygous mutant cells with decreased WNT11 mRNA and protein levels as well as proliferation and mineralization. ( A ) Electropherogram showing the 32-bp deletion leading to a frameshift. fs * : frameshift. ( B ) RT-qPCR analysis of WNT11 mRNA expression in mutant versus control U2OS cells. Normalization was to GAPDH level as a housekeeping gene with ratio of one for control U2OS cells. Ctrl: wild-type U2OS cells, Mut: WNT11 mutant cells, FC: fold change. ( C ) Western blot analysis of WNT11 protein expression, confirming the heterozygosity. ɑ-Tubulin was a control. ( D ) Alizarin red staining showed formation of mineralized nodules after osteogenic differentiation treatment with osteogenic media in control and WNT11 mutant cells. ( E ) Proliferation of control and WNT11 mutant cells ( n = 42). Data are mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Mutant cell lines were treated with recombinant human WNT11 protein (rhWnt11), Chinese hamster ovary derived (CHO derived) (R&D Systems, Minneapolis, MN) for 24 h. Additional experiments were performed in mutant cells cultured with 100 ng/ml recombinant human WNT5A protein (rhWnt5a), CHO derived, and with recombinant human WNT3A protein (rhWnt3a), CHO derived (R&D Systems, Minneapolis, MN) for 24 h. Furthermore, control U2OS cells and mutant WNT11 cells were treated with recombinant human RSPO2 protein (rhRspo2) (from mouse myeloma cell line), rhWnt3a, rhWnt5a and rhWnt11 (100 ng/ml) for 5 h for immunofluorescence analysis.

Techniques: Mutagenesis, Quantitative RT-PCR, Expressing, Control, Western Blot, Staining

Journal: Human Molecular Genetics

Article Title: WNT11, a new gene associated with early onset osteoporosis, is required for osteoblastogenesis

doi: 10.1093/hmg/ddab349

Figure Lengend Snippet: Expression of osteoblast differentiation markers and genes from the canonical and non-canonical Wnt pathways is decreased in WNT11 mutant cells. RT-qPCR analysis of mRNA levels of ( A ) osteoblast differentiation genes and ( B ) genes of the Wnt canonical and non-canonical pathways in control cells and in WNT11 mutant cells with and without Wnt11 recombinant protein treatment. Ctrl: control, Mut: WNT11 mutant cells, Mut + rhWnt11: WNT11 mutant cells treated with rhWnt11. Data are mean ± SEM. * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001.

Article Snippet: Mutant cell lines were treated with recombinant human WNT11 protein (rhWnt11), Chinese hamster ovary derived (CHO derived) (R&D Systems, Minneapolis, MN) for 24 h. Additional experiments were performed in mutant cells cultured with 100 ng/ml recombinant human WNT5A protein (rhWnt5a), CHO derived, and with recombinant human WNT3A protein (rhWnt3a), CHO derived (R&D Systems, Minneapolis, MN) for 24 h. Furthermore, control U2OS cells and mutant WNT11 cells were treated with recombinant human RSPO2 protein (rhRspo2) (from mouse myeloma cell line), rhWnt3a, rhWnt5a and rhWnt11 (100 ng/ml) for 5 h for immunofluorescence analysis.

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Control, Recombinant

Journal: Human Molecular Genetics

Article Title: WNT11, a new gene associated with early onset osteoporosis, is required for osteoblastogenesis

doi: 10.1093/hmg/ddab349

Figure Lengend Snippet: WNT11 mutation severely impedes β-catenin translocation and activation of the canonical Wnt pathway. ( A – J ) Immunofluorescence detection of β-catenin (green), in wild-type control (A–E) and WNT11 mutant (F–J) U2OS cells; blue is DAPI counterstaining for cell nucleus. The cells were untreated (A, F) or treated with rhWnt3a (B, G), rhWnt5a (C, H), rhWnt11 (D, I) or rhWnt3a and rhWnt11 (E, J). ( K ) Control and mutant cells were transfected with the TCF–LEF TOPflash vector and treated with rhWnt3a, rhWnt11 or rhWnt3a + rhWnt11 simultaneously. Data represent normalized luciferase activity relative to untreated cells. Data are mean ± SEM. * * P < 0.01.

Article Snippet: Mutant cell lines were treated with recombinant human WNT11 protein (rhWnt11), Chinese hamster ovary derived (CHO derived) (R&D Systems, Minneapolis, MN) for 24 h. Additional experiments were performed in mutant cells cultured with 100 ng/ml recombinant human WNT5A protein (rhWnt5a), CHO derived, and with recombinant human WNT3A protein (rhWnt3a), CHO derived (R&D Systems, Minneapolis, MN) for 24 h. Furthermore, control U2OS cells and mutant WNT11 cells were treated with recombinant human RSPO2 protein (rhRspo2) (from mouse myeloma cell line), rhWnt3a, rhWnt5a and rhWnt11 (100 ng/ml) for 5 h for immunofluorescence analysis.

Techniques: Mutagenesis, Translocation Assay, Activation Assay, Immunofluorescence, Control, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Human Molecular Genetics

Article Title: WNT11, a new gene associated with early onset osteoporosis, is required for osteoblastogenesis

doi: 10.1093/hmg/ddab349

Figure Lengend Snippet: Wnt non-canonical pathway controls RSPO complex, which controls Wnt canonical pathway. RT-qPCR analysis of mRNA level of ( A ) RSPO2 and ( B ) LGR5 with rhWnt3a, rhWnt5a and rhWnt11 treatment. Data are mean ± SEM. * P < 0.05, * * P < 0.01. ( C ) Immunofluorescence assay of cells treated with rhRspo2; blue (DAPI) and green (β-catenin). ( D ) Luciferase activity of control and WNT11 mutant cells with rhRspo2 treatment. Ctrl: control, Mut: WNT11 mutant cells. ( E ) Scheme summarizing the effect of WNT11 in normal cells (left) and heterozygous WNT11 mutation with red symbols (right): WNT11 is a ligand of the Wnt non-canonical pathway; thus, WNT11 mutation decreased the expression of genes in the non-canonical Wnt pathway as well as RSPO2 and LGR5 . LGR5 expression is rescued upon Wnt non-canonical but not canonical ligand treatment. Lack of WNT11 decreased the expression and activation of genes in the canonical Wnt pathway, which was reversed by treatment with rhRspo2.

Article Snippet: Mutant cell lines were treated with recombinant human WNT11 protein (rhWnt11), Chinese hamster ovary derived (CHO derived) (R&D Systems, Minneapolis, MN) for 24 h. Additional experiments were performed in mutant cells cultured with 100 ng/ml recombinant human WNT5A protein (rhWnt5a), CHO derived, and with recombinant human WNT3A protein (rhWnt3a), CHO derived (R&D Systems, Minneapolis, MN) for 24 h. Furthermore, control U2OS cells and mutant WNT11 cells were treated with recombinant human RSPO2 protein (rhRspo2) (from mouse myeloma cell line), rhWnt3a, rhWnt5a and rhWnt11 (100 ng/ml) for 5 h for immunofluorescence analysis.

Techniques: Quantitative RT-PCR, Immunofluorescence, Luciferase, Activity Assay, Control, Mutagenesis, Expressing, Activation Assay