Journal: Experimental & Molecular Medicine

Article Title: Differential effects of type 1 diabetes mellitus and subsequent osteoblastic β-catenin activation on trabecular and cortical bone in a mouse model

doi: 10.1038/s12276-018-0186-y

Figure Lengend Snippet: a WNT16 and b WNT5a protein expressions were detected by immunohistochemistry in longitudinal sections of femoral trabecular and cortical bone. The quantitative results are shown. n = 9. Scale bars, 50 μm. c mRNA expression levels of WNT16 and WNT5a in trabecular and cortical bone of control mice. n = 8. d mRNA expression levels of WNT16 and WNT5a in trabecular bone and cortical bone in T1DM and T1-CA mice. n = 8. e mRNA expression levels and protein concentrations of WNT16 and WNT5a in primary osteoblasts from Catnblox(ex3) mice treated with high glucose plus Cre adenovirus or vehicle. n = 8. Ob osteoblast. Data are expressed as the mean ± SD. ** P < 0.01 versus trabecular bone ( a – c ), T1DM group ( d ) or Catnblox(ex3) osteoblasts ( e ) by an unpaired t -test. NS not significant, P > 0.05

Article Snippet: The primary antibodies used in the study included a rabbit monoclonal anti-β-catenin antibody (1:400; #8814; Cell Signaling), a rabbit polyclonal anti-Dickkopf-1 antibody (1:100; sc-25516; Santa Cruz), a mouse monoclonal anti-WNT16 antibody (1:100; sc-271897; Santa Cruz), and a rabbit polyclonal anti-WNT5a antibody (1:100; ab72583; Abcam).

Techniques: Immunohistochemistry, Expressing, Control

Journal: Molecular Brain

Article Title: NMDA receptor activation stimulates transcription-independent rapid wnt5a protein synthesis via the MAPK signaling pathway

doi: 10.1186/1756-6606-5-1

Figure Lengend Snippet: NMDAR activation rapidly increases Wnt5a in cortical cultures . A. Cellular localization of Wnt5a in neurons. Shown are confocal images of primary cortical neurons after double-fluorescent immunostaining with anti-Wnt5a (red) and anti-synapsin I (green) antibodies. The nucleus was stained by DAPI (blue). B. MSG or NMDA stimulation increased Wnt5a protein. Primary cortical neurons (10 DIV) were treated with 10 μΜ MSG or 50 μΜ NMDA for 15 min. Wnt5a protein was detected by Western blotting. Data in the summary graphs (mean ± SEM) were from three independent experiments (*, p < 0.05; **, p < 0.01; One-way ANOVA). C. NMDA receptor-regulated Wnt5a increase. Primary cortical neurons (10 DIV) were pre-treated with vehicle (Control) or 100 μΜ DAP5 for 30 min, then incubated with 50 μΜ NMDA for 15 min. Wnt5a and p-P70S6K(included as a marker for translation activation) were detected by Western blotting summarized in the graph (n = 3; *, p < 0.05; **, p < 0.01; One-way ANOVA). D. Dynamic expression of Wnt5a protein after NMDA stimulation. Primary cortical neurons (10 DIV) were treated with 50 μΜ NMDA for 0, 5, 15, 30 and 60 min followed by Western blotting analysis of Wnt5a (n = 3; *, P < 0.05; **, p < 0.01; One-way ANOVA). E. NMDA-induced Wnt5a protein secretion. Primary cortical neurons (10 DIV) were treated with 50 μΜ NMDA for 0, 2, 4, 8, 16 and 32 min, and Wnt5a protein in the media was concentrated and detected on immunoblots.

Article Snippet: Anti-Wnt5a antibody was purchased from R&D Systems (AF654); anti-p-P70S6K (Thr389) antibody from Cell Signaling Technology (#9206); anti-GAPDH antibody from Santa Cruz (SC-32233); anti-Synapsin I from Millipore (AB1543P); and FITC-conjugated donkey anti-rabbit secondary antibody (711-097-020) and Rhodamine-conjugated rabbit anti-goat secondary antibody (305-297-003) from Jackson.

Techniques: Activation Assay, Immunostaining, Staining, Western Blot, Control, Incubation, Marker, Expressing

Journal: Molecular Brain

Article Title: NMDA receptor activation stimulates transcription-independent rapid wnt5a protein synthesis via the MAPK signaling pathway

doi: 10.1186/1756-6606-5-1

Figure Lengend Snippet: NMDAR-elicited Wnt5a increase requires translation but not transcription . A. Primary cortical neurons (10 DIV) were pre-treated with vehicle (Control) or 20 μΜ anisomycin for 30 min, and then incubated with 50 μΜ NMDA for 15 min, followed by Wnt5a immunoblotting. The Graph is a summary of three independent experiments (**, p < 0.01; One-way ANOVA). B. Primary cortical neurons (10 DIV) were pre-treated with vehicle (Control) or 20 μΜ actinomycin D for 30 min, followed by addition of 50 μΜ NMDA for 15 min. Wnt5a protein was detected by Western blotting and quantified. The Graph is a summary of four independent experiments (*, p < 0.05; **, p < 0.01; One-way ANOVA). C. Primary cortical neurons (10 DIV) were treated with vehicle (Control) or 50 μΜ NMDA for 15 min. Wnt5a mRNA was quantified by Real-time RT-PCR (qPCR). The summary graph is from three independent experiments (40 cycles, CT values: 25.1 ± 0.5/control vs. 25.6 ± 0.3/NMDA; p > 0.05; two-tailed Student's tests). D. Melt curve of Wnt5a qPCR on control cells. The melt curve on NMDA-stimulated cells was similar (not shown). E. RT-PCR results of Wnt5a in control and NMDA-treated cells.

Article Snippet: Anti-Wnt5a antibody was purchased from R&D Systems (AF654); anti-p-P70S6K (Thr389) antibody from Cell Signaling Technology (#9206); anti-GAPDH antibody from Santa Cruz (SC-32233); anti-Synapsin I from Millipore (AB1543P); and FITC-conjugated donkey anti-rabbit secondary antibody (711-097-020) and Rhodamine-conjugated rabbit anti-goat secondary antibody (305-297-003) from Jackson.

Techniques: Control, Incubation, Western Blot, Quantitative RT-PCR, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Brain

Article Title: NMDA receptor activation stimulates transcription-independent rapid wnt5a protein synthesis via the MAPK signaling pathway

doi: 10.1186/1756-6606-5-1

Figure Lengend Snippet: mTOR signaling pathway is not required for the NMDAR-dependent Wnt5a protein synthesis . Primary cortical neurons (10 DIV) were pre-treated with vehicle (Control) or 25nΜ Rapamycin for 30 min, followed by addition of 50 μΜ NMDA for 15 min. Western blotting analysis of Wnt5a and phosphor-P70S6K proteins were performed. Graphs (mean ± SEM) are from four independent experiments (*, p < 0.05; **, p < 0.01; #, p > 0.05; One-way ANOVA).

Article Snippet: Anti-Wnt5a antibody was purchased from R&D Systems (AF654); anti-p-P70S6K (Thr389) antibody from Cell Signaling Technology (#9206); anti-GAPDH antibody from Santa Cruz (SC-32233); anti-Synapsin I from Millipore (AB1543P); and FITC-conjugated donkey anti-rabbit secondary antibody (711-097-020) and Rhodamine-conjugated rabbit anti-goat secondary antibody (305-297-003) from Jackson.

Techniques: Control, Western Blot

Journal: Molecular Brain

Article Title: NMDA receptor activation stimulates transcription-independent rapid wnt5a protein synthesis via the MAPK signaling pathway

doi: 10.1186/1756-6606-5-1

Figure Lengend Snippet: NMDAR activation stimulates Wnt5a protein synthesis via the MAPK signaling pathway . A. Primary cortical neurons (10 DIV) were pre-treated with vehicle (Control) or 20 μM PD98059 (PD98) for 30 min, and then stimulated with 50 μΜ NMDA for 15 min. Wnt5a protein was measured by Western blotting and quantified data were presented in graphs (mean ± SEM; n = 3, *p < 0.05; One-way ANOVA). B. Primary cortical neurons (10 DIV) were pre-treated with vehicle (Control) or 20 μM U0126 for 30 min, followed by 50 μΜ NMDA for 15 min. Graphs (mean ± SEM) are from three independent experiments (*, p < 0.05; **, p < 0.01; One-way ANOVA).

Article Snippet: Anti-Wnt5a antibody was purchased from R&D Systems (AF654); anti-p-P70S6K (Thr389) antibody from Cell Signaling Technology (#9206); anti-GAPDH antibody from Santa Cruz (SC-32233); anti-Synapsin I from Millipore (AB1543P); and FITC-conjugated donkey anti-rabbit secondary antibody (711-097-020) and Rhodamine-conjugated rabbit anti-goat secondary antibody (305-297-003) from Jackson.

Techniques: Activation Assay, Control, Western Blot

Journal: Molecular Brain

Article Title: NMDA receptor activation stimulates transcription-independent rapid wnt5a protein synthesis via the MAPK signaling pathway

doi: 10.1186/1756-6606-5-1

Figure Lengend Snippet: A working model for NMDAR-regulated Wnt5a protein synthesis . Stimulation of NMDAR activates MAPK signaling to elicit de novo translation from pre-existing Wnt5a mRNAs.

Article Snippet: Anti-Wnt5a antibody was purchased from R&D Systems (AF654); anti-p-P70S6K (Thr389) antibody from Cell Signaling Technology (#9206); anti-GAPDH antibody from Santa Cruz (SC-32233); anti-Synapsin I from Millipore (AB1543P); and FITC-conjugated donkey anti-rabbit secondary antibody (711-097-020) and Rhodamine-conjugated rabbit anti-goat secondary antibody (305-297-003) from Jackson.

Techniques:

Journal: Oncotarget

Article Title: Wnt5a / planar cell polarity signaling pathway in urothelial carcinoma, a potential prognostic biomarker

doi: 10.18632/oncotarget.15877

Figure Lengend Snippet: (A) Immunohistochemical analysis of human specimens of urothelial carcinoma of the bladder for the expression of Wnt5a, ROR2, CTHRC1 and E-cadherin. Left column, tissue sections from a representative case of low grade urothelial carcinoma; the right column, tissue sections from a representative case of high grade urothelial carcinoma. The middle column represents the trend of expression for each protein in all 15 samples. The expression of Wnt5a, ROR2, and CTHRC1 increases in high grade tumors while E-cadherin shows an opposite trend. Bar=50 μm. (B) Statistical analysis was performed to investigate the correlation between tumor histological grade and immunostaining for Wnt5a, Ror2, CTHRC1, and E-cadherin. Statistical significance was tested at an alpha of 0.05. The software PASW Statistics 18 was used for data analysis (Pearson Education, New York City, NY).

Article Snippet: To one of each of the two sections the following antihuman primary antibodies were applied: mouse monoclonal antibody against Wnt5a (Abcam, Cambridge, MA) applied at 8.33 μg/ml; rabbit polyclonal antibody against Ror2 (Abnova, Taipei City, Taiwan) applied at 1/100 dilution, and mouse monoclonal antibody against CTHRC1 (Abnova, Taipei City, Taiwan) applied at 4.0 μg/ml.

Techniques: Immunohistochemical staining, Expressing, Immunostaining, Software

Journal: Oncotarget

Article Title: Wnt5a / planar cell polarity signaling pathway in urothelial carcinoma, a potential prognostic biomarker

doi: 10.18632/oncotarget.15877

Figure Lengend Snippet: Wnt5a (A) , Ror2 and CTHRC1 (B) , E-cadherin and vimentin (C) , in RT4, J82 and T24 urothelial carcinoma cell lines.

Article Snippet: To one of each of the two sections the following antihuman primary antibodies were applied: mouse monoclonal antibody against Wnt5a (Abcam, Cambridge, MA) applied at 8.33 μg/ml; rabbit polyclonal antibody against Ror2 (Abnova, Taipei City, Taiwan) applied at 1/100 dilution, and mouse monoclonal antibody against CTHRC1 (Abnova, Taipei City, Taiwan) applied at 4.0 μg/ml.

Techniques:

Journal: Oncotarget

Article Title: Wnt5a / planar cell polarity signaling pathway in urothelial carcinoma, a potential prognostic biomarker

doi: 10.18632/oncotarget.15877

Figure Lengend Snippet: Top row: H&E stain shows morphological differences between each cell line. Middle row: confocal microscopy images show the expression of Wnt5a (green) for each cell line. Bottom row: confocal microscopy image of the merge expression of Ror2 (green) and CTHRC1 (red) for each cell line. Although the co-expression and colocalization of Ror2 and CTHRC1 is present in all cell lines, it is clearest for RT4 and J82 cell lines.

Article Snippet: To one of each of the two sections the following antihuman primary antibodies were applied: mouse monoclonal antibody against Wnt5a (Abcam, Cambridge, MA) applied at 8.33 μg/ml; rabbit polyclonal antibody against Ror2 (Abnova, Taipei City, Taiwan) applied at 1/100 dilution, and mouse monoclonal antibody against CTHRC1 (Abnova, Taipei City, Taiwan) applied at 4.0 μg/ml.

Techniques: Staining, Confocal Microscopy, Expressing

Journal: Oncotarget

Article Title: Wnt5a / planar cell polarity signaling pathway in urothelial carcinoma, a potential prognostic biomarker

doi: 10.18632/oncotarget.15877

Figure Lengend Snippet: Primer sequences used for real-time RT-PCR analyses

Article Snippet: To one of each of the two sections the following antihuman primary antibodies were applied: mouse monoclonal antibody against Wnt5a (Abcam, Cambridge, MA) applied at 8.33 μg/ml; rabbit polyclonal antibody against Ror2 (Abnova, Taipei City, Taiwan) applied at 1/100 dilution, and mouse monoclonal antibody against CTHRC1 (Abnova, Taipei City, Taiwan) applied at 4.0 μg/ml.

Techniques: Quantitative RT-PCR, Ubiquitin Proteomics

Journal: PLOS Biology

Article Title: Hyperglycemia increases SCO-spondin and Wnt5a secretion into the cerebrospinal fluid to regulate ependymal cell beating and glucose sensing

doi: 10.1371/journal.pbio.3002308

Figure Lengend Snippet: ( A ) Immunohistochemical staining of Wnt5a (red) and SCO-Spondin (green) in frontal brain sections from normoglycemic animals. Focal Wnt5a immunoreactivity was detected most in the basal area of the cells. N = 3. Ependymal cells were negative for Wnt5a and SCO-spondin. Scale bar: 20 μm. ( B ) Immunohistochemical staining of Wnt5a (red) and SCO-spondin (green) in frontal brain sections from hyperglycemic animals (CSF glucose concentration of 5 mM glucose). Wnt5a immunoreactivity was mainly observed in the apical part of the cells (yellow arrows). N = 3. Immunoreactivity was also observed by DIC microscopy (Bˋ). Ependymal cell cilia were positive for Wnt5a and very weakly positive for SCO-spondin (B`). Scale bar: 20 μm. ( C ) Immunohistochemical staining of Wnt5a (red) and SCO-spondin (green) in frontal brain sections from hyperglycemic animals (CSF glucose concentration of 10 mM). Wnt5a immunoreactivity was observed mainly in the apical area of the cells. N = 3. Ependymal cell cilia were strongly positive for Wnt5a (Cˋ). Immunoreactivity was also observed by DIC microscopy. Ependymal cell cilia were strongly positive for Wnt5a and SCO-spondin (Cˋˋ). Scale bar: 20 μm. ( D ) Immunofluorescence staining of SCO-spondin and Wnt5a in ependymal cells cilia. Increased colocalization was observed (white arrows). Scale bar: 20 μm. ( E ) No immunoreactivity was detected when the primary antibody was omitted. Scale bar: 20 μm. ( F ) Quantitative analysis of Wnt5a immunoreactivity under different glycemic conditions. The graph shows data from 4 biologically independent samples. The error bars represent the SD; *** P < 0.001 (two-tailed Student t test). ( G ) Quantification of Mander’s overlap coefficient for SCO-spondin and Wnt5a in SCO cells and ependymal cell cilia under different glycemic conditions. The graph shows data from 4 biologically independent samples (Normo in ependymal cells, N = 3). The error bars represent the SD; *** P < 0.001, n.s. = not significant (two-tailed Student t test). ( H ) TEM analysis. Ependymal cells with aggregated secretions in the apex of cilia. Exosome-like vesicles were also detected (arrowheads and inset). Scale bars: H, 0.3 μm; I, inset, 0.08 μm. Data used to generate graphs can be found in . CSF, cerebrospinal fluid; DIC, differential interference contrast; d3v, dorsal third ventricle; SCO, subcommissural organ; TEM, transmission electron microscopy.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-bovine RF compounds (SCO-spondin) (1:1,000 dilution; produced in-house at Málaga University), chicken anti-human vimentin (AB5733, 1:400 dilution, Millipore, Billerica, MA, USA), rabbit anti-human GFAP (MAB360, 1:500 dilution, Millipore), mouse anti-βIII tubulin (G712A; 1:1,000 dilution; Promega, Madison, Wisconsin, USA), rat anti-mouse frizzled-2 (sc-74019, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human Wnt5a (sc-365370, 1:100 dilution; Santa Cruz Biotechnology), rat anti-mouse Wnt5a (MAB645, 1:200, R&D System, Minneapolis, MN, USA), rabbit anti-human GLUT1 (C110491, 1:100 dilution, EMD Millipore, Burlington, MA, USA), rabbit anti-rat GLUT2 (GT21-A, 1:100 dilution; Alpha Diagnostic, San Antonio, TX, USA), rabbit anti-human GLUT6 (GT62-A, 1:100 dilution; Alpha Diagnostic), mouse anti-human β-catenin (sc-7963, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human ROR2 (sc-374174, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human CD63 (sc-5275, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human aggrecan (4F4) (sc-33695, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human Syndecan-2 (sc-365624, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human HS6ST1 (sc-398231, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human testican 2 (sc-515691, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human glypican-1 (sc-365000, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human MCT2 (sc-166925, 1:200 dilution; Santa Cruz Biotechnology), rabbit anti-Cx43 (C6219, 1:300 dilution: Millipore, Billerica, MA, USA), mouse anti-KDEL (sc-58774, 1:200 dilution; Santa Cruz Biotechnology).

Techniques: Immunohistochemical staining, Staining, Concentration Assay, Microscopy, Immunofluorescence, Two Tailed Test, Transmission Assay, Electron Microscopy

Journal: PLOS Biology

Article Title: Hyperglycemia increases SCO-spondin and Wnt5a secretion into the cerebrospinal fluid to regulate ependymal cell beating and glucose sensing

doi: 10.1371/journal.pbio.3002308

Figure Lengend Snippet: ( A and B ) Immunohistochemical staining of SCO-spondin (red) and acetylated α-tubulin (green) in ependymal cells from the adult human brain. Scale bar: 15 μm. ( C ) Immunohistochemical staining of Frizzled-2 and GLUT1 in human ependymal cells. Scale bar: 20 μm. ( D ) Immunohistochemical staining of acetylated α-tubulin/Cx43 and vimentin/Wnt5a in human ependymal cells. Scale bar: 20 μm. ( E ) Immunohistochemical staining of vimentin/CD63 and vimentin/ROR2 in human ependymal cells. Scale bar: 20 μm. ( F ) Dot-blot analysis with anti-SCO-spondin. The samples were SCO-spondin and R-spondin-4. ( G ) Overview of hyperglycemic conditions in the third ventricle, SCO cell activation, and the effect of SCO-spondin and Wnt5a on ependymal cells. Similar to β-pancreatic cells, SCO cells express GLUT2, a low-affinity glucose transporter. Under hyperglycemic conditions, the increase in the intracellular glucose concentration is expected to raise the ATP content, stimulating the secretion of SCO-spondin into the CSF (number 1). In ependymal cells, the increase in the glucose concentration changes ciliary beating (number 2). Additionally, it may reduce Cx43-mediated functional coupling. SCO-spondin (soluble in CSF) preferentially binds to the apex of dorsal ependymal cells, further preventing normal ciliary beating. CSF flow shows a transient decline, promoting glucose sensing in the basal hypothalamus (bottom pink area). When the glucose level in the CSF is high, SCO cells also release Wnt5a, possibly via CD63-positive MVB-like EVs (number 3). Wnt5a bound to ROR2 (very likely anchored to testican or glypican) can be internalized (number 4) [ , ] and probably activating the noncanonical β-catenin signaling pathway (number 4) . CX43 is uncoupled under hyperglycemic conditions, and this alteration may be reversed when the intracellular calcium increases . The original blot for this figure (F) can be found in Images. BV, blood vessel; CSF, cerebrospinal fluid; Cx43, connexin-43; EV, extracellular vesicle; MVB, multivesicular body; ROR2, Frizzled 2/receptor tyrosine kinase-like orphan receptor-2; SCO, subcommissural organ.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-bovine RF compounds (SCO-spondin) (1:1,000 dilution; produced in-house at Málaga University), chicken anti-human vimentin (AB5733, 1:400 dilution, Millipore, Billerica, MA, USA), rabbit anti-human GFAP (MAB360, 1:500 dilution, Millipore), mouse anti-βIII tubulin (G712A; 1:1,000 dilution; Promega, Madison, Wisconsin, USA), rat anti-mouse frizzled-2 (sc-74019, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human Wnt5a (sc-365370, 1:100 dilution; Santa Cruz Biotechnology), rat anti-mouse Wnt5a (MAB645, 1:200, R&D System, Minneapolis, MN, USA), rabbit anti-human GLUT1 (C110491, 1:100 dilution, EMD Millipore, Burlington, MA, USA), rabbit anti-rat GLUT2 (GT21-A, 1:100 dilution; Alpha Diagnostic, San Antonio, TX, USA), rabbit anti-human GLUT6 (GT62-A, 1:100 dilution; Alpha Diagnostic), mouse anti-human β-catenin (sc-7963, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human ROR2 (sc-374174, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human CD63 (sc-5275, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human aggrecan (4F4) (sc-33695, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human Syndecan-2 (sc-365624, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human HS6ST1 (sc-398231, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human testican 2 (sc-515691, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human glypican-1 (sc-365000, 1:200 dilution; Santa Cruz Biotechnology), mouse anti-human MCT2 (sc-166925, 1:200 dilution; Santa Cruz Biotechnology), rabbit anti-Cx43 (C6219, 1:300 dilution: Millipore, Billerica, MA, USA), mouse anti-KDEL (sc-58774, 1:200 dilution; Santa Cruz Biotechnology).

Techniques: Immunohistochemical staining, Staining, Dot Blot, Activation Assay, Concentration Assay, Functional Assay

Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A, E) Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.1 µM, 0.5 µM, 1.0 µM) for 24 hours. Expression of WNT-5A mRNA(A) and collagen IαI and fibronectin mRNA (E) was determined by qRT-PCR, corrected for 18S rRNA and expressed relative to vehicle basal. Data represent mean ± SEM of 4-5 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, # p<0.05, ## p<0.01, ### p<0.001 compared to TGF-β-stimulated cells; 2-way ANOVA followed by Bonferroni multiple comparisons test. (B) Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 48 hours. Western analysis was performed on whole cells extracts for WNT-5A protein. Expression of GAPDH was analyzed as loading control. (C–D, F) Airway smooth muscle cells were transfected with TAK1-specific siRNA or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours and analyzed for the expression of TAK1 mRNA (C), WNT-5A mRNA (D) and collagen IαI and fibronectin mRNA (F) by qRT-PCR and expressed relative to non-targeting siRNA-transfected, untreated control. Data represent mean ± SEM of 4 independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to non-targeting siRNA-transfected untreated control, #p<0.05, ## p<0.01 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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

Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A) TAK1 activates p38 and JNK. Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 30 and 60 minutes. Whole cells extracts were immunoblotted for phospho-p38 and phospho-JNK using specific antibodies. Equal protein loading was verified by the analysis of β-actin. (B–D) p38 and JNK involvement in WNT-5A expression. Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of SB203580 (10 µM) or SP600125 (10 µM) or combination of both SB203580 and SP600125 (10 µM each) for 24 hours. RNA was isolated and WNT-5A mRNA expression was determined by qRT-PCR, corrected for 18S rRNA and expressed relative to vehicle basal. Data represent mean ± SEM of 4–6 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, ### p<0.001 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A) De novo protein synthesis is required for TGF-β-induced WNT-5A expression. Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of the protein synthesis inhibitor cycloheximide (5 µg/ml) for 24 hours. WNT-5A mRNA induction was evaluated by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, ## p<0.01 compared to TGF-β-stimulated cells; 2-tailed Student's t test for paired observations. (B-D) β-Catenin silencing reduces TGF-β-induced WNT-5A expression. Airway smooth muscle cells were transfected with β-catenin-specific siRNA or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours (mRNA; B,C) or 48 hours (protein; D). (B,C) Expression of β-catenin mRNA (B) and WNT-5A mRNA (C) was determined by qRT-PCR and expressed relative to non-targeting siRNA transfected, untreated control. Data represent mean ± SEM of 5 independent experiments. *p<0.05, **p<0.01 compared to non-targeting siRNA-transfected, untreated control, # p<0.05, ### p<0.001 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 2-tailed Student's t test for paired observations. (D) Western blot analysis was performed to analyze WNT-5A and β-catenin protein expression in whole cell extracts. Equal protein loading was verified by the analysis of GAPDH. (E) Forced increase in β-catenin abundance elevates WNT-5A protein level. Cells were transfected with S33Y-β-catenin mutant or a GFP expression vector as control. Subsequently, cells were either left untreated or stimulated with TGF-β (2 ng/ml) for 48 hours. Western blot analysis was performed to determine the abundance of WNT-5A and total β-catenin at protein level. GAPDH expression assessed as loading control. (F) Canonical WNT ligand stimulation increases WNT-5A gene expression. Cells were stimulated with L-cells-derived WNT-3A conditioned medium or control conditioned medium for 24 hours. Expression of WNT-5A mRNA was evaluated by qRT-PCR and expressed relative to control conditioned medium. Data represent mean ± SEM of 5 independent experiments. **p<0.01 compared to control conditioned medium; 2-tailed Student's t test for paired observations.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot, Mutagenesis, Plasmid Preparation, Gene Expression, Derivative Assay

Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A) In silico analysis of WNT-5A promoter. Schematic representation of WNT-5A promoter A indicating the transcription factor binding sites as predicted by PROMO version 3. Only selective transcription factors are depicted here. The schematic is not to scale. TSS: Transcriptional Start Site. (B–G) Silencing of various transcription factors and WNT-5A gene expression. Airway smooth muscle cells were transfected with a non-targeting siRNA as control or with CUTL1-specific (B, C), TCF4-specific (D, E) or ETS1-specific (F, G) siRNA. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours and analyzed for the expression of genes as indicated in panels by qRT-PCR, corrected for 18S rRNA and expressed relative to non-targeting siRNA transfected, untreated control. Data represent mean ± SEM of 3-5 independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to non-targeting transfected, untreated control; 1-way ANOVA followed by Newman-Keuls multiple comparisons test.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Silico, Binding Assay, Gene Expression, Transfection, Control, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A-B) Mithramycin A attenuates WNT-5A mRNA and protein expression. (A) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. WNT-5A mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. **p<0.01 compared to vehicle basal, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (B) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 48 hours. Whole cell extracts were prepared and WNT-5A protein abundance was evaluated by western analysis. GAPDH was assessed as loading control. (C, D) Cells were transfected with Sp1-specific or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours and analyzed for the expression of Sp1 mRNA (C) and WNT-5A mRNA (D) by qRT-PCR. Data represent mean ± SEM of 5 independent experiments. *p<0.05, ***p<0.001 compared to non-targeting siRNA-transfected untreated control, #p<0.05, ### p<0.001 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (E) Mithramycin A attenuates TGF-β-induced extracellular matrix expression. Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. Collagen IαI and fibronectin mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. *p<0.05, **p<0.01 compared to vehicle basal, #p<0.05, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (F) Sp1 is recruited to WNT-5A promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) for 16 hours. Chromatin was prepared and ChIP analysis was performed as described in the Materials and Methods section. PCR was carried out using primers specific for Sp1 binding region on WNT-5A promoter A after immunoprecipitation with anti-Sp1 or control IgG antibody. Input DNA from chromatin preparation before immunoprecipitation was amplified to ascertain the loading. Resulting PCR products were analyzed by DNA PAGE. (G) TAK1 mediates recruitment of Sp1 to WNT-5A promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 16 hours. ChIP analysis was performed as described above.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Western Blot, Control, Transfection, Binding Assay, Immunoprecipitation, Amplification