Journal: Stem cells (Dayton, Ohio)

Article Title: Activation of Wnt signaling in hematopoietic regeneration.

doi: 10.1634/stemcells.2007-0768

Figure Lengend Snippet: Figure 2. Wnt10b is temporally regulated in the bone marrow microenvironment after injury. (A): Bone sections of control or Cy/G-injured mice were stained for the expression of Wnt3a, Wnt5a, or Wnt10b (red) and 4,6-diamidino-2-phenylindole (blue) (n 2 for Wnt3a and Wnt5a and n 5 for Wnt10b). (B): Kinetics of Wnt10b expression levels were monitored for 1 week following injury by analyzing bone sections at days 3 (n 3), 5 (n 2), and 7 (n 3) after Cy/G delivery. All images were acquired with a 40 objective.

Article Snippet: Primary antibodies were as follows: Wnt3a-specific antisera from immunized rabbits [28] (generous gift of Roel Nusse), anti-Wnt5a (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http:// www.scbt.com), anti-Wnt10b (Santa Cruz), and/or biotin-conjugated mouse panendothelial cell antigen (MECA)-32 (BD Pharmingen) or the appropriate isotype controls; normal rabbit serum (Jackson Immunoresearch Laboratories), normal goat IgG (Santa Cruz), or biotin-conjugated rat IgG2a,k (BD Pharmingen).

Techniques: Control, Staining, Expressing

Journal: Food Science and Human Wellness

Article Title: Nystose attenuates bone loss and promotes BMSCs differentiation to osteoblasts through BMP and Wnt/β-catenin pathway in ovariectomized mice

doi: 10.1016/j.fshw.2022.07.066

Figure Lengend Snippet: Fig. 8 NST activated the Wnt/β-catenin signaling pathway in BMSCs. (A1) Representative imagines of Western-blot for the expression of key proteins of Wnt/ β-catenin pathway in BMSCs and (A2), (A3), (A4) and (A5) quantitative analysis of P-β-catenin/β-catenin, LRP-5, P-GSK-3β/GSK-3β and Wnt3a, respectively. DKK-1, an inhibitor of Wnt/β-catenin pathway reversed the effects of NST on osteoblast differentiation of BMSCs as evidenced by (B1) proliferation, (B2) ALP activity and (B3) ALP staining. DKK-1 also reversed the regulatory effects of NST on Wnt/β-catenin pathway in BMSCs as showed by (C1) expression of key proteins in representative images of Western-blot and (C2), (C3) and (C4) quantitative analysis of LRP-5, P-β-catenin/β-catenin and P-GSK-3β/GSK-3β, respectively. Data are presented as the mean ± SD. #P < 0.05, ##P < 0.01, compared with NST group; *P < 0.05, **P < 0.01, compared with Ctrl group.

Article Snippet: Antibodies against Wnt3a were purchased from Boster Biological Technology (Wuhan, China).

Techniques: Western Blot, Expressing, Activity Assay, Staining

Journal: Turkish Journal of Medical Sciences

Article Title: Wnt signaling pathway activities may be altered in primary Sjogren’s syndrome

doi: 10.3906/sag-2102-367

Figure Lengend Snippet: Wnt1 and Wnt3a expressions in the salivary glands of patients with primary SS.

Article Snippet: The following antibodies were used for immunohistochemistry: Wnt1 (Mouse Wnt1 primary antibody (10C8), NBP1-51575, Novus biologicals, USA) and Wnt3a (Mouse Wnt3a primary antibody, NBP1-74183, Novus biologicals, USA).

Techniques:

Journal: Turkish Journal of Medical Sciences

Article Title: Wnt signaling pathway activities may be altered in primary Sjogren’s syndrome

doi: 10.3906/sag-2102-367

Figure Lengend Snippet: Differences between Wnt1- and Wnt3a-positive and Wnt1- and Wnt3a-negative patients with primary SS.

Article Snippet: The following antibodies were used for immunohistochemistry: Wnt1 (Mouse Wnt1 primary antibody (10C8), NBP1-51575, Novus biologicals, USA) and Wnt3a (Mouse Wnt3a primary antibody, NBP1-74183, Novus biologicals, USA).

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: Significance of the Wnt signaling pathway in coronary artery atherosclerosis

doi: 10.3389/fcvm.2024.1360380

Figure Lengend Snippet: Coronary plaques have greater mRNA and protein expression of Wnt signaling proteins compared with healthy tissues. ( A ) An increased mRNA expression of Wnt3a and Wnt5b in diseased coronary arteries. The data are represented as mean ± SEM. * P < 0.05 compared with healthy tissues; n = 11 for coronary plaques and n = 7 for healthy tissues. Student’s t -test was used for statistical analysis. ( B ) A representative Western blot of Wnt proteins in diseased and healthy coronary arteries. ( C ) A densitometry analysis of the Western blot of Wnt protein expression in diseased and healthy coronary arteries. The data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with normal tissues; n = 9 for coronary plaques and n = 5 for normal tissues. Student’s t -test was used for statistical analysis. ( D ) An increased mRNA expression of FZD2, FZD9, and FZD10 in diseased coronary arteries. The data are represented as mean ± SEM. * P < 0.05 compared with healthy tissues; n = 11 for coronary plaques and n = 7 for healthy tissues. Student’s t -test was used for statistical analysis. CAD, coronary artery disease.

Article Snippet: The following primary antibodies and concentrations were used: mouse antihuman Wnt3a (Novus, at 1/100), mouse antihuman Wnt5a (Sigma Aldrich, at 1/100), rabbit antihuman Wnt5b (Abcam ab115563, 1/500), α -smooth muscle actin ( α SMA) (Cedarlane, at 1/500), and cluster of differentiation 68 (CD68) (Sigma Aldrich, at 1:2,000).

Techniques: Expressing, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Significance of the Wnt signaling pathway in coronary artery atherosclerosis

doi: 10.3389/fcvm.2024.1360380

Figure Lengend Snippet: Coronary plaques have greater immunostaining of Wnt proteins compared with histologically normal tissues. ( A ) A negative stain control of the coronary artery. Very little Wnt3a ( B ), Wnt5a ( C ), and Wnt5b ( D ) immunostaining is seen in histologically normal coronary arteries. A strong Wnt3a staining is seen in the shoulder of the lesion around the lipid core and in the fibrous cap ( E ), and colocalized with αSMA ( H ) Wnt3a is colocalized with CD68 immunostaining around the lesion ( I , L , respectively). Some Wnt5a immunostaining around the lipid core and in the fibrous cap ( F ) colocalized with αSMA ( H ) no significant colocalization of Wnt5a and CD68 immunostaining ( J , L , respectively). A strong Wnt5b staining is seen in and around the lesion and in the fibrous cap ( G ) and colocalized with αSMA ( H ) colocalization of Wnt5b and CD68 immunostaining around the lesion ( K , L , respectively). Scale bar = 200 μm. *Lipid core, ℓ = lumen. The arrows refer to significant immunostaining in and around the lesion.

Article Snippet: The following primary antibodies and concentrations were used: mouse antihuman Wnt3a (Novus, at 1/100), mouse antihuman Wnt5a (Sigma Aldrich, at 1/100), rabbit antihuman Wnt5b (Abcam ab115563, 1/500), α -smooth muscle actin ( α SMA) (Cedarlane, at 1/500), and cluster of differentiation 68 (CD68) (Sigma Aldrich, at 1:2,000).

Techniques: Immunostaining, Staining, Control

Journal: Journal of Neurotrauma

Article Title: Intranasally Delivered Wnt3a Improves Functional Recovery after Traumatic Brain Injury by Modulating Autophagic, Apoptotic, and Regenerative Pathways in the Mouse Brain

doi: 10.1089/neu.2016.4871

Figure Lengend Snippet: Activation of the Wnt/β-catenin pathway reduces autophagic activity following traumataicbrain injury (TBI) in mice. TBI was induced in a mouse model, followed by intranasal Wnt3a (2 μg/kg) delivery 60 min after the TBI insult. (A and B) Representative immunohistochemical images of LC3 (red) and NeuN (blue) fluorescence and co-localization (arrowheads) following TBI at 24 h and 48 h post-injury in the peri-contusion region. (C) Sham injury did not result in noticeable accumulation of LC3. (D) High magnification (100 × ) images of LC3 puncta (arrowheads) in brain sections of the TBI+Saline group at 24 h post-injury. (E) Quantified numbers of LC3+/NeuN+ cells at 24 h post-injury, and at 48 h post-injury. Only cells containing LC3 signal in the soma was considered to be LC3+. *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001. A total of 600–700 images were quantified. n = 3 for Sham group, n = 5 for both TBI+Saline and TBI+Wnt3a groups for both time-points (24 h and 48 h post-TBI). (F–J) Western blotting was performed on peri-contusion tissue at 24 h post-TBI to evaluate the canonical Wnt signaling and autophagic activity. (F) Representative immunoblots of markers for Wnt signaling and autophagy. All immunoblots were normalized to the β-actin control, and then to sham within group. (G) Quantification of normalized immunoblot intensity of Wnt3a. (H) Quantification of normalized immunoblot intensity of β-catenin. (I) Quantification of normalized immunoblot intensity of Beclin-1. (J) Quantification of normalized immunoblot intensity of LC3-II. *p < 0.05, compared with Sham. #p < 0.05, compared with TBI+Saline. n = 3 for Sham group, n = 5 for both TBI+Saline and TBI+Wnt3a groups. Color image is available online at www.liebertpub.com/neu

Article Snippet: The primary antibodies used and the dilutions for each were rat anti-Wnt3a antibody (R&D Systems) at 1:400, mouse anti-β-catenin antibody (R&D Systems) at 1:1000, rabbit anti-cleaved caspase 3 antibody (Cell Signaling) at 1:400, rabbit anti-Bcl-2 (Cell Signaling) at 1:1000, rabbit anti-matrix metalloproteinases 9 (MMP-9) antibody (Millipore) at 1:2500, rabbit anti-MMP-2 antibody (Millipore) at 1:2500, rabbit anti-glial-derived growth factor (GDNF) antibody (Santa Cruz Biotechnology) at 1:1000, and anti-vascular endothelial growth factor (VEGF) antibody (Sigma) at 1:5000.

Techniques: Activation Assay, Activity Assay, Immunohistochemical staining, Fluorescence, Saline, Western Blot, Control

Journal: Journal of Neurotrauma

Article Title: Intranasally Delivered Wnt3a Improves Functional Recovery after Traumatic Brain Injury by Modulating Autophagic, Apoptotic, and Regenerative Pathways in the Mouse Brain

doi: 10.1089/neu.2016.4871

Figure Lengend Snippet: Neuroprotective effects of Wnt3a against autophagic cell death and brain damage following mild traumatic brain injury (TBI) (A–C) The presence of autophagic cell death was visualized by immunohistochemistry at 2 days post-TBI. (A) Representative images of immunofluorescence (red: Beclin-1, green: TUNEL, blue: DAPI). (B) Quantification of Beclin-1+ cells per 40 × field. (C) Quantification of Beclin-1+/TUNEL+ co-labeled cells per 40 × field. *p < 0.05, compared with Sham. #p < 0.05, compared with TBI+Saline. A total of 600–700 images were quantified. n = 4 for Sham group, n = 7 for both TBI+Saline and TBI+Wnt3a groups. (D) Representative immunofluorescence images are shown (green: TUNEL, red: NeuN, blue: DAPI) revealed significantly greater levels of TUNEL+ cells, as well as TUNEL+/NeuN+ co-labeled cells (arrows). (E) Quantification of total number of TUNEL+ cells. (F) Quantification of total number of NeuN+ cells. (G) Quantification of total number of TUNEL+/NeuN+ colocalized cells. *p < 0.05, compared with Sham. #p < 0.05, compared with TBI+Saline. A total of 500–600 images were quantified. n = 5 per group. (H) Representative images of Nissl-stained sections for post-TBI animals receiving either intranasal vehicle saline injection or Wnt3a injection at 1 h post-injury and 1 day post-injury. Tissue loss can be observed in the ipslateral hemisphere (bordered by dashed line). (I) Volumetric analysis by the Cavalieri method was used to calculate total injury volumes from cumulative summation of injury areas of adjacent sections spanning the length of the contusion. (J) Hemispheric tissue loss was calculated by comparing the difference in volume between the ipsilateral hemisphere and the contralateral hemisphere. *p < 0.05, compared with Sham. #p < 0.05, compared with TBI+Saline. n = 5 per group. Color image is available online at www.liebertpub.com/neu

Article Snippet: The primary antibodies used and the dilutions for each were rat anti-Wnt3a antibody (R&D Systems) at 1:400, mouse anti-β-catenin antibody (R&D Systems) at 1:1000, rabbit anti-cleaved caspase 3 antibody (Cell Signaling) at 1:400, rabbit anti-Bcl-2 (Cell Signaling) at 1:1000, rabbit anti-matrix metalloproteinases 9 (MMP-9) antibody (Millipore) at 1:2500, rabbit anti-MMP-2 antibody (Millipore) at 1:2500, rabbit anti-glial-derived growth factor (GDNF) antibody (Santa Cruz Biotechnology) at 1:1000, and anti-vascular endothelial growth factor (VEGF) antibody (Sigma) at 1:5000.

Techniques: Immunohistochemistry, Immunofluorescence, TUNEL Assay, Labeling, Saline, Staining, Injection

Journal: Journal of Neurotrauma

Article Title: Intranasally Delivered Wnt3a Improves Functional Recovery after Traumatic Brain Injury by Modulating Autophagic, Apoptotic, and Regenerative Pathways in the Mouse Brain

doi: 10.1089/neu.2016.4871

Figure Lengend Snippet: Anti-apoptotic and blood–brain barrier (BBB) protective effects of Wnt3a/β-catenin signaling in vitro and after TBI in vivo. (A) Primary neuronal cultures were dissected from E16 mice and then plated until day 12 in vitro (12 DIV). Groups exposed to oxygen-glucose deprivation (OGD), concurrently with either Wnt3a administration or phosphate-buffered saline (PBS) vehicle control administration. Western blot evaluation of salient protein levels were performed. (B) Quantification of normalized intensity of β-catenin. (C) Levels of cell death as measured by LDH release after OGD, and cultures were treated with either just Wnt3a, Wnt3a + Wnt pathway inhibitor, XAV939, non-targeting (NT)-siRNA, β-catenin siRNA, or β-catenin + Wnt3a. *p < 0.05, compared with Control group. p < 0.05, compared with β-catenin siRNA group. Δp < 0.05, compared with Wnt3a-only group. (D-H) Mice were sacrificed at 2 days post-TBI after having been given two intranasal injections of either saline or Wnt. (D) Representative immunoblots for markers related to cell death and BBB disruption. All immunoblots were normalized to the β-actin control, and then to Sham within group. (E) Quantification of the normalized intensity of cleaved caspase-3. (F) Quantification of the normalized intensity of Bcl-2. (G) Quantification of the normalized intensity of matrix metalloproteinases 9 (MMP-9). (H) Quantification of the normalized intensity of MMP-2. *p < 0.05, compared with Sham. #p < 0.05, compared with TBI+Saline. n = 3 for Sham group, n = 7 for both TBI+Saline and TBI+Wnt3a groups.

Article Snippet: The primary antibodies used and the dilutions for each were rat anti-Wnt3a antibody (R&D Systems) at 1:400, mouse anti-β-catenin antibody (R&D Systems) at 1:1000, rabbit anti-cleaved caspase 3 antibody (Cell Signaling) at 1:400, rabbit anti-Bcl-2 (Cell Signaling) at 1:1000, rabbit anti-matrix metalloproteinases 9 (MMP-9) antibody (Millipore) at 1:2500, rabbit anti-MMP-2 antibody (Millipore) at 1:2500, rabbit anti-glial-derived growth factor (GDNF) antibody (Santa Cruz Biotechnology) at 1:1000, and anti-vascular endothelial growth factor (VEGF) antibody (Sigma) at 1:5000.

Techniques: In Vitro, In Vivo, Saline, Control, Western Blot, Disruption

Journal: Journal of Neurotrauma

Article Title: Intranasally Delivered Wnt3a Improves Functional Recovery after Traumatic Brain Injury by Modulating Autophagic, Apoptotic, and Regenerative Pathways in the Mouse Brain

doi: 10.1089/neu.2016.4871

Figure Lengend Snippet: Regenerative effects of Wnt3a/β-catenin signaling during the chronic phase after traumatic brain injury (TBI). (A-E) At 14 days post-TBI, western blot analysis was performed using peri-contusion tissue from animals given a 7-day treatment regimen. (B) Quantification of the normalized intensity of Wnt3a. (C) Quantification of the normalized intensity of β-catenin. (D) Quantification of the normalized intensity of glial-derived growth factor (GDNF). (E) Quantification of the normalized intensity of vascular endothelial growth factor (VEGF). *p < 0.05. n = 3 for Sham group, n = 9 for both TBI+Saline and TBI+Wnt3a groups. (F-I) Mice received intraperitoneal (i.p.) injections of BrdU starting at 3 days post-TBI and continuing until sacrifice at 14 days post-TBI. (F) Immunohistochemical examination (green: NeuN, red: BrdU:, blue: GLUT1) of the cortical region surrounding the injury site shows that Wnt3a treatment resulted in higher levels of total BrdU+ cells, BrdU+/NeuN+ co-labeled cells (arrow), and BrdU+/GLUT1+ co-labeled cells (arrowheads). (G-I) Distinct subtypes of proliferating cells were quantified and analyzed. Wnt3a treatment resulted in significantly greater levels of overall proliferation (total BrdU+ cells), neurogenesis (BrdU+/NeuN+ co-labeled cells), and angiogenesis (BrdU+/GLUT1+ co-labeled cells) following TBI. *p < 0.05, **p < 0.01. n = 3 for Sham group, n = 6 for both TBI+Saline and TBI+Wnt3a groups. Color image is available online at www.liebertpub.com/neu

Article Snippet: The primary antibodies used and the dilutions for each were rat anti-Wnt3a antibody (R&D Systems) at 1:400, mouse anti-β-catenin antibody (R&D Systems) at 1:1000, rabbit anti-cleaved caspase 3 antibody (Cell Signaling) at 1:400, rabbit anti-Bcl-2 (Cell Signaling) at 1:1000, rabbit anti-matrix metalloproteinases 9 (MMP-9) antibody (Millipore) at 1:2500, rabbit anti-MMP-2 antibody (Millipore) at 1:2500, rabbit anti-glial-derived growth factor (GDNF) antibody (Santa Cruz Biotechnology) at 1:1000, and anti-vascular endothelial growth factor (VEGF) antibody (Sigma) at 1:5000.

Techniques: Western Blot, Derivative Assay, Saline, Immunohistochemical staining, Labeling

Journal: Journal of Neurotrauma

Article Title: Intranasally Delivered Wnt3a Improves Functional Recovery after Traumatic Brain Injury by Modulating Autophagic, Apoptotic, and Regenerative Pathways in the Mouse Brain

doi: 10.1089/neu.2016.4871

Figure Lengend Snippet: Intranasally delivered Wnt3a promoted sensorimotor functional recovery following traumatic brain injury (TBI). (A) All animals were pre-trained in the adhesive removal task prior to the injury to ensure competent removal. Baseline latencies (day 0) were measured to ensure no significant differences across groups. After injury, Wnt3a preserved the mouse forelimb sensorimotor function, as indicated by the significantly shorter latency to removal in the contralateral forepaw of the Wnt3a-treated animals. (B) Mice were pre-screened and pre-trained on the rotarod task to ensure adequate performance of ≥90% maximum running time (i.e., ≥270 sec, day 0). Wnt3a-treated animals were able to remain on the accelerating beam significantly longer than the Saline animals, whereas there were no significant differences between the Wnt3a-treated animals and the Sham animals. *p < 0.05, compared with Sham. #p < 0.05, compared with TBI+Saline. n = 6 for Sham group, n = 12 for TBI+Saline group, and = 11 for TBI+Wnt3a groups.

Article Snippet: The primary antibodies used and the dilutions for each were rat anti-Wnt3a antibody (R&D Systems) at 1:400, mouse anti-β-catenin antibody (R&D Systems) at 1:1000, rabbit anti-cleaved caspase 3 antibody (Cell Signaling) at 1:400, rabbit anti-Bcl-2 (Cell Signaling) at 1:1000, rabbit anti-matrix metalloproteinases 9 (MMP-9) antibody (Millipore) at 1:2500, rabbit anti-MMP-2 antibody (Millipore) at 1:2500, rabbit anti-glial-derived growth factor (GDNF) antibody (Santa Cruz Biotechnology) at 1:1000, and anti-vascular endothelial growth factor (VEGF) antibody (Sigma) at 1:5000.

Techniques: Functional Assay, Adhesive, Saline

Journal: Journal of Neurotrauma

Article Title: Intranasally Delivered Wnt3a Improves Functional Recovery after Traumatic Brain Injury by Modulating Autophagic, Apoptotic, and Regenerative Pathways in the Mouse Brain

doi: 10.1089/neu.2016.4871

Figure Lengend Snippet: Concerted interactivity among three distinct systems: Wnt/β-catenin pathway (green), autophagy (gray), and traumatic brain injury (TBI) pathophysiology (red). Under normal conditions, cytoplasmic β-catenin and autophagic activity are mutually regulated. β-catenin inhibits the initiation and elongation of the phagophore, whereas LC3, which is upregulated upon induction of autophagy, will target β-catenin for autolysosomal degradation. TBI is a pathological event that results in hypoxia and oxidative stress that trigger autophagy induction. Further, due to unknown mechanisms, TBI also results in lysosomal impairment, preventing the degradation of autophagosomes. The accumulation of autophagosomes then contributes to greater levels of neuronal death. Wnt3a can stabilize cytoplasmic β-catenin through its downstream inhibition of the Axin-APC-GSK3β complex, which would otherwise ubiquitinate β-catenin for degradation. Upregulation of β-catenin results in suppression of autophagic induction and amelioration of cell death. Color image is available online at www.liebertpub.com/neu

Article Snippet: The primary antibodies used and the dilutions for each were rat anti-Wnt3a antibody (R&D Systems) at 1:400, mouse anti-β-catenin antibody (R&D Systems) at 1:1000, rabbit anti-cleaved caspase 3 antibody (Cell Signaling) at 1:400, rabbit anti-Bcl-2 (Cell Signaling) at 1:1000, rabbit anti-matrix metalloproteinases 9 (MMP-9) antibody (Millipore) at 1:2500, rabbit anti-MMP-2 antibody (Millipore) at 1:2500, rabbit anti-glial-derived growth factor (GDNF) antibody (Santa Cruz Biotechnology) at 1:1000, and anti-vascular endothelial growth factor (VEGF) antibody (Sigma) at 1:5000.

Techniques: Activity Assay, Inhibition