wnt5a treatment  (Addgene inc)

Journal: bioRxiv

Article Title: Wnt5a-mediated Adipo-Cardiac Interorgan Communication in HFpEF

doi: 10.1101/2025.10.29.685456

Figure Lengend Snippet: (A) Schematic outline of study. (B) Cellular localization of differentially expressed genes (DEGs) in Nprc -AKO visceral adipose tissue (VAT). (C) Increased and decreased gene ontologies in Nprc -AKO vs control VAT. (D) Volcano plot of DEGs in Nprc -AKO vs control VAT (red indicates p < 0.01 and 50%-fold-change compared to control). (E) Reverse transcriptase quantitative PCR of top 5 Wnt isoforms in Nprc -AKO vs control VAT. (F) Plasma circulating levels of Wnt5a in global (GKO), cardiomyocyte-specific (CKO), adipocyte-specific (AKO), and control (WT) mice after L-NAME and high fat diet. (G) Cell size in H9C2 cells after treatment with Wnt5a. (H) RT-qPCR for hypertrophic markers NPPA, NPPB, and MYH7 in H9C2 cells after Wnt5a treatment.

Article Snippet: For studies investigating the effects of Wnt5a treatment, cells were transfected with either pcDNA3.2-Wnt5a plasmid (Addgene 43813) or empty vector control (Addgene 29496).

Techniques: Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Clinical Proteomics, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Wnt5a-mediated Adipo-Cardiac Interorgan Communication in HFpEF

doi: 10.1101/2025.10.29.685456

Figure Lengend Snippet: (A) Schematic outline of study. (B) Histologic and wheat germ agglutinin-based hypertrophy, (C) plasma Wnt5a levels, (D) left ventricular systolic pressure, (E) left ventricular end-diastolic pressure, (F) diastolic relaxation of the left ventricle, (G) right ventricular systolic pressure, (H) right ventricular end-diastolic pressure, (I) diastolic relaxation of the right ventricle, (J) left atrial end-systolic volume by echocardiography, and (K) E/e’ ratio by echocardiography in C57/BL6J mice treated with 8 weeks of L-NAME and high fat diet, followed by LGK974 or carrier control intraperitoneal injections.

Article Snippet: For studies investigating the effects of Wnt5a treatment, cells were transfected with either pcDNA3.2-Wnt5a plasmid (Addgene 43813) or empty vector control (Addgene 29496).

Techniques: Clinical Proteomics, Control

Journal: Oral oncology

Article Title: Critical roles of Wnt5a-Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via ΔNp63β-mediated epithelial-mesenchymal transition.

doi: 10.1016/j.oraloncology.2017.03.019

Figure Lengend Snippet: Fig. 1. Expression of DNp63, Wnt5a, Ror1, and Ror2 in tongue SCC cell lines. (A) RT- PCR analysis shows that DNp63 is not expressed in high metastatic potential SQUU- B cells, but is expressed in other cell lines. Contrastingly, Wnt5a and Ror2 are strongly expressed in SQUU-B cells, but not the other cell lines. Ror1 is equally expressed in all cell lines. (B, C) Real-time PCR and western blotting analyses. DNp63 is expressed in SQUU-BO, but not in SQUU-BC cells. Wnt5a and Ror2 expression in SQUU-BO cells is lower than that in SQUU-BC cells (Mann–Whitney U test, p < 0.05). No significant difference in Ror1 expression between SQUU-BC and SQUU-BO cells is found. (D, E) Real-time PCR and western blotting analyses. DNp63 expression is low in SQUU-A cells transfected with DNp63 siRNA. Wnt5a and Ror2 expression is up-regulated by DNp63 knockdown (Mann–Whitney U test, p < 0.05). However, Ror1 expression is unchanged.

Article Snippet: Exogenous Wnt5a treatment Recombinant human Wnt5a protein (rhWnt5a) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY, Transfection, Knockdown

Journal: Oral oncology

Article Title: Critical roles of Wnt5a-Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via ΔNp63β-mediated epithelial-mesenchymal transition.

doi: 10.1016/j.oraloncology.2017.03.019

Figure Lengend Snippet: Fig. 2. Effects of Wnt5a knockdown on expression of EMT-associated molecules and capacities for migration and invasion. (A) RT-PCR and western blotting analyses demonstrate Wnt5a inhibition following transfection of Wnt5a siRNA into SQUU-B cells. (B) Real-time PCR analyses demonstrate downregulation of CK19 and upregulation of vimentin (Mann–Whitney U test, p < 0.05). (C) Wound healing assay demonstrates low migration capacity 48 h after transfection of Wnt5a siRNA (Mann–Whitney U test, p < 0.05). Scale bar, 200 lm. (D) Invasion assay shows that Wnt5a-knockdown cells have low invasion capacity at 22 h after transfection of Wnt5a siRNA (Mann–Whitney U test, p < 0.05). Scale bars, 100 lm.

Article Snippet: Exogenous Wnt5a treatment Recombinant human Wnt5a protein (rhWnt5a) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Knockdown, Expressing, Migration, Reverse Transcription Polymerase Chain Reaction, Western Blot, Inhibition, Transfection, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Wound Healing Assay, Invasion Assay

Journal: Oral oncology

Article Title: Critical roles of Wnt5a-Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via ΔNp63β-mediated epithelial-mesenchymal transition.

doi: 10.1016/j.oraloncology.2017.03.019

Figure Lengend Snippet: Fig. 3. Effects of Ror2 knockdown on expression of EMT-associated molecules and capacities for migration and invasion. (a) RT-PCR and western blotting analyses show inhibition of Ror2 expression following transfection of Ror2 siRNA into SQUU-B cells. (b) Real-time PCR analyses demonstrate downregulation of CK19 and upregulation of Wnt5a (Mann–Whitney U test, p < 0.05). (c) Wound healing assay shows that Ror2-depleted cells have low migration ability at 48 h after transfection of siRNA (Mann– Whitney U test, p < 0.05). Scale bar, 200 lm. (d) Invasion assay shows that Ror2-depleted cells have low invasion ability at 22 h after transfection of siRNA (Mann–Whitney U test, p < 0.05). Scale bars, 100 lm.

Article Snippet: Exogenous Wnt5a treatment Recombinant human Wnt5a protein (rhWnt5a) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Knockdown, Expressing, Migration, Reverse Transcription Polymerase Chain Reaction, Western Blot, Inhibition, Transfection, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Wound Healing Assay, Invasion Assay

Journal: Oral oncology

Article Title: Critical roles of Wnt5a-Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via ΔNp63β-mediated epithelial-mesenchymal transition.

doi: 10.1016/j.oraloncology.2017.03.019

Figure Lengend Snippet: Fig. 4. Immunohistochemical detection and cause-specific survival curves in tongue SCC patients. Anti-Wnt5a and -Ror2 antibodies were used to stain normal tongue epithelial tissues and tongue SCC specimens. In the normal epithelial tissues, Wnt5a and Ror2 are almost undetectable (Aa, b). In tongue SCC tissues, Wnt5a and Ror2 are frequently strongly present in the cytoplasm and at the membrane of cancer cells (Ac, d), but are undetectable in some tongue SCC tissues (Ae, f). Scale bars, 50 lm. Patients with high Wnt5a or Ror2 expression have lower cause-specific 5-year survival rates than each low expression group (log-rank test) (B).

Article Snippet: Exogenous Wnt5a treatment Recombinant human Wnt5a protein (rhWnt5a) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Staining, Membrane, Expressing

Journal: Oral oncology

Article Title: Critical roles of Wnt5a-Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via ΔNp63β-mediated epithelial-mesenchymal transition.

doi: 10.1016/j.oraloncology.2017.03.019

Figure Lengend Snippet: Fig. 5. Expression and gelatinolytic activity of MMP-2 in tongue SCC cells. (A) RT-PCR analyses demonstrate that MMP-2 is more strongly expressed in SQUU-B cells expressing Wnt5a and Ror2 compared with other OSCC cells. (B) Real-time PCR analyses show downregulation of MMP-2 expression following DNp63b overexpression, Wnt5a knockdown, or Ror2 knockdown, and upregulation by DNp63 knockdown (Mann–Whitney U test, p < 0.05). (C) Gelatin zymography assay with upper bands indicating preformed MMP-2 protein and lower bands indicating active MMP-2 protein. Active MMP-2 expression is present in SQUU-B cells but not SQUU-A cells. Expression of preformed and active MMP-2 is increased by DNp63 knockdown, and decreased by DNp63b overexpression, Wnt5a knockdown, or Ror2 knockdown.

Article Snippet: Exogenous Wnt5a treatment Recombinant human Wnt5a protein (rhWnt5a) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Over Expression, Knockdown, MANN-WHITNEY, Zymography Assay

Journal: Oral oncology

Article Title: Critical roles of Wnt5a-Ror2 signaling in aggressiveness of tongue squamous cell carcinoma and production of matrix metalloproteinase-2 via ΔNp63β-mediated epithelial-mesenchymal transition.

doi: 10.1016/j.oraloncology.2017.03.019

Figure Lengend Snippet: Fig. 6. Effects of rhWnt5a protein on migration, invasion, and activity of MMP-2 in tongue SCC cells. Tongue SCC cells were treated with increasing concentrations of rhWnt5a (0, 0.01, 0.1, and 1 lg/mL). (A) Wound healing assay shows that addition of rhWnt5a for 24 h increases migration abilities of SQUU-B cells transfected with Wnt5a siRNA (Dunnett’s test, p < 0.05). Stimulation with rhWnt5a has no effect on the migration abilities of SQUU-A, SQUU-BO, or Ror2-depleted SQUU-B cells. Scale bars, 200 lm (B) Matrigel TM invasion assay, addition of rhWnt5a for 22 h increases the number of invading SQUU-B cells transfected Wnt5a siRNA (Dunnett’s test, p < 0.05). Stimulation with rhWnt5a has no effect on the invasion abilities of SQUU-A, SQUU-BO, or Ror2-depleted SQUU-B cells. Scale bars, 100 lm.(C) Gelatin zymography assay shows that preformed and active MMP-2 are increased in Wnt5a-depleted SQUU-B cells following stimulation with rhWnt5a.

Article Snippet: Exogenous Wnt5a treatment Recombinant human Wnt5a protein (rhWnt5a) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Activity Assay, Wound Healing Assay, Transfection, Invasion Assay, Zymography Assay

Journal: Oncogene

Article Title: The Orphan Tyrosine Kinase Receptor, ROR2, Mediates Wnt5A Signaling in Metastatic Melanoma

doi: 10.1038/onc.2009.305

Figure Lengend Snippet: ROR2 mRNA is highly expressed in Wnt5A high lines (UACC903 and M93-047), but not Wnt5A low UACC1273EV and G361 cells (A). ROR2 protein levels are also higher in Wnt5A high lines than Wnt5A-low G361 cells, as shown by Western analysis (B). Treating ROR2-low UACC1273EV cells with rWnt5A increases ROR2 expression (C). Immunofluorescent analysis demonstrates that in Wnt5A-low lines, ROR2 is expressed in a diffuse cytoplasmic pattern in most cells. In Wnt5A-high lines, ROR2 is expressed in both a diffuse pattern as well as in perinuclear/ nuclear foci in the vast majority of the cells (D). Treatment of Wnt5A-low cells with rWnt5A increases the focal localization of ROR2 (E). Wnt5A and ROR2 expression are both expressed in the perinuclear foci, as shown by immunolocalization analysis (F).

Article Snippet: For recombinant Wnt5A treatment, 200ng/ml of rWnt5A (R & D Systems, Minneapolis MN) was added for 16 hours.

Techniques: Western Blot, Expressing

Journal: Oncogene

Article Title: The Orphan Tyrosine Kinase Receptor, ROR2, Mediates Wnt5A Signaling in Metastatic Melanoma

doi: 10.1038/onc.2009.305

Figure Lengend Snippet: Immunofluorescent analysis indicates that ROR2 (green) co-localizes with clathrin (red, A, arrows). Wnt5A treatment causes an immediate internalization of ROR2 and co-localization of ROR2 and clathrin (B). Mono-dansyl cadaverine (MDC) inhibits the internalization of ROR2 via clathrin and causes its redistribution from foci to the periphery of the cell (C, arrows). MDC inhibition also decreases the invasion of melanoma cells through Matrigel (D). Clathrin siRNA decreases clathrin expression as demonstrated by Western blot analysis (E) and decreased clathrin inhibits the motility of melanoma cells as shown in a wound-healing assay (F). In Wnt5A-high cells, PKC inhibition results in the movement of ROR2 (green) from foci to a diffuse cytoplasmic distribution in cells, upon PKC inhibition (G). Foci reappear at 6h after treatment. Co-staining with clathrin (red) indicates that PKC inhibition may affect ROR2 internalization, as in the presence of PKC inhibitor, ROR2 does not co-localize with clathrin (H). Re-emergence of ROR2-positive foci at 6h of PKC inhibitor treatment corresponds to increased co-localization with the golgi (I, arrows).

Article Snippet: For recombinant Wnt5A treatment, 200ng/ml of rWnt5A (R & D Systems, Minneapolis MN) was added for 16 hours.

Techniques: Inhibition, Expressing, Western Blot, Wound Healing Assay, Staining

Journal: Oncogene

Article Title: The Orphan Tyrosine Kinase Receptor, ROR2, Mediates Wnt5A Signaling in Metastatic Melanoma

doi: 10.1038/onc.2009.305

Figure Lengend Snippet: WNT5A knockdown results in a decrease in ROR2 mRNA (A, solid lines) in both UACC903 and M93-047 cells as analyzed by real time PCR (A) and in ROR2 protein as analyzed by immunofluorescence (B). Wnt5A protein is maximally decreased at 72hours, coincident with the greatest levels of ROR2 knockdown (A, inset). ROR2 knockdown results in a decrease in ROR2 mRNA (C) and protein (D), but not WNT5A 28 mRNA (C, dashed lines). However ROR2 knockdown does inhibit Wnt5A signaling as demonstrated by a decrease in PO 4 -PKC (E) and an increase in MART-1 expression by Western analysis (F).

Article Snippet: For recombinant Wnt5A treatment, 200ng/ml of rWnt5A (R & D Systems, Minneapolis MN) was added for 16 hours.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Immunofluorescence, Expressing, Western Blot

Journal: Oncogene

Article Title: The Orphan Tyrosine Kinase Receptor, ROR2, Mediates Wnt5A Signaling in Metastatic Melanoma

doi: 10.1038/onc.2009.305

Figure Lengend Snippet: Using a Matrigel invasion assay, ROR2 knockdown inhibits the invasion of UACC903 and M93-047 cells by over 50% (A). Two different siRNAs were used to demonstrate the requirement for ROR2, see also . G361 cells are similarly affected in a matrigel invasion assay, where Wnt5A treatment can significantly (**=p<0.01, ***=p<0.001) increase their migration through Matrigel, but not in the presence of ROR2 siRNA (B). When M93-047 29 cells are injected via the tail vein into nude mice, ROR2 knockdown results in a decreased ability of M93-047 cells to form pulmonary metastases (C). Two lungs with evidence of macroscopic metastases are shown and a histology section from a third mouse (C, arrow) is shown in order to demonstrate the hemorrhaging seen in these tumors.

Article Snippet: For recombinant Wnt5A treatment, 200ng/ml of rWnt5A (R & D Systems, Minneapolis MN) was added for 16 hours.

Techniques: Invasion Assay, Knockdown, Migration, Injection