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: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: The sequence of primer used for RT-PCR in this study

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Sequencing

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Wnt2 and Wnt7b were targets of miR-503-3p. (* p < 0.05, there were significant differences between these two groups) a The sequences of miR-503-3p are highly conservative across species. b miR-503-3p might bind to Wnt2 3′-UTR. c miR-503-3p might bind to Wnt7b 3′-UTR. d The luciferase activity of hASCs co-transfected with miR-503-3p mimic and wt-Wnt2 significantly reduced, compared to the miR-Ctrl mimic and wt-Wnt2 group. The luciferase activity of hASCs co-transfected with miR-503-3p mimic and mut-Wnt2 had no significance, compared to miR-Ctrl mimic and mut-Wnt2 group. e The luciferase activity of hASCs co-transfected with miR-503-3p mimic and wt-Wnt7b significantly reduced, compared to the miR-Ctrl mimic and wt-Wnt7b group. The luciferase activity of hASCs co-transfected with miR-503-3p mimic and mut-Wnt7b had no significance, compared to miR-Ctrl mimic and mut-Wnt7b group. f Compared to the miR-Ctrl mimic group, Wnt2 mRNA levels decreased in hASCs transfected with miR-503-3p mimic as shown by real-time PCR. g Compared to the miR-Ctrl mimic group, Wnt7b mRNA levels decreased in hASCs transfected with miR-503-3p mimic as shown by real-time PCR. e Protein blots were listed in the upper column. Compared to the miR-Ctrl mimic group, Wnt2 protein levels decreased in hASCs transfected miR-503-3p mimic as shown by Western blot. f Protein blots were listed in the upper column. Compared to the miR-Ctrl mimic group, Wnt7b protein levels decreased in hASCs transfected miR-503-3p mimic as shown by western blot

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effect of Wnt2 on osteogenic differentiation of hASCs under cyclic strain. (* p < 0.05, there were significant differences between these two groups) a The results of real-time PCR showed that the Wnt2b expression in EX-Wnt2b group increased compared to the EX-Ctrl group; Wnt2 expression in the siWnt2 group significantly decreased, compared to the siR-Ctrl group. b Protein blots were listed in the left column. The results of western blot showed that Wnt2 expression in the EX-Wnt2 group increased, compared to the EX-Ctrl group; Wnt2 expression in the siWnt2 group significantly decreased, compared to the siR-Ctrl group. c After EX-Wnt2 transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt7b, and β-catenin significantly enhanced, compared to the EX-Ctrl group. d After siWnt2 transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt7b, and β-catenin significantly decreased, compared to the siR-Ctrl group. e Comparing to the EX-Ctrl group, both the ALP activity and the content of Ca 2+ were significantly increased when EX-Wnt2 transfected. f Comparing to the siR-Ctrl group, both the ALP activity and the content of Ca 2+ were significantly decreased when siWnt2 transfected

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effect of Wnt7b on osteogenic differentiation of hASCs under cyclic strain. (* p < 0.05, there were significant differences between these two groups) a The results of real-time PCR showed that the Wnt7b expression in EX-Wnt7b group increased, compared to the EX-Ctrl group; Wnt7b expression in the siWnt7b group significantly decreased, compared to the siR-Ctrl group. b Protein blots were listed in the left column. The results of western blot showed that Wnt7b expression in the EX-Wnt7b group increased, compared to the EX-Ctrl group; Wnt7b expression in the siWnt7b group significantly decreased, compared to the siR-Ctrl group. c After EX-Wnt7b transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt7b, and β-catenin significantly enhanced, compared to the EX-Ctrl group. d After siWnt7b transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin significantly decreased, compared to the siR-Ctrl group. e Comparing to the EX-Ctrl group, ALP activity and the content of Ca 2+ significantly increased when EX-Wnt7b transfected. Comparing to the siR-Ctrl group, ALP activity and the content of Ca 2+ significantly decreased when siWnt7b transfected

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effect of β-catenin on osteogenic differentiation of hASCs under cyclic strain. (* p < 0.05, there were significant differences between these two groups) a The results of real-time PCR showed that the β-catenin expression in EX-β-catenin group increased, compared to the EX-Ctrl group; β-catenin expression in the siβ-catenin group significantly decreased, compared to the siR-Ctrl group. b Protein blots were listed in the left column. The results of western blot showed that cytoplasmic β-catenin and nuclear β-catenin expression in the EX-β-catenin group increased, compared to the EX-Ctrl group; cytoplasmic β-catenin and nuclear β-catenin expression in the siβ-catenin group significantly decreased, compared to the siR-Ctrl group. c After EX-β-catenin transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin significantly enhanced, compared to the EX-Ctrl group. d After siβ-catenin transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin significantly decreased, compared to the siR-Ctrl group. e Comparing to the EX-Ctrl group, ALP activity and the content of Ca 2+ significantly increased when EX-β-catenin transfected. Comparing to the siR-Ctrl group, ALP activity and the content of Ca 2+ significantly decreased when siβ-catenin transfected

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effects of miR-503-3p overexpression and inhibition on osteogenic differentiation of hASCs under cyclic strain. (* p < 0.05, there were significant differences between these two groups) a The expression of miR-503-3p in hASCs transfected with miR-503-3p mimic markedly increased, compared to the miR-Ctrl mimic group. b The expression of miR-503-3p in hASCs transfected with miR-503-3p inhibitor decreased, compared to the miR-Ctrl inhibitor group. c After miR-503-3p mimic transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin decreased, compared to the miR-Ctrl mimic group. d Protein blots were listed in the left column. After miR-503-3p mimic transfection, the results of western blot showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, cytoplasmic β-catenin, and nuclear β-catenin decreased. e Both ALP activity and the content of Ca 2+ significantly decreased when miR-503-3p mimic transfected. f After miR-503-3p mimic transfection, the immunofluorescence intensity of β-catenin in the hASC nucleus was decreased. g After miR-503-3p inhibitor transfection, the results of real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin increased, compared to the miR-Ctrl inhibitor group. h Protein blots were listed in the left column. After miR-503-3p inhibitor transfection, the results of western blot showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, cytoplasmic β-catenin, and nuclear β-catenin decreased, compared to the miR-Ctrl inhibitor group. i Both ALP activity and the content of Ca 2+ significantly increased when miR-503-3p inhibitor transfected. j After miR-503-3p inhibitor transfection, the immunofluorescence intensity of β-catenin in the hASC nucleus was increased

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Over Expression, Inhibition, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Immunofluorescence

Journal: Stem Cell Research & Therapy

Article Title: MicroRNA-503-3p affects osteogenic differentiation of human adipose-derived stem cells by regulation of Wnt2 and Wnt7b under cyclic strain

doi: 10.1186/s13287-020-01842-0

Figure Lengend Snippet: Effect of Wnt2 and Wnt7b regulated by miR-503-3p on osteogenic differentiation of hASCs under cyclic strain (* p < 0.05, there were significant differences between these two groups) a After co-transfecting miR-503-3p mimic with EX-Wnt2 and EX-Wnt7b, real-time PCR showed RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin were increased, than the miR-503-3p mimic and EX-Ctrl group. b Protein blots were listed in the left column. After co-transfecting miR-503-3p mimic with EX-Wnt2 and EX-Wnt7b, western blot showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, cytoplasmic, and nuclear β-catenin increased, than the miR-503-3p mimic and EX-Ctrl group. c Both ALP activity and the content of Ca 2+ significantly increased when co-transfecting miR-503-3p mimic with EX-Wnt2 and EX-Wnt7b. d After co-transfecting miR-503-3p mimic with EX-Wnt2 and EX-Wnt7b, the immunofluorescence intensity of nucleus β-catenin was enhanced. e After co-transfecting miR-503-3p inhibitor with siWnt2 and siWnt7b, real-time PCR showed that RUNX2, ALP, SPARC, Wnt2, Wnt7b, and β-catenin decreased, than the miR-503-3p inhibitor and siR-Ctrl group. f Protein blots were listed in the left column. After co-transfecting miR-503-3p inhibitor with siWnt2 and siWnt7b, western blot showed RUNX2, ALP, SPARC, Wnt2, Wnt7b, cytoplasmic, and nuclear β-catenin decreased, than the miR-503-3p inhibitor and siR-Ctrl group. g Both ALP activity and the content of Ca 2+ significantly decreased when co-transfecting miR-503-3p inhibitor with siWnt2 and siWnt7b. h After co-transfecting miR-503-3p inhibitor with siWnt2 and siWnt7b, the immunofluorescence intensity of nucleus β-catenin was decreased. i After co-transfecting miR-503-3p inhibitor with siβ-catenin, real-time PCR showed that RUNX2, ALP, and SPARC decreased; Wnt2, Wnt7b and β-catenin decreased, than the miR-503-3p inhibitor and siR-Ctrl group. j Protein blots were listed in the left column. After co-transfecting miR-503-3p inhibitor with siβ-catenin, western blot showed that RUNX2, ALP, and SPARC decreased; Wnt2, Wnt7b, cytoplasmic and nuclear β-catenin decreased, than the miR-503-3p inhibitor and siR-Ctrl group. k Both ALP activity and the content of Ca 2+ significantly decreased when co-transfecting miR-503-3p inhibitor with siβ-catenin. l After co-transfecting miR-503-3p inhibitor with siβ-catenin into hASCs, the immunofluorescence intensity of nucleus β-catenin was decreased. m After co-transfecting miR-503-3p mimic with EX-β-catenin, real-time PCR showed that RUNX2, ALP, and SPARC increased; Wnt2, Wnt7b, and β-catenin increased, than the miR-503-3p mimic and EX-Ctrl group. n Protein blots were listed in the left column. After co-transfecting miR-503-3p mimic with EX-β-catenin, western blot showed that RUNX2, ALP, and SPARC increased; Wnt2, Wnt7b, cytoplasmic, and nuclear β-catenin increased, than the miR-503-3p mimic and EX-Ctrl group. o Both ALP activity and the content of Ca 2+ significantly increased when co-transfecting miR-503-3p mimic with EX-β-catenin. p After co-transfecting miR-503-3p mimic with EX-β-catenin, the immunofluorescence intensity of nucleus β-catenin was increased

Article Snippet: The primary antibodies used in western blot were listed as follows: RUNX2 (Abcam, USA), ALP (Abcam, USA), SPARC (Abcam, USA), Wnt7b (R&D Systems, USA), Wnt2 (R&D Systems, USA), GAPDH (Cell Signaling Technology, USA), and β-catenin (Abcam, USA) and followed by washing of membranes thrice in TBS-0.05% Tween 20, followed by incubation at room temperature with the corresponding secondary antibodies for 1 h. Blots were then incubated in the dark with ECL (Thermo Fisher Scientific, Germany) and visualized by exposing to enhanced chemiluminescence reagents (GE Healthcare, USA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Distinct Roles of Glycogen Synthase Kinase (GSK)-3? and GSK-3? in Mediating Cardiomyocyte Differentiation in Murine Bone Marrow-derived Mesenchymal Stem Cells *

doi: 10.1074/jbc.M109.019109

Figure Lengend Snippet: GSK-3β induces cardiomyocyte differentiation more potently than Wnt agonists. MSCs were transduced with adenoviruses harboring LacZ, Wnt3a, Wnt11, GSK-3α, GSK-3β, and DN-GSK-3β. A–C, protein expression of Wnt3a (A), Wnt11 (B), β-catenin (C), and phosphorylated pan-protein kinase C (Phospho-PKC (Pan)) (C) was evaluated by immunoblot analyses. The level of β-catenin protein expression was quantitated (C, right). GAPDH expression was evaluated as an internal control. D, the effects of LacZ, Wnt3a, Wnt11, GSK-3α, GSK-3β, and DN-GSK-3β expression upon branched myofibril formation were evaluated. Left, representative photos are shown. Right, the extent of branched myofibrils/cell surface (%) was quantitated. ***, p < 0.001. Pictures with a higher magnification are shown in supplemental Fig. S5. Error bars in B and D show S.E. E and F, mRNA expression of Flk-1, Nkx2.5, cTnC, and α-MHC in MSCs after transduction with Wnt11 (E) and Wnt3a (F) was evaluated by RT-PCR analyses. mRNA expression of GAPDH is shown as an internal control. Experiments were repeated at least 4 times.

Article Snippet: For immunoblot analyses, polyvinylidene difluoride membranes were incubated with 5% nonfat milk buffer containing primary antibody overnight, followed by incubation with anti-mouse IgG or anti-rabbit IgG (Cell Signaling Technology, 1:2500 dilution) for 3 h. The following antibodies were used as primary antibodies: GSK-3α (Cell Signaling Technology, 1:5000), GSK-3β (BD Biosciences, 1:5000), phospho-GSK-3α/β (Cell Signaling Technology, 1:5000), phospho-GSK-3β (Cell Signaling Technology, 1:5000), β- catenin (BD Biosciences, 1:10000), Wnt3a (Santa Cruz Biotechnology, 1:2500), Wnt11 (R&D Systems, 1:2000), phospho-protein kinase C (Pan) (Cell Signaling Technology, 1:2500), sarcomeric α-actinin (Sigma, 1:10000), GATA4 (Santa Cruz Biotechnology, 1:5000), c-Jun (Cell Signaling Technology, 1:2500), troponin I (Santa Cruz Biotechnology, 1:5000), β-actin (Sigma, 1:10000), and GAPDH (Sigma, 1:5000).

Techniques: Transduction, Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility

doi: 10.1186/s12885-016-2943-4

Figure Lengend Snippet: Quantitative real-time PCR primers

Article Snippet: For mouse sections, Alexa Fluor 594 conjugated monoclonal anti-mouse CD31 (1:100, Clone MEC13.3, BioLegend, San Diego, USA), Alexa Fluor 488 conjugated monoclonal anti-mouse CD105 (1:100, Clone MJ7/18, BioLegend, San Diego, USA), purified monoclonal anti-mouse VEGF-A (1:100, Clone 1 F07-2C01, BioLegend, San Diego, USA) and purified monoclonal anti-mouse Wnt5a (1:50, Clone 442625, R&D Systems, Minneapolis, USA) primary antibodies were applied.

Techniques: Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility

doi: 10.1186/s12885-016-2943-4

Figure Lengend Snippet: Transcript analysis of Wnt5a, miRNA and VEGF-A in primary human lung cancer samples of AC and SCC and 3D in vitro lung aggregate cultures. a, Wnt5a mRNA is significantly upregulated in SCC compared to both normal lung and AC specimens. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Immunohistochemical staining of Wnt5a in primary resected AC and SCC samples, n = 3 per groups. c, miR-27b and miR-200b expression levels are significantly lower in AC compared to SCC. Error bars; SEM. Independent samples t -test, n = 5 per groups. d, miR-27b is up-regulated by rhWnt5a in 3D lung aggregate cultures, while neither miR-27b nor miR-200b was affected by rhWnt11. Error bars; SEM. One-way ANOVA, post hoc Bonferroni, n = 6. e, VEGF-A and miR-27b expression levels after 10 μM RSG (PPARgamma agonist) and 10 μM GW9662 (PPARgamma antagonist) and combination treatment with rhWnt5a. Error bars; SEM. Independent samples t -test n = 3. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For mouse sections, Alexa Fluor 594 conjugated monoclonal anti-mouse CD31 (1:100, Clone MEC13.3, BioLegend, San Diego, USA), Alexa Fluor 488 conjugated monoclonal anti-mouse CD105 (1:100, Clone MJ7/18, BioLegend, San Diego, USA), purified monoclonal anti-mouse VEGF-A (1:100, Clone 1 F07-2C01, BioLegend, San Diego, USA) and purified monoclonal anti-mouse Wnt5a (1:50, Clone 442625, R&D Systems, Minneapolis, USA) primary antibodies were applied.

Techniques: In Vitro, Immunohistochemical staining, Staining, Expressing

Journal: BMC Cancer

Article Title: Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility

doi: 10.1186/s12885-016-2943-4

Figure Lengend Snippet: The effect of Wnt5a on VEGF-A induced endothelial cell activation and motility. a, CD105 mRNA expression is significantly higher in primary AC compared to SCC samples. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Flow cytometric analysis of CD105 protein expression in CD31 positive endothelial cells in primary AC and SCC samples. n = 6 per groups. c, Flow cytometric analysis of CD105 levels in normal and high VEGF-A microenvironment in 3D lung aggregate tissues has also shown an increase of activation marker CD105 in VEGF-A high tissues. The double positive (CD105/CD31) cell population was considered as activated endothelial cells. Independent samples t -test, n = 6. 1 μg/ml rhWnt5a treatment had no effect on the VEGF-A induced endothelial cell activation measured by the double positive (CD105/CD31) cell population identified by flow cytometric analysis. Independent samples t -test, n = 6. d, Localization of endothelial cells was identified by immunoflurescent staining of CD31 and analyzed by confocal microscopy in 3D lung tissue aggregates. In VEGF-A normal microenvironment endothelial cells remained diffuse in the tissue. Under VEGF-A excess endothelial cell migrated towards the source (VEGF-A high fibroblasts) of the signal in the center of the aggregate tissue. 1 μg/ml rhWnt5a treatment of VEGF-A high tissue aggregates inhibited endothelial cell accumulation in the center of the aggregate. Bar chart represents the quantification of endothelial cell distribution. Relative area of CD31+ endothelial cells are compared to total field. Percentages were calculated as relative area of endothelial cells/area of total field *100. Error bars; SEM. Independent samples t -test n = 3. Representative images of three independent experiments are shown. Scale bars, 50 μm. e , HMVEC-L transwell migration assay. Endothelial cells migrate significantly faster towards VEGF-A high fibroblast, while 1 μg/ml rhWnt5a can reverse the effect of elevated VEGF-A level. Scale bar 100 μm. One-way ANOVA, n = 3. P < 0.05 was considered as significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For mouse sections, Alexa Fluor 594 conjugated monoclonal anti-mouse CD31 (1:100, Clone MEC13.3, BioLegend, San Diego, USA), Alexa Fluor 488 conjugated monoclonal anti-mouse CD105 (1:100, Clone MJ7/18, BioLegend, San Diego, USA), purified monoclonal anti-mouse VEGF-A (1:100, Clone 1 F07-2C01, BioLegend, San Diego, USA) and purified monoclonal anti-mouse Wnt5a (1:50, Clone 442625, R&D Systems, Minneapolis, USA) primary antibodies were applied.

Techniques: Activation Assay, Expressing, Marker, Staining, Confocal Microscopy, Transwell Migration Assay