recombinant wnt5b protein  (R&D Systems)

Journal: JCI Insight

Article Title: DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells

doi: 10.1172/jci.insight.171821

Figure Lengend Snippet: ( A ) Quantification of the relative branch length of a tube formation assay with LECs cultured in conditioned media (CM) from monotypic LECs, WM852 cells, or LEC+WM852 coculture for 24 hours and analyzed by a 16-hour tube formation assay. Experiment was performed 2 independent times. ( B ) qRT-PCR of WNT5B mRNA levels in the indicated monotypic or LEC-cocultured melanoma cell lines from 3 independent experiments. ( C ) Immunofluorescence images of monotypic WM852 and WM852+LEC cultures labeled with an antibody against WNT5B. Small inserts identify the GFP-expressing melanoma cells. Nuclei were counterstained with Hoechst 33342. Representative images from 3 independent experiments are shown in the left panel, and quantification of WNT5B relative signal intensity from at least 100 cells/experiment/condition is shown in the right panel. Scale bar: 50 μm. ( D ) Quantification of the spheroid-sprouting assay of monotypic LECs and LECs cocultured with the indicated melanoma cells (LEC*). Prior to the coculture, melanoma cells were pretreated with control (siCtrl) or WNT5B -targeting siRNAs (si WNT5B ) for 24 hours. Graph shows the mean of 3 independent experiments, each with at least 4 spheroids/condition quantified. WM852 and WM165 melanoma cell–LEC cocultures were performed at the same time and therefore the same LEC control spheroids were used for analysis. ( E ) Quantification of the tube formation assay with LECs cultured and treated as in D ( n = 3). WM165 and WM793 melanoma cell–LEC cocultures were performed at the same time and therefore the same LEC control samples were used for analysis. ( F ) The electrical cell impedance assay with LECs cultured and treated as in D . Data are presented as mean ± SD for each sample. Representative experiment of 2 experiments is shown. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple-comparison test ( A , B , and D ), unpaired, 2-tailed t test ( B and C ), or AUC analysis followed by 1-way ANOVA with Tukey’s multiple-comparison test ( F ).

Article Snippet: Where indicated, γ-secretase inhibitor DAPT (Sigma-Aldrich) at 10 μM concentration and recombinant WNT5B protein (7347, R&D Systems) at 1000 ng/mL were applied.

Techniques: Tube Formation Assay, Cell Culture, Quantitative RT-PCR, Immunofluorescence, Labeling, Expressing, Control, Comparison

Journal: JCI Insight

Article Title: DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells

doi: 10.1172/jci.insight.171821

Figure Lengend Snippet: ( A ) Schematic of the workflow. WM852 melanoma cells were treated with siRNAs for 24 hours and cultured as monotypic cultures (siCtrl) or with LEC (siCtrl*, si WNT5B *). After 2 days, the 2 cell types were separated and melanoma cells were injected intradermally into mouse ear pinna. After 1 week, mice were sacrificed and the ears, lungs, liver, and superficial and inguinal lymph nodes were harvested and processed for analyses. Schematics generated with BioRender.com. ( B ) Representative images of the GFP-expressing WM852 melanoma cells (siCtrl, siCtrl*, si WNT5B *) in mouse ear pinna epidermis. Dashed line indicates the boundaries of injected melanoma cells and arrowheads show the diffuse growth phenotype of the siCtrl* melanoma cells. The relative size of areas occupied by GFP + melanoma cells was quantified from each mouse ear. Relative size for the GFP + area of each mouse ear is shown (siCtrl, n = 4; siCtrl*, n = 8; si WNT5B *, n = 8; where n refers to number of ears quantified). Scale bar: 200 μm. ( C ) qPCR for the relative human Alu sequences from the mouse superficial cervical lymph nodes. Mouse genomic actin was used as a control. Single values for each mouse are shown. Day 7 (d7): siCtrl, n = 3; siCtrl*, n = 5; si WNT5B *, n = 5. d14: siCtrl, n = 1, siCtrl*, n = 3, si WNT5B *, n = 3. ( D ) Mouse ear xenografts were generated from WM852 cells as described in A . Superficial cervical lymph nodes were harvested after 8 and 14 days and analyzed by FACS for the presence of GFP + tumor cells. Lymph nodes from untreated mice (no cells) were used as controls. Single values for each mouse are shown (siCtrl, n = 1; siCtrl*, n = 3; si WNT5B *, n = 3). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple-comparison test ( B and C , d7 samples) or 1-tailed t test (panel C d14 samples and D ).

Article Snippet: Where indicated, γ-secretase inhibitor DAPT (Sigma-Aldrich) at 10 μM concentration and recombinant WNT5B protein (7347, R&D Systems) at 1000 ng/mL were applied.

Techniques: Cell Culture, Injection, Generated, Expressing, Control, Comparison

Journal: JCI Insight

Article Title: DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells

doi: 10.1172/jci.insight.171821

Figure Lengend Snippet: ( A ) WM852 cells were pretreated with the indicated siRNAs and subjected to monotypic or LEC cocultures (*). After cell sorting, WNT5B mRNA levels in melanoma cells were measured by qRT-PCR. Graph shows results from 3 independent experiments. ( B ) WM852 cells were cultured as monotypic cultures or in coculture with LECs (*) and treated with vehicle (EtOH) or DAPT. WNT5B mRNA level was measured in the sorted melanoma cells by qRT-PCR ( n = 3). ( C ) Top: Schematic presentation of the WNT5B promoter area amplified by qPCR following ChIP. Numbers indicate the nucleotides upstream of the WNT5B transcription start site. Bottom: ChIP from WM852 cells expressing ectopic NICD3 for 24 hours. ChIP was performed with 2 different anti-Notch3 antibodies and respective control IgGs and DNA was amplified from the indicated promoter regions of the WNT5B gene ( n = 2). Data in A – C are presented as mean ± SD. ( D and E ) Representative images of human primary melanoma tumor ( D ) and metastasis samples ( E ) labeled for the indicated proteins. Percentage below the images indicates the proportion of samples with Notch3 and WNT5B signal colocalization. Scale bars: 20 μm. ( F ) Statistical analysis of co-distribution in primary tumors and metastatic samples. ( G ) RSEM-quantitated mRNA abundance comparing NOTCH3 and WNT5B shows a significant, positive correlation between the 2 gene transcripts. The P value was obtained using the 2-tailed t test for Pearson’s correlation coefficient, r . ( H ) Survival statistics were computed for the entire cohort of 442 melanoma patients (left panel), or for the cases with no distant metastases at the time of diagnosis (right panel). Patients were divided by their NOTCH3 mRNA level expression into 3 groups based on RSEM-quantitated mRNA abundance percentiles: Low, first to 33rd percentile; Medium, 34th to 67th percentile; High, 68th to 100th percentile. Log-rank P values for the left panel: 0.004, right panel: 0.013. * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA followed by Tukey’s multiple-comparison test ( A and B ) or 2-tailed t test ( F ).

Article Snippet: Where indicated, γ-secretase inhibitor DAPT (Sigma-Aldrich) at 10 μM concentration and recombinant WNT5B protein (7347, R&D Systems) at 1000 ng/mL were applied.

Techniques: FACS, Quantitative RT-PCR, Cell Culture, Amplification, Expressing, Control, Labeling, Biomarker Discovery, Comparison

Journal: JCI Insight

Article Title: DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells

doi: 10.1172/jci.insight.171821

Figure Lengend Snippet: ( A ) Indicated melanoma cell lines cultured on dishes precoated with the indicated chimeric Fc fused with Notch ligands or Fc alone as a control for 2 days, and analyzed by RT-qPCR for NOTCH3 , HEY1 , and HES1 ( n = 3). ( B ) Immunoblotting of WM852 cells cultured as in A for the indicated targets. FL, full length. A representative blot of 3 independent experiments is shown. ( C ) A 3D fibrin droplet invasion assay of WM852 cells cultured on DLL4-Fc and Fc as in A . GFP-expressing melanoma cells were stained with Phalloidin 594 and nuclei were counterstained with Hoechst 33342. Maximum intensity of z projections of the confocal stacks are shown. Graph shows quantification of the relative invasive index from 3 independent experiments with at least 50 cell clusters quantified/condition. Scale bar: 20 μm. ( D ) qRT-PCR of WNT5B levels in melanoma cell lines cultured as in C . Experiment was performed 3 independent times. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001 by 1-way ANOVA followed by Dunnett’s multiple-comparison test ( A ) or 2-tailed t test ( C and D ).

Article Snippet: Where indicated, γ-secretase inhibitor DAPT (Sigma-Aldrich) at 10 μM concentration and recombinant WNT5B protein (7347, R&D Systems) at 1000 ng/mL were applied.

Techniques: Cell Culture, Control, Quantitative RT-PCR, Western Blot, Invasion Assay, Expressing, Staining, Comparison

Journal: JCI Insight

Article Title: DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells

doi: 10.1172/jci.insight.171821

Figure Lengend Snippet: Schematic model of the bidirectional melanoma cell crosstalk with LECs and the role of Notch3 in the LEC functional changes through induction of WNT5B.

Article Snippet: Where indicated, γ-secretase inhibitor DAPT (Sigma-Aldrich) at 10 μM concentration and recombinant WNT5B protein (7347, R&D Systems) at 1000 ng/mL were applied.

Techniques: Functional Assay

Journal: Nature Communications

Article Title: WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion

doi: 10.1038/s41467-020-18951-2

Figure Lengend Snippet: a Heatmap displaying enrichment scores for differentially expressed EMT and metastasis-related signatures in amoeboid A375M2 cells compared to A375P cells or to A375M2 cells treated with ROCKi (H1152 and Y27632) or blebbistatin using ssGSEA. b Representative phase-contrast images of A375M2 cells on top of collagen I matrix (left) and immunoblots of p-MLC2 in A375M2 and WM1361 cells (right) after 4 h treatment with ROCKi (H1152) ( n = 3). Scale bar, 50 μm. c Fold regulation of EMT-related gene expression from EMT-directed qPCR array in A375M2 and WM1361 cells treated with ROCKi (H1152) for 4 h ( n = 4). d Representative confocal images of p-MLC2 and F-actin staining in WM1361 cells on collagen I matrix after WNT11 knockdown ( n = 3). Scale bar, 20 μm. e Quantification of cell morphology (>280 cells pooled from n = 3) and f p-MLC2 immunofluorescence signal normalized by cell area (>85 cells pooled from n = 3) in WM1361 cells on collagen I matrix after depletion of indicated genes. g Representative confocal images (left) and quantification (right) of adhesion of WM1361 cells to a monolayer of keratinocytes after depletion of indicated genes ( n = 5 for SERPINE1 , n = 4 for TCF4 , n = 3 for WNT11 , WNT5B , AHNAK and CAV2 ). Scale bar, 20 μm. h Representative confocal images (left) and quantification (right) of 3D invasion index through a collagen I matrix of WM1361 cells after depletion of indicated genes ( n = 3). Scale bar, 50 μm. i Summary table of amoeboid functional assays on WM1361 cells after SERPINE1 , WNT11 , WNT5B , AHNAK , TCF4 and CAV2 knockdown. “+” sign indicates a significant phenotype of the corresponding gene knockdown. c , g , h Graphs show mean ± s.e.m. e , f Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. b – h n means number of independent biological experiments. c Two-tailed t -test with Benjamini, Krieger and Yekutieli correction for multiple comparisons. e, f Kruskal–Wallis test with Benjamini, Krieger and Yekutieli correction. g , h One-way ANOVA with Benjamini, Krieger and Yekutieli correction. For all graphs, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The exact significant p values for * p , ** p and *** p are provided in Supplementary Table .

Article Snippet: WNT11 (200 ng/ml; 6179-WN-010, R&D Systems,) and WNT5B (500 ng/ml; 7347-WN-025, R&D Systems) were added in serum-free media for 24 h.

Techniques: Western Blot, Gene Expression, Staining, Knockdown, Immunofluorescence, Functional Assay, Two Tailed Test

Journal: Nature Communications

Article Title: WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion

doi: 10.1038/s41467-020-18951-2

Figure Lengend Snippet: a mRNA expression of stem cell-related markers by qRT-PCR in A375M2 cells treated with ROCKi (GSK269962A) for 24 h compared to control A375M2 cells ( n = 4 for ALCAM , ALDH1A3 , ALDH1A1 ; n = 3 for CD44 , OCT4 , JARID1B , SOX2 ). b Schematic of treatment (top), representative phase-contrast images (bottom left) and quantification of sphere formation index (bottom right) and c cell viability of A375M2 cells and A375M2 cells treated with one dose of ROCKi (H1152 or GSK269962A) or blebbistatin ( n = 3). Scale bar, 250 μm. d – f After ROCK1/2 , MYL9 or MYL12B knockdown in A375M2 and WM1361 cells, quantification of d sphere formation index ( n = 4 for A375M2, n = 3 for WM1361) and e , f cell viability after e 7 days ( n = 3 for A375M2, n = 4 for WM1361) or f 3 days ( n = 3) of cell seeding. g – j After WNT11 or WNT5B knockdown in A375M2 and WM1361 cells, g , h representative phase-contrast images (left) and quantification of sphere formation index (right) ( n = 3) and i , j cell viability ( n = 4 for A375M2, n = 3 for WM1361). Scale bar, 250 μm. k Representative phase-contrast images (left) and quantification of cell morphology (right) of A375P cells after 24 h of WNT11 or WNT5B stimulation (>225 cells pooled from n = 3). Scale bar, 100 μm. a – j Graphs show mean ± s.e.m. k Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. a – k n means number of independent biological experiments. a – c , h , j Two-tailed t -test. d – g , i One-way ANOVA with Dunnett post-hoc test. k Kruskal–Wallis with Dunn’s multiple comparison test. For all graphs, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The exact significant p values for * p , ** p and *** p are provided in Supplementary Table .

Article Snippet: WNT11 (200 ng/ml; 6179-WN-010, R&D Systems,) and WNT5B (500 ng/ml; 7347-WN-025, R&D Systems) were added in serum-free media for 24 h.

Techniques: Expressing, Quantitative RT-PCR, Control, Knockdown, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion

doi: 10.1038/s41467-020-18951-2

Figure Lengend Snippet: a – i Representative images (left) and quantification (right) of a melanoma cell shape score, b H-score of p-MLC2 staining, c ki-67 positive cells, H-score of d DAAM1, e WNT11, f WNT5B, g ALDH1A1, h CD44 and i NANOG staining in matched TB and IF from primary melanomas. j Principal component analysis (PCA) based on the expression of amoeboid (cell shape and p-MLC2), proliferative (ki-67), non-canonical Wnt pathway (WNT11, WNT5B and DAAM1) and cancer stem cell-related (ALDH1A1, CD44 and NANOG) markers assessed by immunohistochemistry in matched TB and IF from primary melanomas. Percentage of variation explained by each component is given in the axis labels. k , l Kaplan–Meier survival curves of k overall survival and l disease-free survival according to ALDH1A1 protein expression in the IF from our cohort of primary melanomas. ALDH1A1 expression was categorized as low or high using the median expression. a – l 53 primary melanomas. a , e – i Scale bar, 100 μm; inset, 50 μm. b – d Scale bar, 300 μm. a – i Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show the minimum and maximum range of values. a , b , d – f Two-tailed paired t -test. c , g – i Two-tailed Wilcoxon test. j Scatter plot showing principal components 1 (PC1) and 2 (PC2). k , l Log-rank test. Human schematic in this figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License ( https://smart.servier.com ).

Article Snippet: WNT11 (200 ng/ml; 6179-WN-010, R&D Systems,) and WNT5B (500 ng/ml; 7347-WN-025, R&D Systems) were added in serum-free media for 24 h.

Techniques: Staining, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion

doi: 10.1038/s41467-020-18951-2

Figure Lengend Snippet: a – i Representative images (left) and quantification (right) of a melanoma cell shape score, b H-score of p-MLC2 staining, c ki-67 positive cells, H-score of d DAAM1, e WNT11, f WNT5B, g ALDH1A1, h CD44 and i NANOG staining in matched TB and IF from melanoma metastases. j Model summarizing the findings of this study. The IF of human primary melanomas is enriched in cells with rounded-amoeboid morphology, high levels of Myosin II, proliferative, non-canonical Wnt and cancer stem cell-related markers. This phenotype is recapitulated and further enriched in melanoma metastasis. WNT11/5B activate FZD7 and DAAM1 to control Rho activity and then ROCK1/2-Myosin II levels. All these signalling components have an impact on amoeboid features and tumour initiation in melanoma both in vitro and in vivo. Specifically, DAAM1 plays an essential role in tumour and metastasis initiation and subsequent metastatic outgrowth by sustaining the amoeboid phenotype. a – i 45 metastatic melanomas. a , e – i Scale bar, 100 μm; inset, 50 μm. b – d Scale bar, 300 μm. a – i Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. a , b , d – f Two-tailed paired t -test. c , g – i Two-tailed Wilcoxon test. Human schematic in this figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License ( https://smart.servier.com ).

Article Snippet: WNT11 (200 ng/ml; 6179-WN-010, R&D Systems,) and WNT5B (500 ng/ml; 7347-WN-025, R&D Systems) were added in serum-free media for 24 h.

Techniques: Staining, Control, Activity Assay, In Vitro, In Vivo, Two Tailed Test

Journal: Journal of Biomedical Materials Research. Part a

Article Title: Three‐dimensional cultured tissue constructs that imitate human living tissue organization for analysis of tumor cell invasion

doi: 10.1002/jbm.a.36319

Figure Lengend Snippet: Migration assays for SAS‐Venus ( A ) and HSC3‐Venus ( B ) cells stimulated with Wnt5b (500 ng/mL, 48 h). C : Migration assay for SAS‐Venus cells relative to SAS‐LM8 cells. SAS‐Venus, HSC3‐Venus, and SAS‐LM8 cells stably overexpressing green fluorescent Venus protein. Wnt5b(±): recombinant human Wnt5b was added to the culture solution at a concentration of 500 ng/mL and cultured for 48 h at 37°C. The area occupied by the cells on the lower side of the filter was measured under a fluorescence microscope in 10 randomly selected fields at 200× magnification and quantified using the public domain software ImageJ. Each column indicates the mean ± SD of 3 separate experiments. * p < 0.05, ** p < 0.005.

Article Snippet: Cells were cultured with recombinant human Wnt5b (500ng/mL) (R&D Systems, Minneapolis, MN, USA) for 48 h at 37°C and then used for experiments.

Techniques: Migration, Stable Transfection, Recombinant, Concentration Assay, Cell Culture, Fluorescence, Microscopy, Software

Journal: Journal of Biomedical Materials Research. Part a

Article Title: Three‐dimensional cultured tissue constructs that imitate human living tissue organization for analysis of tumor cell invasion

doi: 10.1002/jbm.a.36319

Figure Lengend Snippet: Invasion assay using a Matrigel chamber for SAS‐Venus ( A ) and HSC3‐Venus ( B ) cells stimulated with Wnt5b. C : Invasion assay using a Matrigel chamber for SAS‐Venus cells relative to SAS‐LM8 cells. SAS‐Venus, HSC3‐Venus, and SAS‐LM8 cells stably overexpressing green fluorescent Venus protein. Wnt5b(±): recombinant human Wnt5b was added to the culture solution at a concentration of 500 ng/mL and cultured for 48 h at 37°C. The measurement method was the same as that in Figure . * p < 0.05, ** p < 0.005.

Article Snippet: Cells were cultured with recombinant human Wnt5b (500ng/mL) (R&D Systems, Minneapolis, MN, USA) for 48 h at 37°C and then used for experiments.

Techniques: Invasion Assay, Stable Transfection, Recombinant, Concentration Assay, Cell Culture

Journal: Journal of Biomedical Materials Research. Part a

Article Title: Three‐dimensional cultured tissue constructs that imitate human living tissue organization for analysis of tumor cell invasion

doi: 10.1002/jbm.a.36319

Figure Lengend Snippet: Invasion assay using 3‐D tissue constructs. The number of cells that reached the membrane at the bottom of the 3‐D tissue was counted in each view manually. SAS (A) and HSC‐3 ( B ) cells stimulated by Wnt5b. C : SAS‐LM8 cells relative to SAS‐Venus cells. Three randomly selected views were imaged for each of the three specimens. * p ≤ 0.05.

Article Snippet: Cells were cultured with recombinant human Wnt5b (500ng/mL) (R&D Systems, Minneapolis, MN, USA) for 48 h at 37°C and then used for experiments.

Techniques: Invasion Assay, Construct, Membrane