recombinant human wnt5b rwnt5b 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: Figure 4. WNT5B facilitates melanoma cell escape into draining lymph nodes.

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

Techniques:

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: Figure 5. Notch3 regulates WNT5B expression in melanoma cells and its expression correlates

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

Techniques: Expressing

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: American journal of human genetics

Article Title: Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

doi: 10.1016/j.ajhg.2021.11.018

Figure Lengend Snippet: Figure 1. SNP rs2887571 correlates with WNT5B expression in primary human osteoblasts (A) Nucleotide sequence near SNP rs2887571 with predicted NFAT and ERa binding motifs. (B) Diagram representing the location between SNP rs2887571 and nearby genes in chromosome 12. (C–G) Expression of (C) ERC1, (D) LINC00942, (E) LOC107984507, (F) FBXL14, and (G) WNT5B were measured by qPCR and compared with its genotype for SNP rs2887571 in human osteoblasts (AA ¼ 52, AG ¼ 49, and GG ¼ 9). Bars indicate median with interquartile range. Mann-Whitney U-test: **p ¼ 0.0016.

Article Snippet: Recombinant mouse WNT5B (rWNT5B; Cat. # 3006-WN) was acquired from R&D Systems.

Techniques: Expressing, Sequencing, Binding Assay, MANN-WHITNEY

Journal: American journal of human genetics

Article Title: Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

doi: 10.1016/j.ajhg.2021.11.018

Figure Lengend Snippet: Figure 2. E2 suppresses WNT5B expres- sion via ERa (A–D) Human osteoblasts (hOBs) and oste- oblast-like U2OS-ERa cells were treated with EtOH or 10 nM E2 for 4 h then levels of (A, C) WNT5B mRNA expression were detected by qPCR. Immunoblotting anal- ysis of WNT5B protein expression after treatment with EtOH or 10 nM E2 for 24 h and normalized to b-actin (BA) and quantified by ImageJ in (B) hOBs and (D) U2OS-ERa cells. Bar ¼ mean with SEM. n ¼ 3, Student’s t test: *p<0.05. Three different biological replicates of hOBs and U2OS-ERa cells were tested and a represen- tative picture from one biological replicate is presented. (E) Mouse osteoblasts (mOBs) were differ- entiated for 3 and 7 days then treated with EtOH or 10 nM E2 for 24 h, and levels of Wnt5b mRNA expression were detected by qPCR. The level of WNT5B was detected by IHC in mice from sham-treated, ovariec- tomized (OVX), and OVX then treated with E2. (F) The number of WNT5B-positive osteo- blasts were counted and divided by the to- tal osteoblasts from three different mice in each group. Bar ¼ mean with SEM. n ¼ 3, Student’s t test: ****p < 0.0001, **p < 0.01, *p < 0.05. (G) The regions of interest (red square) are shown below at 403 magnification. Black scale bar ¼ 200 mM, red scale bar ¼ 20 mM, red arrow ¼ osteoblast, and asterisk ¼ osteoid.

Article Snippet: Recombinant mouse WNT5B (rWNT5B; Cat. # 3006-WN) was acquired from R&D Systems.

Techniques: Expressing, Western Blot

Journal: American journal of human genetics

Article Title: Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

doi: 10.1016/j.ajhg.2021.11.018

Figure Lengend Snippet: Figure 3. ERa and NFATc1 interact and bind at SNP rs2887571 (A–D) U2OS-ERa cells were treated with or without 10 nM E2 for 45 min, then ChIP was performed using an antibody to (A) ERa, (B) H3K27ac, (C) SIN3A, or (D) NFATc1. qPCR was performed with primers for SNP rs2887571 in comparison with b-actin (ACTB) promoter or hemoglobin (HBB) promoter. (E) U2OS-ERa cells were treated with 10 nM E2, then ChIP-reChIP was performed with antibodies to ERa, NFATc1 and/or IgG, and primers for SNP rs2887571. (F) Human genomic DNA around SNP rs2887571 was amplified and inserted into the pGL3 vector, then U2OS-ERa cells were transfected with pGL3-rs2887571, in the presence or absence of EGFPC1-huNFATc1EE-WT. Cells were treated with Dox. to induce ERa. 24 h later, cells were treated with EtOH or 10 nM E2 for an additional 24 h. Relative luciferase activity was measured using dual-luciferase reporter assays. (G) U2OS-ERa cells were transfected with pGL3-rs2887571 and EGFPC1-huNFATc1EE-WT, together with or without pCS2þMT-mSin3A, before being induced or uninduced to express ERa. Cells were treated with EtOH or 10 nM E2 and the relative luciferase activity was detected at 24 h. Luciferase activity was normalized with pRL-SV40 (Renilla). (H and I) U2OS-ERa cells were transfected with siRNA directed against SIN3A mRNA, before being treated with or without 10 nM E2. The mRNA levels of (H) SIN3A and (I) WNT5B were detected by qPCR. n ¼ 3, Student’s t test: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: Recombinant mouse WNT5B (rWNT5B; Cat. # 3006-WN) was acquired from R&D Systems.

Techniques: Comparison, Plasmid Preparation, Transfection, Luciferase, Activity Assay

Journal: American journal of human genetics

Article Title: Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

doi: 10.1016/j.ajhg.2021.11.018

Figure Lengend Snippet: Figure 4. SNP rs2887571 has allelic spec- ificities to alter WNT5B expression (A) U2OS-ERa cells with either homozy- gous AA and GG at SNP rs2887571 were lysed and immunoblotting was performed with an antibody to WNT5B. The level of protein was normalized with b-actin and quantified by ImageJ. Bar ¼ mean with SEM. n ¼ 3, Student’s t test: *p < 0.05. (B–D) Three different biological replicates of U2OS-ERa cells were tested and a repre- sentative picture from one biological repli- cate is presented. ChIP was performed us- ing an antibody to (B) ERa, (C) SIN3A and (D) NFATc1, and qPCR was done with primers for SNP rs2887571 in compar- ison with the ACTB promoter. n ¼ 3, Stu- dent’s t test: **p < 0.01, *p < 0.05.

Article Snippet: Recombinant mouse WNT5B (rWNT5B; Cat. # 3006-WN) was acquired from R&D Systems.

Techniques: Expressing, Western Blot

Journal: American journal of human genetics

Article Title: Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

doi: 10.1016/j.ajhg.2021.11.018

Figure Lengend Snippet: Figure 5. WNT5B suppresses osteoblast differentiation and promotes adipogenesis (A–C) mOBs were isolated from calvaria then treated with various doses of rWNT5B at day 0 (D0), day 7 after differentiation (D7) or day 14 after differentiation (D14) for 24 h. The levels of (A) Sp7, (B) Alpl, and (C) Bglap were detected by qPCR. (D) mOBs were treated with or without 50 ng/mL rWNT5B and stained for alkaline phosphatase (ALP) and Alizarin Red. (E) The levels of Alizarin Red in Ctrl and WNT5B groups were quantified at O.D. 570 nm and shown as % of the control. (F) mOBs were infected with lentivirus containing sh-Ctrl or sh-Wnt5b, before collecting mRNA or protein. The levels of WNT5B mRNA and protein were normalized with b-actin and quantified by ImageJ. (G and H) Cells were differentiated for 7 days in the presence or absence of 50 ng/mL rWNT5B, then (G) expression of Bglap was detected by qPCR and (H) alkaline phosphatase (ALP) activity was detected at O.D. 405 nm then was normalized with total protein. (I) Bone marrow-derived MSCs were treated with or without 50 ng/mL rWNT5B in osteogenic differentiation media for 12 days and stained with Alizarin Red. CFU-OB was reported as the number of colonies. (J) Bone marrow-derived MSCs were treated with or without 50 ng/mL rWNT5B in adipogenic differentiation medium for 14 days and stained with Oil Red O. (K) The levels of Oil Red O between Ctrl and WNT5B groups were quantified as % Area by ImageJ. (L and M) Expression of (L) Pparg and (M) Adipoq in MSC-derived adipocytes treated with or without rWNT5B for 24 h were detected by qPCR. Three different biological replicates were obtained and a representative image or graph from one replicate is presented. n ¼ 3, Student’s t test: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: Recombinant mouse WNT5B (rWNT5B; Cat. # 3006-WN) was acquired from R&D Systems.

Techniques: Isolation, Staining, Control, Infection, Expressing, Activity Assay, Derivative Assay

Journal: American journal of human genetics

Article Title: Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

doi: 10.1016/j.ajhg.2021.11.018

Figure Lengend Snippet: Figure 6. WNT5B activates WNT-PCP signaling pathway via ROR1/2 and inhibits b-catenin activity in osteoblasts (A) U2OS-ERa cells were transfected with Super8XTOPFlash (a luciferase reporter for b-catenin activity) together with empty vector (pcDNA3), WNT5B, WNT10B, or WNT5B with WNT10B vectors. After 48 h, relative luciferase activity was measured using dual-luciferase reporter assays. Firefly luciferase activity was normalized to pRL-SV40 (Renilla). (B) mOBs were differentiated for 7 days before treatment with or without 50 ng/mL rWNT5B for 30 min and the levels of active b-catenin (ABC) was detected by immunofluorescence. Scale bar ¼ 10 mM. Three different mOBs were tested in biological replicates and a repre- sentative image from one replicate is presented. (C) Differentiated mOBs at D7 were treated with 50 ng/mL rWNT5B and total cell lysate was collected at different time points before detecting the level of phospho-b-catenin (Ser552), ABC, total b-catenin, and b-actin by immunoblotting. (D and E) mOBs were differentiated for 7 days before treatment with or without 50 ng/mL rWNT5B for 30 min and localization of WNT5B with (D) ROR1 or (E) ROR2 were detected by immunofluorescence. Scale bar ¼ 10 mM. (F and G) mOBs were differentiated for 7 days and treated with 50 ng/mL rWNT5B, and protein was collected at 0, 15, 30, and 60 min. The levels of downstream effectors were detected with the indicated antibodies. (H–J) mOBs (H), MSCs (I), and MSC-derived adipocytes (J) were treated with or without 50 ng/mL rWNT5B for 24 h and qPCR was per- formed for Il6 mRNA. (K) mOBs were treated with 50 ng/mL rWNT5B for 1 h and then ChIP was performed using a specific antibody to phospho-c-Fos (Ser32) and qPCR using primers to mouse negative control binding site and the Il6 promoter. n ¼ 3, Student’s t test: ****p < 0.0001, **p < 0.01, *p < 0.05.

Article Snippet: Recombinant mouse WNT5B (rWNT5B; Cat. # 3006-WN) was acquired from R&D Systems.

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Western Blot, Derivative Assay, Negative Control, Binding Assay

Journal: American journal of human genetics

Article Title: Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

doi: 10.1016/j.ajhg.2021.11.018

Figure Lengend Snippet: Figure 7. WNT5B inhibits E2-induced ALPL expression via SIN3A (A and B) mOBs (A) or U2OS-ERa cells (B) were treated with EtOH or 10 nM E2 in the presence or absence of 50 ng/mL rWNT5B for 4 days. Then, alkaline phos- phatase (ALP) activity was detected and was normalized with total protein. (C) Expression of ALPL also was measured in U2OS-ERa cells. (D) Diagram represents ALPL enhancers in U2OS-ERa cells. U2OS-ERa cells were treated with PBS/EtOH (Ctrl), 10 nM E2, 50 ng/mL rWNT5B, or E2 with rWNT5B for 45 min. (E and F) ChIP was performed using an antibody to (E) ERa or (F) SIN3A. qPCR was performed with primers to the ALPL enhancers and normalized to the b-actin (ACTB) promoter. (G) Co-immunoprecipitations were per- formed with an antibody to SIN3A before blotting with an antibody to ERa or IgG in U2OS-ERa cells. (H) mOBs were treated with 50 ng/mL rWNT5B for 0 and 15 min, and then were incubated with or without calf in- testinal protease (CIP) in vitro before detect- ing phospho-CDK5 (Tyr15) and SIN3A by immunoblotting and normalized to b-actin (BA) by ImageJ. Three different bio- logical replicates were tested and a repre- sentative image from one replicate is pre- sented. n ¼ 3, Student’s t test: **p < 0.01, *p < 0.05.

Article Snippet: Recombinant mouse WNT5B (rWNT5B; Cat. # 3006-WN) was acquired from R&D Systems.

Techniques: Expressing, Activity Assay, Incubation, In Vitro, Western Blot

Journal: American journal of human genetics

Article Title: Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

doi: 10.1016/j.ajhg.2021.11.018

Figure Lengend Snippet: Figure 8. Model of the regulation of WNT5B expression at SNP rs2887571 and the signaling pathway of WNT5B in osteoblasts (A) The molecular mechanisms of ERa and NFATc1 at SNP rs2887571 in cells with homozygous AA (right) or homozygous GG (left) at rs2887571. (Top) Homozygous GG has increased ERa, NFATc1 and SIN3A compared to homozygous AA, which re- sults in lower endogenous WNT5B expression due to the repres- sive effects of SIN3A. (Bottom) The presence of E2 recruits SIN3A to suppress the expression of WNT5B in both genotypes, with increased suppression in cells with GG due to increased recruit- ment of SIN3A. (B) WNT5B binds with ROR1/2 then activates DVL2/3, RAC1/ CDC42, p-JNK, and p-c-Fos. Phosphorylation of SIN3A by CDK5 results in proteasome degradation. WNT5B inhibits phosphoryla- tion of CDK5 (Tyr15), resulting in accumulation of SIN3A. WNT5B also inhibits b-catenin activity in osteoblasts. Solid lines indicate the signaling pathways described in this study. The dashed line in- dicates the signaling that has been previously reported. P indicates phosphorylation. Created with BioRender.com

Article Snippet: Recombinant mouse WNT5B (rWNT5B; Cat. # 3006-WN) was acquired from R&D Systems.

Techniques: Expressing, Phospho-proteomics, Activity Assay, Protein-Protein interactions