Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Expressing

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

Journal: Oncotarget

Article Title: Aberrant expression of homeobox gene SIX1 in Hodgkin lymphoma.

doi: 10.18632/oncotarget.5556

Figure Lengend Snippet: Figure 4: WNT-signalling activates SIX1 expression in HL. A. RQ-PCR analysis of L-428 and U-HO1 cells after treatment with FGF2, TGFb and WNT5B demonstrates activation of SIX1 expression by the WNT-pathway. B. RQ-PCR analysis of L-428 and U-HO1 cells after treatment with WNT-signalling inhibitor IWR confirms activation of SIX1 expression by WNT-signalling.

Article Snippet: Treatments of cell lines were performed for 20 h with 10 μg/ml Trichostatin A (TSA) (Sigma, Taufkirchen, Germany), 10 μM XMD8–92 (Biozol, Eching, Germany), 10 μM IWR1 (R&D Systems, Wiesbaden, Germany), 20 ng/ml recombinant human Bone Morphogenic Protein 4 (BMP4) protein, 20 ng/ml Fibroblast Growth Factor 2 (FGF2) protein, 20 ng/ml Transforming Growth Factor beta (TGFb), or 20 ng/ml WNT5B protein (R&D Systems).

Techniques: Expressing, Activation Assay