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

Journal: Disease models & mechanisms

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway.

doi: 10.1242/dmm.019083

Figure Lengend Snippet: Fig. 5. The receptor tyrosine kinase-like orphan receptor ROR2 colocalises and interacts with TMEM67, and is dependent on this interaction for phosphorylation. (A) Four-colour IF imaging showing that endogenous ROR2 (green) colocalizes with TMEM67 (blue) and RPGRIP1L (red) at the ciliary transition zone. Arrowheads indicate regions shown in magnified insets. DAPI is pseudocoloured in grey. Scale bar: 10 μm. (B) Anti-HA co-immunoprecipitations (IPs) demonstrating interaction between full-length exogenous HA-tagged TMEM67 (size 115 kDa) and FLAG-tagged ROR2 (size 105 kDa). Input whole-cell extracts (WCE) for the indicated transfected constructs are on the left. IP of an irrelevant protein (HA-tagged MCPH1) was a negative control. Results are shown for immunoblotting (IB) for anti-FLAG (upper panel) and anti-TMEM67 (lower panel). * indicates a non-specific band in IPs; see supplementary material Fig. S6 for full unprocessed images. (C) Upper panel: IPs demonstrating interaction between FLAG-tagged ROR2 and endogenous TMEM67. Input WCE is shown on the left, and negative control IPs include a no antibody (Ab) control and goat (Gt) and rabbit (Rb) irrelevant (irr.) polyclonal antibodies (PAb). Immunoblotting (IB) for anti-FLAG shows pulldown of FLAG-ROR2 by Gt anti-ROR2 and Rb anti-TMEM67. Lower panel: IPs with irrelevant protein (FLAG-MCPH1, size 93 kDa). (E) Loss of the active phosphorylated ROR2 isoform (labelled P) in mutant Tmem67−/−cells following Wnt5a treatment, compared with strong induction of the active isoform (upper band, as indicated) in wild-type Tmem67+/+ cells. Loading control is for β-actin.

Article Snippet: Protein expression and in vitro binding assay Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer.

Techniques: Phospho-proteomics, Imaging, Transfection, Construct, Negative Control, Western Blot, Control, Mutagenesis

Journal: Disease models & mechanisms

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway.

doi: 10.1242/dmm.019083

Figure Lengend Snippet: Fig. 6. Loss of Wnt5a-induced branching morphogenesis during Tmem67−/−embryonic lung ex vivo organogenesis. (A) Embryonic (E12.5) lungs were explanted and treated for 0, 6 and 24 h with either control-conditioned medium or medium containing Wnt5a. Magnified insets (black frames) under high power are shown for 24-h treatments. Epithelial branching is significantly induced by Wnt5a in Tmem67+/+ lungs, but this response is absent in Tmem67−/−lungs. The bar graph shows quantification of the total number of branches in one lung for each genotype. Values shown are means of three independent replicates and error bars indicate ±s.e.m. The statistical significance of the pair-wise comparisons are represented as *P<0.05 and n.s. for non-significant, Student’s two-tailed t-test. (B) H&E staining of ex-vivo-cultured embryonic lung sections, showing normal acini (ac) and mesenchymal tissue (ms, in green) for wild-type Tmem67+/+ lung, and the stimulation of normal epithelial branching by Wnt5a (green asterisk and arrowheads). In contrast, Tmem67−/−lungs have abnormal mesenchymal cell condensates (red arrowheads), suggesting defective epithelial-mesenchymal induction. The red asterisks indicate abnormal bronchiolar formation; cl indicates the direction of the central lung. (C) Rho activation pull-down assays of whole-cell extracts from wild-type Tmem67+/+ and mutant Tmem67−/−embryonic (E15.5) lungs. Total RhoA in input material is shown as the loading control, with the ratio indicating active:total RhoA levels. A positive control for the assay (+GTPγS; loading with non-hydrolyzable GTPγS) and a negative control (+GDP; loading with GDP) are also shown. (D) Quantitative real-time PCR assays of transcript expression levels in wild-type Tmem67+/+ and mutant Tmem67−/−embryonic (E15.5) lungs for Shh, downstream effectors of the Shh signalling pathway (Gli1 and Ptch1) and a downstream effector of the canonical Wnt signalling pathway (Axin2). Levels of transcripts were all significantly increased in Tmem67−/−embryonic lungs, with the indicated pair-wise comparisons represented as **P<0.01, Student’s two-tailed t-test for n=3 independent assays. Error bars indicate ±s.e.m.

Article Snippet: Protein expression and in vitro binding assay Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer.

Techniques: Ex Vivo, Control, Two Tailed Test, Staining, Cell Culture, Activation Assay, Mutagenesis, Positive Control, Negative Control, Real-time Polymerase Chain Reaction, Expressing

Journal: Disease models & mechanisms

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway.

doi: 10.1242/dmm.019083

Figure Lengend Snippet: Fig. 7. Rescue of normal embryonic lung-branching morphogenesis and polarity in mutant Tmem67−/−tissue by ex vivo treatment with the RhoA activator calpeptin. (A) Embryonic lungs (age E11.5) grown in culture for the indicated times after treatment with either vehicle control (0.1% DMSO) or calpeptin at final concentration 1 unit/ml for 3 h. Tmem67−/−lungs had abnormally dilated branches (arrowheads) surrounded by areas of condensed mesenchyme, in contrast to the fine distal branches visible in Tmem67+/+ lungs. Calpeptin treatment of mutant Tmem67−/−lungs resulted in more developed branch development and a general morphology that was similar to the wild-type lungs. Magnified insets are indicated by the black frames and shown on the right. (B) The bar graph shows the quantification of the total number of terminal branches per lung (total n=3) for each genotype and treatment condition. The statistical significance of the indicated pair-wise comparisons is *P<0.05 and **P<0.01, Student’s two-tailed t-test. Error bars indicate ±s.e.m. (C) The polarity of mitotic cell division is rescued by treatment with calpeptin from predominantly parallel (para.) in mutant alveoli to predominantly perpendicular (perp.) divisions, as observed in wild-type epithelia. The statistical significance of the indicated pair-wise comparisons is ***P<0.001, chi-squared test, with the total number of cells counted in ten fields of view indicated above each bar. Representative examples of mitotic divisions, visualised by γ-tubulin (green) and indicated by the fine dotted lines, are shown on the right. Apical surfaces are highlighted by the broad dotted lines, with asterisks indicating the alveolar lumen. Scale bar: 20 μm. (D) Schematic in which signalling through the Wnt5a-TMEM67-ROR2 axis normally represses Shh and canonical Wnt (Wnt3a) signalling to moderate levels (small green arrow) between embryonic ages E9.5 and E11.5. Loss or mutation of any component in this axis (red cross) causes loss of repression (dashed line) with Shh and canonical Wnt pathway de-regulation and ectopic expression of Shh at later gestation ages (large red arrow). This contributes to pulmonary hypoplasia with condensed mesenchyme and impaired development of the alveolar system in the ciliopathy disease state.

Article Snippet: Protein expression and in vitro binding assay Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer.

Techniques: Mutagenesis, Ex Vivo, Control, Concentration Assay, Two Tailed Test, Expressing

Journal: Oncotarget

Article Title: Targeting the ROR1 and ROR2 receptors in epithelial ovarian cancer inhibits cell migration and invasion.

doi: 10.18632/oncotarget.5643

Figure Lengend Snippet: Figure 6: Knockdown of WNT5A in serous ovarian cancer decreases migration and invasion. A. WNT5A is decreased at the mRNA level following siRNA (A) induced knockdown in serous ovarian cancer (OVCAR3) cells. No effect on ROR1 or ROR2 mRNA level. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, RPL13A). Results represent an average of three experiments. Error bars represent the s.d of the mean. **P < 0.01. B. Cell proliferation decreases following WNT5A knockdown in OVCAR3 cells over a 48–72 hour period, however did not come to significance (P = 0.076). Results represent the average of three independent experiments. Error bars represent the s.d of the mean. C. Relative cell migration performed using the transwell migration assay is significantly decreased following WNT5A knockdown in OVCAR3 cells. Results represent an average of three experiments. Error bars represent the s.d of the mean. **P < 0.01. D. Relative cell invasion performed using the matrigel pre coated transwell assay is significantly decreased following WNT5A knockdown in OVCAR3 cells. Results represent the average of three experiments. Error bars represent the s.d of the mean. **P < 0.01. E. Representative picture of OVCAR3 cells invading matrigel over 48 hours. F. Luciferase assay determined no change in β-catenin dependent signalling after WNT5A knockdown in OVCAR3. Relative β-catenin driven transcription activity was calculated as a TOP/FOP ratio in triplicate wells. Results represent an average of three experiments. Error bars represent the s.d of the mean.

Article Snippet: Triplicate wells were then stimulated with 40 ng/ul of Wnt3a (#5036-WN-010/CF R&D Systems, Minneapolis, USA) and Wnt5a (#645-WN-010, R&D Systems, Minneapolis, USA) alongside an un-stimulated control over night.

Techniques: Knockdown, Migration, Quantitative RT-PCR, Transwell Migration Assay, Transwell Assay, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Targeting the ROR1 and ROR2 receptors in epithelial ovarian cancer inhibits cell migration and invasion.

doi: 10.18632/oncotarget.5643

Figure Lengend Snippet: Figure 7: Simultaneous knockdown of WNT5A and ROR2 in serous ovarian cancer decreases proliferation, migration and invasion. A. WNT5A and ROR2 are decreased at the mRNA level following siRNA (A) induced knockdown in serous ovarian cancer (OVCAR3) cells. No effect on ROR1 mRNA level. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, RPL13A). Results represent an average of three experiments. Error bars represent the s.d of the mean. *P < 0.05, **P < 0.01. B. Cell proliferation decreases following WNT5A and ROR2 knockdown in OVCAR3 cells after 72 hours. Results represent the average of three independent experiments. Error bars represent the s.d of the mean. *P < 0.05. C. Relative cell migration performed using the transwell migration assay is significantly decreased following WNT5A and ROR2 knockdown in OVCAR3 cells. Results represent an average of three experiments. Error bars represent the s.d of the mean. *P < 0.05. D. Relative cell invasion performed using the matrigel pre coated transwell assay is significantly decreased following WNT5A and ROR2 knockdown in OVCAR3 cells. Results represent the average of three experiments. Error bars represent the s.d of the mean. ***P < 0.001. E. Representative picture of OVCAR3 cells invading matrigel over 48 hours. F. Luciferase assay determined a slight non-significant increase in WNT3A stimulated β-catenin dependent signalling after WNT5A and ROR2 knockdown in OVCAR3. Relative β-catenin driven transcription activity was calculated as a TOP/FOP ratio in triplicate wells. Results represent an average of three experiments. Error bars represent the s.d of the mean.

Article Snippet: Triplicate wells were then stimulated with 40 ng/ul of Wnt3a (#5036-WN-010/CF R&D Systems, Minneapolis, USA) and Wnt5a (#645-WN-010, R&D Systems, Minneapolis, USA) alongside an un-stimulated control over night.

Techniques: Knockdown, Migration, Quantitative RT-PCR, Transwell Migration Assay, Transwell Assay, Luciferase, Activity Assay