Journal: European Journal of Oral Sciences

Article Title: Higher expression of WNT 5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

doi: 10.1111/eos.12352

Figure Lengend Snippet: Expression of WNT 5A in different tissue regions. (A and B) Nuclear expression of WNT5A in oral mucosa with normal appearance. (C and D) Cytoplasmic and nuclear expression of WNT5A in severe‐grade dysplasia (black arrows). (E and F) Cancer islands; black arrows indicate expression of WNT5A in the cytoplasm in the periphery of cancer islands and red arrows indicate the absence of expression of WNT5A in the central part of the cancer islands. Scale bar = 50 μ m.

Article Snippet: The cells were cultured in six‐well plates until 80% confluence was reached, rinsed with PBS, incubated with serum‐free medium overnight, rinsed again with PBS, and treated either with 0.1% BSA in PBS as the control or with 0.4 μ g/ml of recombinant WNT5A (rWNT5A; 645‐WN; R&D Systems, Minneapolis, MN, USA) in serum‐free medium.

Techniques: Expressing

Journal: European Journal of Oral Sciences

Article Title: Higher expression of WNT 5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

doi: 10.1111/eos.12352

Figure Lengend Snippet: Effect of recombinant WNT 5A ( rWNT 5A) on expression of β ‐catenin and E‐cadherin in SCC 9 and SCC 25. (A, E) Representative western blots of expression of active β ‐catenin, total β ‐catenin, and E‐cadherin after stimulation with rWNT 5A. (B, F) Quantification of relative E‐cadherin; (C, G) quantification of active β ‐catenin; (D, H) quantification of total β ‐catenin. All quantifications were performed on four separate experiments.

Article Snippet: The cells were cultured in six‐well plates until 80% confluence was reached, rinsed with PBS, incubated with serum‐free medium overnight, rinsed again with PBS, and treated either with 0.1% BSA in PBS as the control or with 0.4 μ g/ml of recombinant WNT5A (rWNT5A; 645‐WN; R&D Systems, Minneapolis, MN, USA) in serum‐free medium.

Techniques: Recombinant, Expressing, Western Blot

Journal: European Journal of Oral Sciences

Article Title: Higher expression of WNT 5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

doi: 10.1111/eos.12352

Figure Lengend Snippet: Expression of WNT5A, β ‐catenin, and E‐cadherin in oral mucosa with a normal appearance, dysplasia, and at the invasive front of oral squamous cell carcinoma (OSCC)

Article Snippet: The cells were cultured in six‐well plates until 80% confluence was reached, rinsed with PBS, incubated with serum‐free medium overnight, rinsed again with PBS, and treated either with 0.1% BSA in PBS as the control or with 0.4 μ g/ml of recombinant WNT5A (rWNT5A; 645‐WN; R&D Systems, Minneapolis, MN, USA) in serum‐free medium.

Techniques: Expressing, Staining

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

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: Nature communications

Article Title: Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

doi: 10.1038/s41467-022-35487-9

Figure Lengend Snippet: Fig. 4 | Ptk7 acts through noncanonical Wnt signaling. a Schematic repre- sentation of canonical and noncanonical Wnt signaling pathways. While canonical and noncanonical Wnt signaling pathways share components such as FZD and Dvl, other components such as LRP5/6 and β-Catenin are specific for canonical Wnt signaling. The figure was created with BioRender.com. b Representative images of organoids cultured in corresponding conditioned media with DMSO or inhibitors. Scale bar, 200 μm. c Quantification of data from (b). The percentage of cystic organoids in organoids cultured in conditioned media is shown. d Representative images of organoids treated with DMSO (carrier), Wnt3a or Wnt5a. Scale bar, 200 μm. e Quantification of data from (d). The percentage of cystic organoids in organoids cultured in conditioned media is shown. f Representative images of organoids cultured in Wnt5a with DMSO or inhibitors. Scale bar, 200 μm. g Quantification of data from (f). mean ± s.d. percentage of cystic organoids in organoids cultured in conditioned media. h Representative images of organoids

Article Snippet: Proteins used in ELISAs were obtained from R&D Systems: PTK7-Fc (R&D Systems, #9799-TK), biotinylated Wnt3a (R&D Systems, #BT1324), biotinylated Wnt5a (R&D Systems, #BT645).

Techniques: Protein-Protein interactions, Cell Culture

Journal: Nature communications

Article Title: Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

doi: 10.1038/s41467-022-35487-9

Figure Lengend Snippet: Fig. 5 | Ptk7 modulates cytosolic Ca2+ oscillations in intestinal organoids. a Lgr5- CreERT2-mediated expression of GCaMP6f in this experiment. Cre removes stop cassettes upon addition of 4-OHT, allowing expression of tTA, GCaMP6f, and tdTomato. b Time lapse images showing Ca2+ flashes in the crypt domain of a mouse intestinal organoid. GCaMP6f and tdTomato expression is shown in green and red, respectively. Scale bar, 30 μm. c Representative frames from live imaging of an organoid expressing GCaMP6f before and after Wnt5a treatment. n = 5 d GCaMP3f mean intensity traces of 10-minute live imaging matching the frames shown in (c). Each trace represents one cell. Boxplots showing baselined area under curve of live imaging before and after Wnt5a (e), QCM (f), SCM (g), or SCM and anti- Ptk7 antibody (h) treatment. The graphs show results from a representative experiment. n = 5 in (e), n = 3 in (f), n = 6 in (g), and n = 6 in (h). In (h), organoids were imaged every 30 min. 90 min after the addition of the antibodies, SCM was

Article Snippet: Proteins used in ELISAs were obtained from R&D Systems: PTK7-Fc (R&D Systems, #9799-TK), biotinylated Wnt3a (R&D Systems, #BT1324), biotinylated Wnt5a (R&D Systems, #BT645).

Techniques: Expressing, Imaging