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

Journal: Bioactive Materials

Article Title: Microfluidic chip-integrated vascularized endometrial complexes: Mitochondrial function and paracrine crosstalk enhance regenerative potential

doi: 10.1016/j.bioactmat.2025.08.035

Figure Lengend Snippet: Single-cell RNA sequencing (scRNA-seq) analysis of the dynamically cultured EO and HEO complexes. a. UMAP visualization of 12 samples across four culture conditions: HEOe (HEO + estrogen), HEOp (HEO + estrogen + progesterone + cAMP), EOe (EO + estrogen), EOp (EO + estrogen + progesterone + cAMP). Endometrial epithelial cell clusters were annotated as main Epi (mEpi), ribosome-related Epi (rEpi), proliferative Epi (pEpi), and ciliated Epi (cEpi). Endometrial stromal cell clusters were annotated as main Str (mStr), ribosome-related Str (rStr), proliferative Str (pStr), and a second proliferative Str cluster (pStr2); the endothelial cell clusters included main endothelial cells (mEndo) and ribosome-related endothelial cells (rEndo). b–c. Relative abundance of epithelial (b) and stromal (c) clusters within their respective compartments across groups.]d. Comparison of the percentage of proliferative epithelial cells (pEpi, left panel) and proliferative stromal cells (pStr, right panel) between the dynamically cultured EO and HEO complexes under estrogen culture.e. Validation of enhanced proliferation in HEO complexes by flow cytometry. Significantly higher proportions of EdU + cells in epithelial cells of HEO vs. EO complexes under estrogen culture.f. Ligand-receptor pairs exhibiting significant differences in communication probability between HEOe and EOe complexes. Pathways of biological interest include WNT7A, WNT5A , BMP6 , and LGALS9. Dot size represents the maximum communication probability across compared groups; dot color indicates statistical significance ( P < 0.05). Cluster abbreviations: Epi (endometrial epithelial cells), Str (endometrial stromal cells) and Endo (endothelial cells).g. Circle plot showing the BMP6, LGALS9, WNT7A, WNT5A signaling networks among different cell types in EOe and HEOe complexes.

Article Snippet: The spheroids were stimulated with 100 μL of ECM with different treatments, including WNT5A (200 ng/mL, CSB-EP026138HU, CUSABIO, Wuhan, China), WNT7A (100 ng/mL, P06680 , Solarbio), DKK1 (200 ng/mL, C12B, Novoprotein), BOX5 (HY-123071A, MCE), anti-WNT5A neutralizing antibody (2 μg/mL, MAB645, R&D), anti-WNT7A neutralizing antibody (10 μg/mL, sc-365665, Santa Cruz), or VEGFA (10 ng/mL, C744, Novoprotein) as a positive control, by adding dropwise onto the collagen matrix.

Techniques: RNA Sequencing, Cell Culture, Comparison, Biomarker Discovery, Flow Cytometry

Journal: Bioactive Materials

Article Title: Microfluidic chip-integrated vascularized endometrial complexes: Mitochondrial function and paracrine crosstalk enhance regenerative potential

doi: 10.1016/j.bioactmat.2025.08.035

Figure Lengend Snippet: Endometrial epithelial and stromal cells enhance the formation of vascular networks in HUVECs within the HEO complex through WNT7A and WNT5A signaling pathways. a. Representative images of sprouting assays using HUVEC spheroids treated as indicated. HUVEC spheroids cultured with basic ECM medium are shown as the blank group (Control). VEGFA added to the basic ECM medium served as the positive control. Scale bar: 400 μm. b. Quantification of sprouting number was determined by Image J software. ∗ P < 0.05 versus Control; # P < 0.05 versus WNT7A; $ P < 0.05 versus DKK1; ^ P < 0.05 versus WNT5A; & P < 0.05 versus BOX5; one-way ANOVA was conducted for statistical analysis. c. Representative images of the tube formation of HUVECs on Matrigel surface treated as indicated. HUVECs cultured with basic ECM medium are shown as the blank (Control). VEGFA added to the basic ECM medium served as the positive control. Scale bar: 400 μm. d. Quantification of branch number using Image J software. ∗ P < 0.05 versus Control; $ P < 0.05 versus DKK1; &P < 0.05 versus BOX5; one-way ANOVA was conducted for statistical analysis.e. Vascular networks of complex with HUVECs and ESCs (HE), complex with HUVECs and EEOs (HO), and HEO treated with WNT7A (or its inhibitor DKK1) and WNT5A (or its inhibitor BOX5) as indicated. Vascular networks are depicted in green. Scale bar: 100 μm.f. Quantification of the vessel-positive area within the formed vascular network across different groups. ∗P < 0.05, ∗∗P < 0.01, Student's t-test.g. The electrical resistance of HEO and HE with DKK1 and WNT7A treatments, respectively. ∗∗∗P < 0.001, two-way ANOVA.h. The electrical resistance of HEO and HO with BOX5 and WNT5A treatments, respectively. ∗∗∗P < 0.001, two-way ANOVA.

Article Snippet: The spheroids were stimulated with 100 μL of ECM with different treatments, including WNT5A (200 ng/mL, CSB-EP026138HU, CUSABIO, Wuhan, China), WNT7A (100 ng/mL, P06680 , Solarbio), DKK1 (200 ng/mL, C12B, Novoprotein), BOX5 (HY-123071A, MCE), anti-WNT5A neutralizing antibody (2 μg/mL, MAB645, R&D), anti-WNT7A neutralizing antibody (10 μg/mL, sc-365665, Santa Cruz), or VEGFA (10 ng/mL, C744, Novoprotein) as a positive control, by adding dropwise onto the collagen matrix.

Techniques: Protein-Protein interactions, Cell Culture, Control, Positive Control, Software

Journal: BMC Cancer

Article Title: ATBF1 is a potential diagnostic marker of histological grade and functions via WNT5A in breast cancer

doi: 10.1186/s12885-022-10380-2

Figure Lengend Snippet: Primer sequences for real-time qPCR amplification

Article Snippet: The reagents were purchased from their respective vendors: expose mouse- and rabbit-specific HRP/DAB detection IHC kit (Cat. ab236466, Abcam, Shanghai, China); RNA-Solv Reagent (Cat. R6830, Omega, Guangzhou, China); TRIzolTM (Cat. 15,596,026, Invitrogen, Shanghai, China); BCA Protein Assay Kit (Cat. CW0014S, CWBIO, Jiangsu, China); secondary antibodies (BOSTER, Wuhan, China); Lipofectamine RNAiMAX reagent (Cat. 13,778,100, Invitrogen); recombinant human WNT5A (Cat. CSB-EP026138HU1, CUSABIO, Wuhan, China); cell counting kit (APE×Bio, Shanghai, China); WNT5A primary antibody (Cat. A19133, Abclonal, Wuhan, China); and β-actin primary antibody (Cat. AC026, Abclonal).

Techniques:

Journal: BMC Cancer

Article Title: ATBF1 is a potential diagnostic marker of histological grade and functions via WNT5A in breast cancer

doi: 10.1186/s12885-022-10380-2

Figure Lengend Snippet: WTN5A was a crucial downstream gene of ATBF1. A Venn diagram of genes involved in the top 3 enriched GO (cell communication, extracellular matrix and system development) and the top 1 enriched KEGG (pathways in cancer). Five common genes (WNT5A, WNT6, WNT11, WNT5B and LAMA1) were detected. B The five common genes were listed by the fold change. The color represents the P value. C The expression of WNT5A in MCF7 cells transfected with siControl or siATBF1 was examined by real-time qPCR and western blot. Down-regulation of WNT5A was observed after ATBF1 silencing. Triplicate experiments were performed and P -value between two groups was calculated by student’s t test. D The mRNA expression of WNT5A in paired breast cancer cases, as determined by real-time qPCR. E The protein levels of WNT5A in paired breast cancer cases, quantified by WI index. Paired t test was used to calculate P -value in Fig. 8D&E. F Representative IHC images of WNT5A in paired breast cancer cases. G The correlation between the expression of WNT5A and ATBF1 at protein level, quantified by WI index. H Cell proliferation assay. MCF7 cells were transfected with siControl or siATBF at the concentration of 150 nM. Twenty four hours later, the cells were treated with 200ng/mL recombinant human WNT5A to rescue the down-regulation of WNT5A induced by ATBF1 knockdown. The cell number was determined as OD450 after 24 h of WNT5A treatment with a cell counting kit. One-way ANOVA with Bonferroni was used to determine the statistical differences among the three groups

Article Snippet: The reagents were purchased from their respective vendors: expose mouse- and rabbit-specific HRP/DAB detection IHC kit (Cat. ab236466, Abcam, Shanghai, China); RNA-Solv Reagent (Cat. R6830, Omega, Guangzhou, China); TRIzolTM (Cat. 15,596,026, Invitrogen, Shanghai, China); BCA Protein Assay Kit (Cat. CW0014S, CWBIO, Jiangsu, China); secondary antibodies (BOSTER, Wuhan, China); Lipofectamine RNAiMAX reagent (Cat. 13,778,100, Invitrogen); recombinant human WNT5A (Cat. CSB-EP026138HU1, CUSABIO, Wuhan, China); cell counting kit (APE×Bio, Shanghai, China); WNT5A primary antibody (Cat. A19133, Abclonal, Wuhan, China); and β-actin primary antibody (Cat. AC026, Abclonal).

Techniques: Expressing, Transfection, Western Blot, Proliferation Assay, Concentration Assay, Recombinant, Knockdown, Cell Counting