Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Protein expression of BMDMs pretreated with DMF (5 μM) overnight or not (−), followed by VSV infection, was determined by Western blot. b , Protein expression in BMDMs pretreated overnight with the indicated metabolites followed by VSV infection or not (−) was determined by Western blot. c , HEK293 cells transfected with GST-MAVS plasmids were treated with MMF (20 μM), DMF (10 μM or 20 μM), fumarate (200 μM) respectively or not (−) for 6 h. MAVS succination was analyzed by immunoblotting (left), and densitometry quantified the succinated-to-total MAVS ratio (right). d , Schematic of RIG-I-like Receptor (RLR) signaling. e , MAVS-deficient iBMDMs transduced with human MAVS were transfected with siRNAs targeting RIG-I and MDA5 or vector control (siCtrl), followed by VSV infection and DMF (5 μM) treatment for 6 h. Whole cell lysates and anti-MAVS immunoprecipitants were analyzed by immunoblotting. f , MAVS-deficient iBMDMs transduced with human MAVS were transfected with both siRNAs targeting RIG-I and MDA5 or vector control (siCtrl), followed by VSV infection or not (−) for 6 h. Whole cell lysates were analyzed by immunoblotting. g , MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS (2CS) were transfected with siRNAs targeting RIG-I and MDA5, or vector control (siCtrl, -), followed by VSV infection for 6 h. IFN-β expression(left) and protein levels were measured (n = 3). h , Purified Flag-MAVS protein from HEK293 cells was used in in vitro assays to assess succination and aggregation of MAVS in the presence or absence of fumarate (1 mM) for 6 h. i , Purified Flag-MAVS and MAVS (2CS) proteins from HEK293 cells were used in in vitro assays to assess succination and aggregation of MAVS. Statistical analysis: two-tailed Student’s t -test ( c right) or two-way ANOVA ( g ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Western Blot, Transfection, Transduction, Plasmid Preparation, Control, Mutagenesis, Purification, In Vitro, Two Tailed Test

Journal: Bioactive materials

Article Title: Marine-inspired molecular mimicry generates a drug-free, but immunogenic hydrogel adhesive protecting surgical anastomosis.

doi: 10.1016/j.bioactmat.2020.09.010

Figure Lengend Snippet: Fig. 3. CD206 mediates the recognition of Da-g-Xan and M2 macrophage polarization. (A) The fifteen most significantly changed genes of carbohydrate binding molecules in the GO analysis. (B) The computer simulation by Autodock Vina reveals the moderate affinity between different Da-g-Xan monomers and CD206. (C) The western blotting assay suggests that Da-g-Xan and its reactants, regardless of dopamine and xanthan gum, can improve the protein expression of CD206. (D) The immunofluorescent staining shows the co-localization of Da-g-Xan and CD206 in the cell membrane and cytoplasm of raw264.7 macrophages. (E) The immune dot- blotting assay suggests the specific binding of Da-g-Xan by CD 206. (F) Knockdown of CD206 by shRNA hinders the M2 macrophage polarization induced by Da-g- Xan; n = 3 for each replicate, ***p < 0.001. (G) Activation of ERK signaling is related to the M2 macrophage polarization induced by Da-g-Xan. Knockdown of CD206 slightly reduces the activation of ERK signaling and affects the macrophage polarization.

Article Snippet: One microliter of 1 μg/μL CD206 recombinant proteins (R&D systems, Q2HZ94) was spotted on pre-equilibrated polyvinylidene fluoride (PVDF) membranes, and PBS without CD206 was used as a control.

Techniques: Binding Assay, Western Blot, Expressing, Staining, Membrane, Knockdown, shRNA, Activation 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