Journal: Materials Today Bio

Article Title: Inhalable engineered probiotic outer membrane vesicles co-expressing multiple mpox antigens induce potent specific systemic and mucosal immune responses

doi: 10.1016/j.mtbio.2026.103089

Figure Lengend Snippet: Design, Preparation, and Characterization of ABM@OMV. ( A ) Schematic illustrating the preparation of ABM@OMV. The pThioHisA plasmid, encoding Trx-A9R and ClyA-B6R-M1R fusion proteins, was transformed into Δ lpxM Escherichia coli Nissle 1917 (ΔEcN). OMVs were harvested via ultracentrifugation. ( B ) Bacterial lysates analyzed by SDS-PAGE and stained with Coomassie blue. Lanes: Marker; uninduced ΔEcN; ΔEcN + IPTG; uninduced BMΔEcN; BMΔEcN + IPTG; uninduced ABMΔEcN; ABMΔEcN + IPTG. Black boxes indicate ClyA-B6R-M1R and Trx-A9R fusion proteins. ( C ) Western blot analysis of proteins of interest expressed in ABMΔECN bacteria. Anti-His tag antibody detected Trx-A9R; anti-Flag tag antibody detected ClyA-B6R-M1R. Lane order is the same as in B . ( D ) Dynamic light scattering (DLS) size distribution profiles of Δ lpxM OMVs, BM@OMV, and ABM@OMV. ( E ) Zeta potential measurements of Δ lpxM OMVs, BM@OMV, and ABM@OMV ( n = 3). ( F ) Transmission electron microscopy (TEM) images of Δ lpxM OMVs, BM@OMV, and ABM@OMV. Scale bar: 100 nm. ( G ) Western blot analysis of proteins of interest expressed in ABM@OMV. Lanes: Δ lpxM OMVs, BM@OMV, ABM@OMV. ( H ) Endotoxin levels in Δ lpxM EcN-derived OMV and wild-type EcN-derived OMV, as measured by Limulus amebocyte lysate (LAL) assay ( n = 3). ( I ) Representative western blot showing the stability of proteins of interest in Δ lpxM OMVs, BM@OMV, and ABM@OMV following storage at 4 °C (left) or −80 °C (right) for the indicated time periods for the indicated time periods. ( J, K ) Changes in particle size ( J ) and zeta potential ( K ) of OMVs stored at 4 °C and −80 °C ( n = 3). All data were analyzed with GraphPad Prism 8 and are presented as mean ± SD. For panel H , statistical significance between two groups was determined by an unpaired two-tailed t -test. ∗∗∗ P < 0.001.

Article Snippet: The membrane was blocked with 5% BSA (BSA0020, Biosharp, China), then incubated with anti-His tag antibody (1:10,000, HY-P809476X, Med ChemExpress, USA) and anti-FLAG antibody (1:10,000, HY- P80111 , Med ChemExpress, USA), respectively for A9R and B6R-M1R, followed by HRP-conjugated secondary antibodies.

Techniques: Plasmid Preparation, Transformation Assay, SDS Page, Staining, Marker, Western Blot, Bacteria, FLAG-tag, Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Derivative Assay, LAL Assay, Two Tailed Test

Journal: Tumour Virus Research

Article Title: Oncolytic virus hijacks GOT1 and pyrimidinosomes to fuel pyrimidine synthesis for replication in tumor cells

doi: 10.1016/j.tvr.2026.200342

Figure Lengend Snippet: | NDV infection promotes the assembly of pyrimidinosome involving GOT1 and pyrimidine synthases. (A) H1299 cells were co-transfected with GFP-GOT1 and mCherry-UMPS for 24 h, followed by mock infection or NDV infection (MOI = 0.1, 1, 5, 10, or UV-NDV for 12 hpi). Cells were then analyzed using confocal microscopy. (B) H1299 cells were co-transfected with GFP-GOT1 and mCherry-UMPS for 24 h, followed by mock infection or NDV infection (MOI = 1 for 6, 12, and 18 hpi). Cells were then analyzed using confocal microscopy. (C) Immunofluorescence analysis (IFA) of mock-infected and NDV-infected A549 cells (MOI = 1, 12 hpi), stained for endogenous GOT1, UMPS, and DHODH, alongside the mitochondrial marker protein Tom 20. (D-F) H1299 cells were co-transfected with GFP-GOT1 and mCherry-UMPS (D), GFP-DHODH and mCherry-UMPS (E), or GFP-GOT1 and mCherry-DHODH (F) for 24 h, followed by mock infection or NDV infection (MOI = 1, 12 hpi). Cells were then analyzed using confocal microscopy. (G-H) HEK293T cells were mock-infected or infected with NDV (MOI = 1) for 12 h. The cells were lysed and subjected to immunoprecipitation (IP) using GOT1 (G) and UMPS (H) antibodies, alongside with anti-IgG antibody as a control, followed by WB with anti-CAD, -UMPS, -DHODH and -GOT1 antibodies. (I-J) H1299 cells were co-transfected with Flag-CAD and HA-GOT1 for 24 h, followed by mock infection or NDV infection (MOI = 1, 12 hpi). The cells were lysed and subjected to IP using anti-Flag (I) or anti-HA (J) magnetic beads, followed by WB with anti-Flag and anti-HA antibodies. (K) H1299 cells were transfected with GFP-GOT1, mCherry-UMPS, or GFP-DHODH for 24 h, followed by NDV infection (MOI = 1, 12 hpi). FRAP analysis was then performed using confocal microscopy.

Article Snippet: Anti-Flag Magnetic Beads (Cat# HY-K0207), anti-HA Magnetic Beads (Cat# HY-K0201), Mycophenolate (Cat# HY-B0421), Leflunomide (Cat# HY-B0083), AG 2037 (Cat# HY-14530), Aminooxyacetic acid hemihydrochloride (Cat# HY-107994) were purchased from MedChemExpress (MCE).

Techniques: Infection, Transfection, Confocal Microscopy, Immunofluorescence, Staining, Marker, Immunoprecipitation, Control, Magnetic Beads

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 promotes m 3 C modification of Tcf7 mRNA and its genome-specific loops of Tox in CD8 + T cells. (A) Venn plot illustrates the overlap of downregulated genes from RNA-seq, m 3 C-seq, and Mettl8-binding genes from RIP-seq. (B) Mettl8 occupancy at the Tcf7 gene loci is revealed through m 3 C-seq (WT and Mettl8 −/− ) of EG7-OVA tumor-infiltrating OT-I cells and RIP-seq (Mettl8-tdTomato-Flag) of B16F10 tumor-infiltrating CD44 + CD8 + T cells. The binding peaks on Tcf7 loci are depicted. The m 3 C tracks are all plotted on a consistent scale. (C) The RNA decay assay demonstrates the remaining Tcf7 mRNA of CD8 + T cells from the spleens of WT and Mettl8 −/− mice detected by qRT-PCR, normalized to t = 0. (D) Heatmaps display changes in total Tcf1-targeting genes between WT and Mettl8 −/− EG7-OVA tumor-infiltrating OT-I cells and Mettl8-targeting genes in B16F10 tumor-infiltrating CD44 + CD8 + T cells of Mettl8-tdTomato-Flag mice as detected by CUT&Tag. (E) Diamond graphs exhibit chromatin interactions in WT and Mettl8 −/− tumor-infiltrating OT-I cells at the Tox gene loci (top), with CUT&Tag and ATAC-seq tracks, and gene structures on the bottom. An enlarged view highlights the signal profiles across the Tox gene region. (F) co-IP of Tcf1 by anti-Flag magnetic beads in CD3 + T cells from the spleens of Mettl8-tdTomato-Flag (RPT) and WT mice. IB, immunoblot. (G) co-IP of Tcf1 by Flag-tagged Mettl8 protein with anti-Flag magnetic beads after co-transfection into HEK293T cells. (H) Single-cell transcription levels of representative genes illustrated in the UMAP plot. Transcription levels are color coded: gray, not expressed; blue, expressed. (I) Schematic diagram of the tumor model: Mettl8 fl/fl Cd4 cre mice were subcutaneously injected with 2 × 10 5 B16F10 cells and harvested after 13 days. (J) Representative flow cytometry plots and cumulative data show the frequency of Tcf1 + Tox + cells gated on tumor-infiltrating CD8 + CD44 + T cells (right). n = 6 per group. (K) Schematic diagram of the OT-I–transferred tumor model: CD45.1 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 2 × 10 6 WT or Mettl8 −/− OT-I cells transfer at 9 dpi. Mice were harvested at 21 dpi. Representative flow cytometry plots and cumulative data show the frequency of Tox + cells gated on Tcf1 + OT-I cells. n = 6 per group. (L) The MFI of Tox gated on Tcf1 + OT-I cells of the mice in K. n = 6 per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test; *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available for this figure: .

Article Snippet: In briefly, cells were sorted enriched by ConA-magnetic beads and resuspended in wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCI, 0.5 mM spermidine; 1× protease inhibitor cocktail; 0.05% digitonin) and then incubated overnight with anti-Tcf1 (1:50, C63D9, cat. no. 2203; Cell Signaling Technology), anti-H3K27ac (1:50, cat. no. ab4729; Abcam), or anti-Flag (1:50, D6W5B, cat. no. 14793; Cell Signaling Technology).

Techniques: Modification, RNA Sequencing, Binding Assay, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Cotransfection, Single Cell, Injection, Flow Cytometry, Two Tailed Test

Journal: Genes & Diseases

Article Title: Blockage of SUMO E1 enzyme inhibits ocular lens fibrosis by mediating SMAD4 SUMOylation

doi: 10.1016/j.gendis.2025.101827

Figure Lengend Snippet: ML792 disrupts SMAD4 SUMOylation-dependent nuclear translocation in TGFβ 2 -stimulated lens epithelial cells (LECs). (A – F) FHL124 LECs were treated with or without TGFβ2 (10 ng/mL, 2 h). Triple immunofluorescence staining of SMAD4 (green), SUMO1 (red)/SUMO2/3 (red), and DAPI (nuclei, blue) shows spatiotemporal dynamics of SMAD4-SUMO colocalization. (A, D) SMAD4-SUMO1/SUMO2/3 immunofluorescence staining and colocalization scatterplot. (B, E) Pearson's r analysis of colocalization performed by Image J. n = 9 replicates per group. (C, F) Quantification of nuclear SMAD4 intensity. n = 30 cells in (C) and n = 44 cells in (F). Unpaired Student's t -test; ∗ P < 0.05 and ∗∗∗ P < 0.001. (G, H) Flag-SMAD4 immunoprecipitation in engineered FHL124 LECs overexpressing Flag-SMAD4. Treatments were 0.1% DMSO, TGFβ 2 (10 ng/mL), ML792 (10 μM), or their combination for 2 h. (G, H) Whole-cell lysates were blotted with anti-Flag and anti-SMAD4 (INPUT). Cell lysates were immunoprecipitated with anti-Flag, followed by SUMO1 immunoblotting (G) and SUMO2/3 immunoblotting (H). (I, J) Subcellular fractionation analysis. (I) Immunoblots of cytoplasmic/nuclear SMAD4 after 8 h treatments in FHL12.4 LECs. (J) Quantification was normalized to GAPDH (cytoplasm) and lamin A/C (nucleus). One-way ANOVA with Bonferroni correction; ns, not significant; ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (K, L) SMAD4 nuclear translocation analysis. (K) Triple immunofluorescence staining SMAD4 (red), F-actin (Phalloidin, green), and DAPI (nuclei, blue) in LECs treated as indicated in (I). Scar bar: 20 μm. (L) Nuclear SMAD4 fluorescence intensity quantification. n = 30 cells per group. One-way ANOVA with Bonferroni post-hoc test; ∗ P < 0.05 and ∗∗∗ P < 0.001.

Article Snippet: Cells were lysed in 0.5% NP-40 buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 10% glycerol) containing protease inhibitors (#P2714, Sigma–Aldrich, Missouri, USA) on ice for 5 min. Lysate (2 mg) was precleared with control IgG (#2729, #53484, CST) at 4 °C for 2 h. Immunoprecipitation was performed at 4 °C overnight using anti-Flag antibody/Nano-Agarose beads (#FNM-25-500, NuoyiBio, Tianjin, China), anti-SUMO1 or anti-HA antibody with protein A/G Magnetic beads (#HY-K0202, MedChemExpress, New Jersey, USA).

Techniques: Translocation Assay, Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Fractionation, Fluorescence

Journal: Genes & Diseases

Article Title: Blockage of SUMO E1 enzyme inhibits ocular lens fibrosis by mediating SMAD4 SUMOylation

doi: 10.1016/j.gendis.2025.101827

Figure Lengend Snippet: SUMOylation site mutagenesis abolishes SMAD4-mediated epithelial–mesenchymal transition (EMT) in TGFβ 2 -stimulated lens epithelial cells (LECs). (A) Sanger sequencing validation of SMAD4 mutants. WT, wild-type; K113R, Lys113→Arg; K159R, Lys159→Arg. The black frames indicate WT and mutated codons. (B, C) SUMOylation capacity analysis in SMAD4 mutants. (B) FHL124 LECs stably overexpressed empty vector and flag-SMAD4 variants treated with TGFβ 2 (10 ng/mL, 2 h). Whole-cell lysates were immunoblotted with anti-Flag and anti-SMAD4. β-Tubulin served as the loading control. The cell lysates were immunoprecipitated with anti-Flag nano beads, followed by immunoblotting for SUMO1, SUMO2/3, and Flag antibody. (C) Quantification of SMAD4 expression (Input lysates). One-way ANOVA with Bonferroni correction; ns, not significant; ∗∗∗ P < 0.001. (D, E) SMAD4 nuclear translocation analysis. (D) Triple fluorescence imaging of Flag (SMAD4, red), F-actin (phalloidin, green), and DAPI (nuclei, blue) in engineered LECs treated with TGFβ 2 (10 ng/mL, 2 h). (E) Nuclear SMAD4 intensity quantification ( n = 15–18 cells/group). One-way ANOVA with Bonferroni post-hoc test; ∗∗∗ P < 0.001. (F, G) Functional consequence of double site mutant (K113 plus 159R) SMAD4 protein. (F) EMT marker immunoblotting 24 h after TGFβ 2 treatment in human LECs overexpressing empty vector, WT Flag-tagged SMAD4, or double site mutant Flag-tagged SMAD4. (G) Densitometric analysis from (F). β-Tubulin served as the loading control. One-way ANOVA followed by Bonferroni correction; ns, not significant; ∗ P < 0.05. ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Cells were lysed in 0.5% NP-40 buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 10% glycerol) containing protease inhibitors (#P2714, Sigma–Aldrich, Missouri, USA) on ice for 5 min. Lysate (2 mg) was precleared with control IgG (#2729, #53484, CST) at 4 °C for 2 h. Immunoprecipitation was performed at 4 °C overnight using anti-Flag antibody/Nano-Agarose beads (#FNM-25-500, NuoyiBio, Tianjin, China), anti-SUMO1 or anti-HA antibody with protein A/G Magnetic beads (#HY-K0202, MedChemExpress, New Jersey, USA).

Techniques: Mutagenesis, Sequencing, Biomarker Discovery, Stable Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Expressing, Translocation Assay, Fluorescence, Imaging, Functional Assay, Marker