Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: E0771 cells have reduced expression of the high-affinity reovirus attachment receptor JAM-A. E0771 and L929 cells were harvested using CellStripper, fixed with 4% paraformaldehyde (PFA), and analyzed by flow cytometry following immunostaining with monoclonal antibodies specific for SNA, JAM-A, or β1 integrin. Representative histograms (left) and corresponding quantification (right) show cell surface expression of ( A ) α2,6-linked sialic acids detected using fluorescently labeled SNA lectin in neuraminidase-treated (+) or untreated (−) cells, ( B ) JAM-A detected using murine JAM-A-specific antibodies (+) compared with isotype (Iso) controls, and ( C ) β1 integrin detected using β1 integrin-specific primary antibodies (+) compared with Iso controls. MFIs were normalized to the corresponding negative controls (neuraminidase-treated or isotype-stained cells) from a representative L929 experiment. Data represent mean ± SD ( n = 3). Statistical significance was determined by the two-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism v10.4 (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Expressing, Flow Cytometry, Immunostaining, Bioprocessing, Labeling, Staining

Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: Mutations in the sialic acid-binding domain permit high-affinity, receptor-independent attachment by increasing binding to sialic acids at physiological temperature (37°C). ( A ) Representative western blot analysis of outer capsid proteins µ1C and σ3, detected using anti-reovirus polyclonal serum. ( B and C ) L929 ( B ) and E0771 ( C ) cells were treated with PBS or neuraminidase (+neuraminidase) for 1 h at 37°C to deplete cell surface sialic acids. Viruses indicated in the legend were incubated with cells for 1 h at 4°C or 37°C in the presence of NH 4 Cl, washed, and cells processed for flow cytometric analysis using σ3-specific antibodies. MFI reflects the level of cell-associated virus particles. ( D ) Virus-cell association was measured as in panel C , without neuraminidase treatment, using parental U937 cells or U937 cells deficient in sialic acids (U937-Sia - ). ( E ) Flow cytometric detection of α2,6-linked sialic acids on RBCs using fluorescently labeled SNA lectin. Representative histograms (left) show unstained RBCs (light gray), SNA-stained RBCs (red), and SNA-stained H1299 cells (dark gray). n = 3. ( F ) Flow cytometric detection of JAM-A on RBCs using a primary/secondary antibody system specific for hJAM-A. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and E0771+JAM cells (dark gray). n = 3. ( G ) Flow cytometric detection of β1 integrins on RBCs using primary/secondary antibodies specific for human β1 integrin. Representative histograms (left) show RBCs with secondary antibody only (light gray), primary/secondary-stained RBCs (red), and H1299 cells (dark gray). n = 3. ( H ) RBCs were incubated with particle-normalized T3D PL at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C or 37°C. Unbound virions were removed by PBS washes prior to fixation and immunostaining for outer capsid proteins, followed by flow cytometric analysis. n = 3. ( I–K ) RBCs were incubated with particle-normalized T3D PL or variant viruses at serial dilutions starting at 1.9 × 10 5 particles for 1 h at 4°C ( I and J ) or 37°C ( I and K ). Following removal of unbound virions by PBS washes, cells were fixed, immunostained for outer capsid proteins, and analyzed by flow cytometry. Absolute MFI values ( I ) were used to calculate the AUC for each virus across all independent experiments, normalized to T3D PL at the corresponding temperature for each independent experiment ( n = 3–5). ( L ) Levels of σ1 per virion for full-length T3D PLσ1-G196R were assessed by agarose gel electrophoresis (top) and quantitative serial dilution-based western blot analysis using anti-σ3 and anti-µ1 monoclonal antibodies and anti-σ1 tail polyclonal antibodies (middle). Bottom: relative average σ1 per virion calculated relative to T3D PL from five independent virus preparations based on σ1 to (σ3 + µ1) protein ratios determined by western blot analysis. Data represent mean ± SD. Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test ( E, J, and K ) or the paired t -test ( F, G, and H ) in GraphPad Prism v10.4. (ns = P > 0.05; * P < 0.05; ** P < 0.005; *** P < 0.001; and **** P < 0.0001).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Binding Assay, Western Blot, Incubation, Virus, Labeling, Staining, Immunostaining, Variant Assay, Flow Cytometry, Agarose Gel Electrophoresis, Serial Dilution, Bioprocessing

Journal: Journal of Virology

Article Title: Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors

doi: 10.1128/jvi.00074-26

Figure Lengend Snippet: Mutational enhancement of σ1-mediated receptor binding and structural basis of sialic acid interaction. ( A ) Model depiction of relative binding strengths deduced from experimental mean AUCs between the sialic acid-binding domain (orange) to sialic acids (dark gray, Sia), the JAM-A-binding domain (circular head of σ1) to JAM-A (black), and the RGD domain (red) to β-integrin (light gray, βInt). Where “~” is indicated, relative binding strength was deduced by subtracting the total binding measured in the JAM-A-deficient condition from the domain-specific binding strength. ( B ) Structural models of the T3D PL σ1 body domain were generated using UCSF ChimeraX (v1.9). Wild-type (bordered) and mutant σ1 structures were created by introducing identified substitutions. Predicted hydrogen bonds with α2,3-linked sialic acid (PDB: 3S6X) were assessed using default cutoffs; a representative G196R rotamer shows novel hydrogen bonds between the arg196 and sialic acid (red arrow). ( C ) Amino acid sequence alignment of the σ1 body domain encompassing the sialic acid-binding pocket (NCBI). Mutations identified through passage (G196R, T193M, and N206H) are annotated alongside known sialic acid-binding residues (N198, R202, and P204).

Article Snippet: To quantify cell-surface receptor expression, cells were incubated with monoclonal antibodies specific for murine JAM-A (clone BV11, Millipore Sigma), murine β1 integrin (eBioscience), human JAM-A (CSTEM27, Thermo Fisher Scientific), or human β1 integrin (clone P5D2, DSHB).

Techniques: Binding Assay, Generated, Mutagenesis, Sequencing

Journal: Inflammation

Article Title: A Novel Insight into Chronic Pancreatitis Pathogenesis: the USP1/ITGB5 Axis-Mediated Stellate Cell Activation

doi: 10.1007/s10753-025-02434-x

Figure Lengend Snippet: Proteomics reveals impairment of ECM degradation in PSCs with USP1 knockdown. A , B . USP1 expression was estimated in PSCs treated with different concentration of TGF-β1 (n = 3). C , D . The USP1 expression after lentiviral transfection was detected by real-time PCR ( C ) and western blot ( D ) ( n = 3). E Heap map of differentially expressed proteins (DEPs) in label-free proteomic ( n = 4). F . GO enrichment analysis of DEPs associated with collagen fibers. G . Heat map of collagen protein (n = 4). H . The protein expression of COL1A1, COL1A2, and FN in the PSCs was determined by western blot ( n = 3). I . Immunofluorescence staining of COL1A1 in the PSCs ( n = 3). Bar: 50 μm. J . Immunofluorescence staining of α-SMA. Bar: 50 μm. K . Quantitative analysis of the fluorescence intensity of COL1A1 ( I ) and α-SMA (J) ( n = 3). **, p < 0.01; ***, p < 0.001

Article Snippet: For human TGF-β1 (hTGF-β1) induction, cells were treated with 5 ng/mL hTGF-β1 (HY- P78668 , MCE, New Jersey, USA) for 0, 12, 24, or 48 h. For lentiviral infection, cells were cultured in virus-containing medium for 48 h; subsequently, infected cells were treated with 5 ng/mL hTGF-β1 for 24 h for further analysis.

Techniques: Knockdown, Expressing, Concentration Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Inflammation

Article Title: A Novel Insight into Chronic Pancreatitis Pathogenesis: the USP1/ITGB5 Axis-Mediated Stellate Cell Activation

doi: 10.1007/s10753-025-02434-x

Figure Lengend Snippet: USP1 knockdown promotes ITGB5 ubiquitination-mediated degradation. ( A ) Venn plot showing a total of 2015 proteins bind to USP1 under TGF-β1 stimulation. (B) KEGG pathway analysis and GO enrichment analysis pair of 2015 proteins in ( A ). ( C ) Label-free proteomics combined with IP-LC/MS to analysis the USP1 downstream target. The intersection of differential downregulated expressed protein of label-free proteomics, IP-LC/MS, and the upregulated genes of GSE41418 dataset. ( D ) The ITGB5-associated PPI was established via the String database. E-F. The expression of ITGB5 of the PSCs (E) ( n = 3) and pancreatic tissues ( F ) ( n = 6) was estimated by western blot. G . Co-IP shows the interaction of USP1 and ITGB5 in the PSCs ( n = 3). H . Ubiquitination detection of ITGB5 in the PSCs were measured by Co-IP ( n = 3). **, p < 0.01; ***, p < 0.001.

Article Snippet: For human TGF-β1 (hTGF-β1) induction, cells were treated with 5 ng/mL hTGF-β1 (HY- P78668 , MCE, New Jersey, USA) for 0, 12, 24, or 48 h. For lentiviral infection, cells were cultured in virus-containing medium for 48 h; subsequently, infected cells were treated with 5 ng/mL hTGF-β1 for 24 h for further analysis.

Techniques: Knockdown, Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, Co-Immunoprecipitation Assay

Journal: Bioactive Materials

Article Title: Pre-priming cell sheet therapy enabled by dynamic wrinkled electroactive substrate for muscle reconstruction

doi: 10.1016/j.bioactmat.2026.01.046

Figure Lengend Snippet: Schematic illustration of the NIR-responsive dynamic wrinkle platform for the non-invasive harvesting of pre-primed cell sheets and their application in volumetric muscle loss (VML) repair. (A) The process of obtaining and applying pre-conditioned cell sheets for VML repair. (B) NIR-triggered dynamic reconfiguration of the wrinkle topography remotely switches the interfacial adhesion state. (C) This reconfiguration alters cellular mechanotransduction and focal adhesion density, leading to cell sheet detachment when the interfacial mechanical force (Fm) surpasses the cell-substrate adhesion force (Fc). (D) Immunofluorescence staining of focal adhesion-related markers (integrin β1, talin, pFAK(Y397), paxillin, and YAP/TAZ) and cytoskeleton in cells under control and mechanical stimulation conditions.

Article Snippet: FAK Antibody (sc-271126), pFAK(Y3978556s), talin (sc: 4021s), paxillin (sc: 365379), integrin β1 (sc: 374429), and YAP (cst: 14074s), TAZ (cst: 83669s) were ordered from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Staining, Control

Journal: Bioactive Materials

Article Title: Pre-priming cell sheet therapy enabled by dynamic wrinkled electroactive substrate for muscle reconstruction

doi: 10.1016/j.bioactmat.2026.01.046

Figure Lengend Snippet: Immunofluorescence staining of focal adhesion-related markers and cytoskeleton in cells under control and mechanical stimulation conditions. Left (Control group): Immunofluorescence staining shows focal adhesion-associated proteins (green channels). These correspond to integrin β1(A), talin (B), pFAK/FAK (C), paxillin (D), YAP (E), and TAZ (F) in each row. F-actin and FAK are shown in the red channel. The rightmost column of each row displays merged images. These integrate focal adhesion marker signals (green), F-actin (red), and DAPI-stained nuclei (blue). Yellow indicates co-localization of focal adhesion markers and F-actin. Right (After mechanical stimulation group): Immunofluorescence staining displays the same set of focal adhesion-related proteins and F-actin in cells after mechanical stimulation. Merged images are presented in the same format as the control group.

Article Snippet: FAK Antibody (sc-271126), pFAK(Y3978556s), talin (sc: 4021s), paxillin (sc: 365379), integrin β1 (sc: 374429), and YAP (cst: 14074s), TAZ (cst: 83669s) were ordered from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Staining, Control, Marker

Journal: International Journal of Cancer

Article Title: Arp2/3 complex and β1 integrin drive an invasive front through extracellular matrix adaptation in pancreatic cancer

doi: 10.1002/ijc.70376

Figure Lengend Snippet: β1 integrin signaling facilitates Arp2/3‐dependent migration on type I collagen. (A) Volcano plot of RNAseq results comparing Arpc4 KO versus control in 8025 and R254 cells. Functional annotation of downregulated genes (8025 cells, n = 379; R254 cells, n = 328) after Arpc4 knock‐out in murine PDAC cells. Top 10 annotations are shown. Reference genome: GRCm39. (B) Western blot shows expression levels of Arpc4, β1 integrin and p‐MLC2 in control and Arpc4 KO cells, Hsp90 as a loading control. (C) Panels depict single‐cell trajectories on plates coated with type I collagen with treatment of 100 μg/mL IgG or β1‐Integrin antibody. Quantitative analysis for migrated distance and velocity of cells in each group. (D) Representative phase‐contrast pictures show the morphology of control 8025 cells in type I collagen (left, scale bars: 200 μm) with treatment of 100 μg/mL IgG or β1‐Integrin antibody at various time points. Quantitative analysis of size for the structures formed by PDAC cells (right). p ‐values for β1‐Integrin antibody vs. IgG are 0.0002 (Day 3), <0.0001 (Day 5), and 0.011 (Day 7).

Article Snippet: The cultures were then incubated at 37°C in a 5% CO2 atmosphere for 12 h. For those groups receiving blocking antibody treatments, the media also included 100 μg/mL of IgG (I‐1195, Leinco Technologies, St. Louis, MO) or β1‐integrin antibody (AIIB2, Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: Migration, RNA sequencing, Control, Functional Assay, Knock-Out, Western Blot, Expressing, Single Cell