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: 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

Journal: bioRxiv

Article Title: Integrin Activation Enhances Lesion-Specific Targeting of Monocyte-Mimetic Nanoparticles in Atherosclerosis

doi: 10.64898/2026.03.04.707824

Figure Lengend Snippet: Fluorescence images and quantification of nanoparticle uptake in TNFα-activated ECs under the following conditions: (A) VCAM1 blockade, (B) β1-integrin blockade on nanoparticles, and (C) CPZ treatment. Red: IA@MoNP, MoNP, or bare NP; blue: DAPI-stained nuclei. Scale bar = 50 μm. (A–B) *p < 0.05 vs. IgG antibody; # p < 0.05 vs. MoNP. (C) *p < 0.05 vs. control ECs; # p < 0.05 vs. bare NP. Data are presented as mean ± SD from n = 3 independent experiments.

Article Snippet: Membrane proteins were assessed by Western blot using antibodies against CD11b (Cell Signaling #17800, 1:1000), Na + /K + ATPase (Cell Signaling #3010, 1:1000), α4-integrin (Invitrogen #PA5-20599, 1:1000), and β1-integrin (Cell Signaling #4706S, 1:1000).

Techniques: Fluorescence, Staining, Control

Journal: bioRxiv

Article Title: Integrin Activation Enhances Lesion-Specific Targeting of Monocyte-Mimetic Nanoparticles in Atherosclerosis

doi: 10.64898/2026.03.04.707824

Figure Lengend Snippet: (A) Schematic illustration of the experimental design. (B) IVIS imaging and quantification of nanoparticle accumulation in partially ligated LCA of ApoE -/- mice. (C) Representative cross-sectional fluorescence images of the LCA. Red: IA@MoNP or MoNP; green: elastin fibers; blue: DAPI-stained nuclei. Scale bar = 100 μm. (D–E) IVIS analysis showing (D) biodistribution in major organs across nanoparticle formulations and (E) reduction of IA@MoNP signal in the LCA following β1-integrin blockade on nanoparticles. (B) *p < 0.05 vs. RCA; # p < 0.05 vs. MoNP; (E) *p < 0.05 vs. IgG antibody. Data are presented as mean ± SD from n = 4 mice each group.

Article Snippet: Membrane proteins were assessed by Western blot using antibodies against CD11b (Cell Signaling #17800, 1:1000), Na + /K + ATPase (Cell Signaling #3010, 1:1000), α4-integrin (Invitrogen #PA5-20599, 1:1000), and β1-integrin (Cell Signaling #4706S, 1:1000).

Techniques: Imaging, Fluorescence, Staining