Journal: Virology Journal

Article Title: Replication kinetics of pathogenic Eurasian orthohantaviruses in human mesangial cells

doi: 10.1186/s12985-024-02517-5

Figure Lengend Snippet: Flow cytometry analysis of surface expression of orthohantaviral entry receptors. CIHGM-1 cells were analyzed for the presence of integrin α v β 3 , integrin β 1 , and CD55 on the cell surface. Plots shown are gated on the viable cell population according to scatter profile and the exclusion of Via-Probe™ Cell Viability Solution. Quadrant statistics (Q1-Q4) indicate the percentage of cells in the respective quadrant

Article Snippet: CIHGM-1 cells were washed with PBS, scraped, and stained with phycoerythrin (PE)-conjugated mouse anti-integrin α V β 3 antibody (clone LM609, Millipore) or PE-conjugated mouse anti-integrin β 1 antibody (clone P5D2, R&D Systems, Minneapolis, MN, USA) together with allophycocyanin (APC)-conjugated anti-CD55 (clone IA10, BD Pharmingen, NJ, USA).

Techniques: Flow Cytometry, Expressing

Journal: Advanced Science

Article Title: NaBC1 Boron Transporter Enables Myoblast Response to Substrate Rigidity via Fibronectin‐Binding Integrins

doi: 10.1002/advs.202407548

Figure Lengend Snippet: AlphaFold 3 predictions and experimental validation of NaBC1 interaction with β 1 integrin and vinculin. A) AlphaFold 3 models of the interaction between a NaBC1 dimer (both monomers shown in orange, with monomer A slightly lighter than monomer B for clarity) and the transmembrane domain along with the intracellular C‐terminal tail of β 1 integrin (residues 722–798, both monomers shown in green, with monomer C lighter than monomer D for clarity). The models, viewed from the side, front, and bottom (intracellular side), predict the transmembrane helix of β 1 integrin to sit in a groove on the side of the interacting face of the NaBC1 dimer. The C‐terminal tail of β 1 integrin is predicted to wrap along the underside of the intracellular domain of NaBC1. The model of NaBC1 and vinculin (both monomers shown in purple, with monomer C lighter than monomer D for clarity) suggests that vinculin interacts with both the transmembrane domain (TMD) and the N‐terminal intracellular domain (NTD) of NaBC1. B) Detailed view of the interactions. The C‐terminal tail of each β 1 integrin monomer is predicted to interact with the intracellular domains of both NaBC1 monomers. A section of vinculin's proline‐rich hinge region (residues 839–873) is predicted to insert between the NaBC1 monomers, reaching from the intracellular domains up to the transmembrane domains and interacting with the 10 β1 and 10 β2 β‐sheets of NaBC1. Red asterisk represents the presence of B ion. pLDDT confidence scores: 68.6 for NaBC1/β 1 integrin; 76.1 for NaBC1/vinculin. C) Immunoblots validating the interaction of NaBC1 with β 1 integrin. β 1 integrin was immunoprecipitated from cells cultured on rigid hydrogels in the absence or presence of B (1.47 m m ) for 24 h. Immunodetection was performed using antibodies specific for β 1 integrin and NaBC1. An increase in the interaction of NaBC1 with β 1 integrin was observed in the presence of B. D) Immunoblots showing interaction between NaBC1 and vinculin (VCL). Vinculin was immunoprecipitated using protein‐specific antibodies, and NaBC1 and vinculin were immunodetected. An increase in the interaction of NaBC1 with β 1 integrin was observed in the presence of B. IP, Immunoprecipitated protein; IB, Immunodetected protein; Un, untreated cells; L, protein ladder; B, B‐treated cells. Cell lysate is referred to as input to confirm protein‐specific bands. All immunoblot experiments were duplicated with identical results. Uncropped immunoblots are presented in Figure (Supporting Information).

Article Snippet: Antibodies specific for β 1 integrin (Proteintech) and vinculin (Proteintech) were conjugated to Dynabeads beads coated with protein A or G (ThermoFisher) and then incubated with equal amounts of protein lysates (2 mg) to allow formation of protein–antibody complexes which was then eluted from the beads by using reducing agent (ThermoFisher) and sample buffer (ThermoFisher) at final concentration of 1×, followed by heating at 95 °C for 10 min. Electrophoresis was then performed at 190 V for 50 min at room temperature, and proteins were transferred to PVDF membrane at 20 V and 160 mA for 70 min in a cooled environment, followed by blocking using 5% non‐fat dry milk (NFDM) and immunodetection using protein‐specific antibodies for 1.5 h at room temperature.

Techniques: Biomarker Discovery, Western Blot, Immunoprecipitation, Cell Culture, Immunodetection

Journal: iScience

Article Title: Mechanical control of the alternative splicing factor PTBP1 regulates extracellular matrix stiffness induced proliferation and cell spreading

doi: 10.1016/j.isci.2025.112273

Figure Lengend Snippet: PTBP1 is important for cellular mechanoresponses (A) Mesenchymal stem cells were cultured on plastic (stiff) and transfected with the indicated siRNAs. Osteoblastic differentiation was quantified using alkaline phosphatase staining. Bar represents 100 μm and refers to the bottom images. Data were analyzed using ordinary one-way ANOVA. (B) Mesenchymal stem cells were cultured on collagen coated stiff PAA gels (25 kPa), transfected with the indicated siRNAs and osteoblastic differentiation was quantified as described in (A). Bar represents 100 μm. (C) MCF10A cells transfected with the indicated siRNAs and seeded on stiff (25 kPa) or soft (0.2 kPa) collagen coated PAA gels. Cells were stained with F-actin for measurement of spreading area. Knockdown was confirmed by PTBP1 immunofluorescence. Bar represents 10 μm. Data were analyzed using ordinary one-way ANOVA. (D) MCF10A cells were transfected with the indicated siRNAs followed by a surface biotinylation assay. Total proteins and cell surface proteins were analyzed by western blot using indicated antibodies. Bar diagrams represent the quantification of the experiments. Data were analyzed by ordinary one-way ANOVA. (E) MCF10A cells were transfected with the indicated siRNAs and the surface amount of integrin β1 (CD29) was quantified using flow cytometry. Data were analyzed by ordinary one-way ANOVA. (F) MCF10A cells were cultured on stiff (25 kPa) or soft (0.2 kPa) collagen coated PAA gels and transfected with the indicated siRNAs. Proliferation rate and knockdown was determined by immunofluorescence. Bar diagram shows the quantification of BrdU incorporation. Bar represents 20 μm. Data were analyzed by ordinary one-way ANOVA. (G) MCF10A cells were cultured on stiff (25 kPa) collagen coated PAA gels and treated with 50 μM ATN161 inhibitor overnight. Proliferation rate was quantified using the BrdU assay. Bar represents 50 μm. Data were analyzed by unpaired t-test. (H) MCF10A cells were seeded on stiff (25 kPa) collagen coated PAA gels and transfected with the indicated siRNAs. Knockdown was confirmed by western blot and proliferation rate was quantified by BrdU assay. Bar represents 50 μm. Data were analyzed by unpaired t-test. Three replicates in all experiments. n = numbers of cells analyzed or in the case of BrdU assays refers to the number of analyzed microscopic fields. All values are means ± s.d. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns: not significant.

Article Snippet: Primary antibodies were used as follows: YAP (1:200; Santa Cruz, sc-101199), GFP (1:2000; Invitrogen, A-11122), PTBP1 (1:500; Thermo Fisher Scientific, 32–4800), Pitx2 (1:1000; Capra Science, PA-1020), CCT2 (1:200; Santa Cruz, sc-374152), Nucleolin (1:300; Santa Cruz, sc-8031), Numb (1:1000; Cell Signaling Technology, 2756), Integrin β-1 (1:1000; Santa Cruz, sc-374429), Transferrin receptor 1 (TfR1) (1:1000; Cell Signaling Technology, 13113), β-tubulin (1:5000; Sigma-Aldrich, T4026), γ-adaptin (1:1000; gift from Dr Andrew Peden, University of Sheffield), GAPDH (1:20000; Proteintech, 60004-1-IG), β-catenin (1:2000; BD Biosciences, 610153).

Techniques: Cell Culture, Transfection, Staining, Knockdown, Immunofluorescence, Surface Biotinylation Assay, Western Blot, Flow Cytometry, BrdU Incorporation Assay, BrdU Staining

Journal: iScience

Article Title: Mechanical control of the alternative splicing factor PTBP1 regulates extracellular matrix stiffness induced proliferation and cell spreading

doi: 10.1016/j.isci.2025.112273

Figure Lengend Snippet:

Article Snippet: Primary antibodies were used as follows: YAP (1:200; Santa Cruz, sc-101199), GFP (1:2000; Invitrogen, A-11122), PTBP1 (1:500; Thermo Fisher Scientific, 32–4800), Pitx2 (1:1000; Capra Science, PA-1020), CCT2 (1:200; Santa Cruz, sc-374152), Nucleolin (1:300; Santa Cruz, sc-8031), Numb (1:1000; Cell Signaling Technology, 2756), Integrin β-1 (1:1000; Santa Cruz, sc-374429), Transferrin receptor 1 (TfR1) (1:1000; Cell Signaling Technology, 13113), β-tubulin (1:5000; Sigma-Aldrich, T4026), γ-adaptin (1:1000; gift from Dr Andrew Peden, University of Sheffield), GAPDH (1:20000; Proteintech, 60004-1-IG), β-catenin (1:2000; BD Biosciences, 610153).

Techniques: Recombinant, Selection, Staining, Negative Control, Software

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: EVs from GT16 and CV3 were analyzed by MS. The abundance of proteins was compared after normalization based on the total peptide amount according to the human database of Discoverer 2.4. Perseus analysis was performed after focusing on Master Protein, removing contamination, and taking higher than PSMs 4. In the scatter plot, the X-axis represents the log2-transformed normalized abundance of CV3-derived EVs, while the Y-axis represents the log2-transformed normalized abundance of GT16-derived EVs. The plot includes fold change lines at ±2 log2 unit. These lines indicate a 4-fold increase (+2 log2 unit) or a 0.25-fold decrease (−2 log2 unit) in the expression level of GT16-derived EVs relative to CV3-derived EVs. Integrin binding proteins are marked by red circles based on the GO term. Integrin isoforms are marked by blue dots. Other identified proteins are indicated by gray circles.

Article Snippet: After removing the insoluble cell debris through repeated centrifugation, the supernatants (cell lysates) were used for immunoprecipitation with mouse anti-integrin β 1 mAb (Santa Cruz Biotechnology) at 4 °C overnight with rotation.

Techniques: Transformation Assay, Derivative Assay, Expressing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: The expression of integrins and gangliosides on/in cells and EVs. Cell surface expression of gangliosides, GD3 and GD2, and that of integrins, β 1, β 2, β 3, β 4, β 5, β 6, β 7, β 8, α 2, α 3, α 4, α 5, α 6, and α v , were analyzed by flow cytometry. Anti-GD3 mAb (R24), anti-GD2 mAb (220-51), anti-integrin β 1 ~β 8 Abs, anti-integrin α 2 ~α 6 Abs, or anti-integrin α v Ab were used as primary antibodies, and FITC-labeled secondary antibodies were used ( A ). The expression levels of integrin β 1, α 3, and α v were analyzed in both GD3/GD2(+) and (-) cell-derived EVs by Tim4-bead flow cytometry ( B ). Red lines are results with individual mAbs, and black lines are of negative controls. The expression of integrin β 1 and α 3 was observed in GT16 and CV2 cells and also in EVs derived from them by IB using lysates (1 μg) of both cells and EVs. Anti-integrin β 1 Ab and anti-integrin α 3 Ab were used as primary antibodies, and HRP-labelled secondary Ab was used ( Ci ). The intensity of the obtained bands in Ci was measured and plotted ( Cii,Ciii ). Representative results from repeated experiments (at least 3 times) are presented.

Article Snippet: After removing the insoluble cell debris through repeated centrifugation, the supernatants (cell lysates) were used for immunoprecipitation with mouse anti-integrin β 1 mAb (Santa Cruz Biotechnology) at 4 °C overnight with rotation.

Techniques: Expressing, Flow Cytometry, Labeling, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: Colocalization of GD3 and/or GD2 with integrin β 1 on the cell surface. GD3/GD2(+) GT16 and GD3/GD2(-) CV2 cells (2 × 10 4 ) were seeded on collagen-I-pre-coated glass-bottom dishes and fixed on the bottom of the dish with 4% paraformaldehyde at 70 to 80% confluency. The colocalization of GD3 and integrin β 1 was analyzed by staining with two kinds of primary antibodies, i.e., mouse anti-GD3 mAb (1:100) and rabbit anti-integrin β 1 Ab (1:100), and then by the individual secondary antibodies, Alexa 568-conjugated goat anti-mouse IgG Ab (red) (1:100) and Alexa 488-conjugated goat anti-rabbit IgG (H+L) Ab (1:100) (green), respectively, under a confocal microscope (FLUOVIEW FV10i, Olympus, Tokyo, Japan). ( A ). In the case of the colocalization of GD2 and integrin β 1 on the cell surface, mouse anti-GD2 mAb (1:100) was used with rabbit anti-integrin β 1 Ab (1:100) as primary Abs, and the secondary Abs were as mentioned above ( B ).

Article Snippet: After removing the insoluble cell debris through repeated centrifugation, the supernatants (cell lysates) were used for immunoprecipitation with mouse anti-integrin β 1 mAb (Santa Cruz Biotechnology) at 4 °C overnight with rotation.

Techniques: Staining, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: The close localization of GD3 and/or GD2 with integrin β 1 on EVs as analyzed by double immunostaining. Fresh exosomes (3 μg/200 μL) were added on Tim4-Fc-coated glass-bottom dishes for 30 min at RT. After washing, the bound exosomes were fixed on the bottom of the dish with 4% paraformaldehyde. Fixed exosomes were incubated with primary antibodies (mouse anti-GD3 mAb (1:100) and rabbit anti-integrin β 1 Ab (1:100)) and secondary Abs (Alexa 568-conjugated goat anti-mouse IgG Abs (red) (1:100) and/or Alexa 488-conjugated goat anti-rabbit IgG (H+L) Abs (green) (1:100)) and finally analyzed with a confocal microscope, where the yellow color of the overlapping image indicates the close localization of GD3 and integrin β 1 on GT16-derived EVs ( A upper ) but not on CV2-derived EVs ( A lower ). Colocalization between GD2 and integrin β 1 was examined on EVs using primary antibodies, mouse anti-GD2 mAbs (1:100) and rabbit anti-integrin β 1 Abs (1:100), and the proper secondary Abs as mentioned above. The yellow color of the confocal imaging means close localization between GD2 and integrin β 1 on GT16 cell-derived EVs ( B upper ), but not on CV2-derived EVs ( B lower ).

Article Snippet: After removing the insoluble cell debris through repeated centrifugation, the supernatants (cell lysates) were used for immunoprecipitation with mouse anti-integrin β 1 mAb (Santa Cruz Biotechnology) at 4 °C overnight with rotation.

Techniques: Double Immunostaining, Incubation, Microscopy, Derivative Assay, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: Molecular clustering of gangliosides and integrin β 1 . ( A ) The association of GD3 and integrin β 1 and that of GD2 and integrin β 1 on cells was analyzed by IP with anti-integrin β 1 Ab and subsequent IB with anti-GD3 mAb or anti-GD2 mAb as well as anti-integrin β 1 Ab. ( B ) The molecular clustering between GD3 and integrin β 1 and between GD2 and integrin β 1 in EVs derived from them was examined by IP followed by IB as mentioned above.

Article Snippet: After removing the insoluble cell debris through repeated centrifugation, the supernatants (cell lysates) were used for immunoprecipitation with mouse anti-integrin β 1 mAb (Santa Cruz Biotechnology) at 4 °C overnight with rotation.

Techniques: Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Action Mechanisms of Exosomes Derived from GD3/GD2-Positive Glioma Cells in the Regulation of Phenotypes and Intracellular Signaling: Roles of Integrins

doi: 10.3390/ijms252312752

Figure Lengend Snippet: Effects of anti-integrin Abs and anti-GD3/GD2 mAbs on EV function. ( A ) The adhesion activity of GD3/GD2(-) cells to collagen-I examined by RT-CES. Cells (1 × 10 4 ) were cultured in collagen-I-precoated wells containing 100 μL of culture medium and incubated. The data after 24 h ( A ) and 9 h ( B ) of cell incubation are shown. GT16 cell-derived EVs enhanced the adhesion activity of CV2 cells ( A , B ), but anti-integrin β 1 Ab remarkably suppressed the adhesion activity of CV2 cells ( A ). Anti-GD3 mAb and/or anti-GD2 mAb also significantly suppressed the action of EVs to the adhesion of CV2 cells when either one of these anti-ganglioside mAbs was added to CV2 cells together with GD3/GD2(+) cell-derived EVs ( B ). The data until 24 h of incubation ( A ) and until 9 h of incubation ( B ) were analyzed with an unpaired Student’s two-tailed t test. Not significant (NS) p > 0.05, and *** p < 0.001. The mean values ± SD (n = 3) were plotted for each time point.

Article Snippet: After removing the insoluble cell debris through repeated centrifugation, the supernatants (cell lysates) were used for immunoprecipitation with mouse anti-integrin β 1 mAb (Santa Cruz Biotechnology) at 4 °C overnight with rotation.

Techniques: Activity Assay, Cell Culture, Incubation, Derivative Assay, Two Tailed Test