Journal: Cell Proliferation

Article Title: ITGA5 as a Dual Regulator of Epithelial‐Mesenchymal Transition and Epithelial Cell Anoikis Resistance: Functional Validation and Drug Prediction

doi: 10.1111/cpr.70190

Figure Lengend Snippet: Upregulated ITGA5 expression in the airways of asthma patients and in a TGF‐ β 1‐induced EMT model, and its association with impaired lung function. (A) ITGA5 expression in bronchial brushings from asthma patients compared with healthy volunteers ( GSE41862 ). (B) ITGA5 expression in sputum airway cells from patients with mild, moderate, and severe asthma ( GSE184433 ). (C) ITGA5 expression in bronchial brushing samples from moderate and severe asthma patients ( GSE76226 ). (D) ITGA5 expression in bronchial brushing samples from asthma patients ( GSE164015 ). (E) ITGA5 expression in airway mucosal samples from asthma patients and allergic non‐asthma controls ( GSE193816 ). (F) Differential distribution of ITGA5 in airway mucosal samples from asthma patients and allergic non‐asthma controls shown in a umap plot ( GSE193816 ). (G) ITGA5 expression in an in vitro TGF‐ β 1‐induced EMT model using human bronchial epithelial cells ( GSE40374 ). (H) Correlation heatmap between ITGA5 and mesenchymal markers (Fibronectin, N‐cadherin, Vimentin) and the epithelial marker E‐cadherin ( GSE40374 ). (I) Scatter plots depicting correlations between ITGA5 expression and sputum total leukocyte count, and lung function parameters: ΔFEV 1 , FEV 1 % predicted, FVC % predicted, and FEV 1 /FVC % predicted in asthma patients ( GSE184433 ). Data were presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ITGA5 (TargetMol, TMPJ‐00810) was covalently immobilised on a CM5 sensor chip (Cytiva, BR‐1005‐30) using the standard 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide amine coupling chemistry.

Techniques: Expressing, In Vitro, Marker, Significance Assay

Journal: Cell Proliferation

Article Title: ITGA5 as a Dual Regulator of Epithelial‐Mesenchymal Transition and Epithelial Cell Anoikis Resistance: Functional Validation and Drug Prediction

doi: 10.1111/cpr.70190

Figure Lengend Snippet: TGF‐ β 1 induced EMT and anoikis resistance in human bronchial epithelial cells (HBE135‐E6E7). (Aand B) Western blot analysis of EMT phenotype markers (ZO‐1, E‐cadherin, Occludin, Fibronectin, N‐cadherin, Vimentin) at 48 h and 72 h, respectively. (C and D) Protein expression level of ITGA5 in the TGF‐ β 1‐induced HBE135‐E6E7 cell EMT model. (E and F) Fluorescence expression intensity of ITGA5 in the TGF ‐β 1‐induced HBE135‐E6E7 cell EMT model scale bar = 50 μm. (G) Protein interaction network of ITGA5 predicted by STRING. (H) Pro DIA‐based quantitative proteomics analysis process ( n = 5). (I) PCA analysis of all samples. (J) Volcano plot, red and green dots denote significantly up‐ and down‐regulated differentially expressed proteins (DEPs). DEPs identification criteria: | log 2 FC | ≥ 1 and p < 0.05. (K) GO enrichment analysis of DEPs, enrichment threshold: P < 0.05. (L) KEGG enrichment analysis of DEPs, enrichment threshold: P < 0.05. (M and N) Anoikis assay: Green calcein‐AM fluorescence (Ex = 485 nm, Em = 516 nm) indicates viable cells independent of adhesion, while red PI fluorescence (Ex = 535 nm, Em = 617 nm) indicates anoikis‐induced cell death. Scale bar: 100 μm. (O and P) Fluorescent intensity of the anoikis resistance marker TrkB. Scale bar: 50 μm. (Q and R) Protein levels of the anoikis resistance marker TrkB. Data were presented as mean ± SEM. n = 3 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ITGA5 (TargetMol, TMPJ‐00810) was covalently immobilised on a CM5 sensor chip (Cytiva, BR‐1005‐30) using the standard 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide amine coupling chemistry.

Techniques: Western Blot, Analysis, Expressing, Fluorescence, Quantitative Proteomics, Marker, Sample Prep, Protein Binding, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Immunofluorescence

Journal: Cell Proliferation

Article Title: ITGA5 as a Dual Regulator of Epithelial‐Mesenchymal Transition and Epithelial Cell Anoikis Resistance: Functional Validation and Drug Prediction

doi: 10.1111/cpr.70190

Figure Lengend Snippet: ITGA5 was associated with both TGF‐ β 1‐induced EMT and anoikis resistance in bronchial epithelial cells. (A) Venn plot of DEPs involved in EMT and anoikis resistance gene set identified 45 anoikis resistance‐related genes (ARRGs). (B) Radar plot of protein expression levels of ARRGs. (C) Co‐expression network of ARRGs. The size of the node represents the degree value. The larger the degree, the more genes interact with it. The connecting lines represent the correlation between ARRGs. The red line represents positive correlation and the blue line represents negative correlation. (D) Bubble plot of cell expression of ARRGs in healthy human lungs. (E) Pathway regulatory network of ARRGs. (F) Scatter plots of the correlation between ITGA5 and mesenchymal markers Fibronectin, N‐cadherin, Vimentin and epithelial marker E‐cadherin. (G) Biological process enrichment analysis of ITGA5‐high and ITGA5‐low groups, enrichment threshold: P < 0.05. (H) KEGG enrichment analysis of ITGA5‐high and ITGA5‐low groups, enrichment threshold: P < 0.05.

Article Snippet: ITGA5 (TargetMol, TMPJ‐00810) was covalently immobilised on a CM5 sensor chip (Cytiva, BR‐1005‐30) using the standard 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide amine coupling chemistry.

Techniques: Expressing, Marker, Analysis

Journal: Cell Proliferation

Article Title: ITGA5 as a Dual Regulator of Epithelial‐Mesenchymal Transition and Epithelial Cell Anoikis Resistance: Functional Validation and Drug Prediction

doi: 10.1111/cpr.70190

Figure Lengend Snippet: Protein interaction analyses of ITGA5 with PI3K and TRKB and MD simulation of the Res‐ITGA5 complex. (A and B) Representative docked conformations of ITGA5 with PI3K and ITGA5 with TrkB, respectively. (C and D) Covariance matrices derived from NMA: Red regions denote positively correlated motions between residue pairs, whereas blue regions denote negatively correlated (anti‐correlated) motions. (E and F) Elastic network models illustrating the rigidity of interfacial atoms (grey regions), which resist deformation during collective motions. (G and H) SPR analysis of the protein–protein interaction between ITGA5 and PI3K. (I and K) Cellular expression localisation and fluorescence intensity of ITGA5 (green), PI3K (magenta), and TrkB (yellow). Scale bar: 10 μm. (L and M) Representative docking conformations of the Res‐ITGA5 complex, together with interaction mapping and hydrogen bond analysis. (N) RMSD plot of the Res‐ITGA5 complex. (O) RMSF analysis of the Res‐ITGA5 complex. (P) Binding energy profile of the Res‐ITGA5 complex, in which VDW represents van der Waals and hydrophobic interactions, ELE denotes electrostatic interactions, and Binding corresponds to the sum of VDW and ELE without considering solvation effects. (Q) Rg plot curve characteristics. Data were presented as mean ± SEM. n = 3 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ITGA5 (TargetMol, TMPJ‐00810) was covalently immobilised on a CM5 sensor chip (Cytiva, BR‐1005‐30) using the standard 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide amine coupling chemistry.

Techniques: Derivative Assay, Residue, Analysis, Expressing, Fluorescence, Docking Assay, Binding Assay, SPR Assay, Immunofluorescence

Journal: Cell Proliferation

Article Title: ITGA5 as a Dual Regulator of Epithelial‐Mesenchymal Transition and Epithelial Cell Anoikis Resistance: Functional Validation and Drug Prediction

doi: 10.1111/cpr.70190

Figure Lengend Snippet: Effects of ITGA5 knockdown on TGF‐ β 1‐induced EMT, PI3K/Akt signalling, and anoikis resistance in bronchial epithelial cells. (A–H) Western blot analysis of protein expression levels of EMT markers (Fibronectin, N‐cadherin, E‐cadherin, Vimentin, ZO‐1 and Occludin) in TGF‐ β 1 stimulated cells following ITGA5 knockdown. (I–L) Western blot analysis of PI3K/Akt signalling axis proteins following ITGA5 knockdown. (M–S) Western blot analysis of anoikis resistance related proteins (XIAP, MCL‐1, TrkB, Caspase 3, Cleaved‐caspase 3, Caspase 9 and Cleaved‐caspase 9) after ITGA5 knockdown. (T–U) Anoikis assay post‐ITGA5 knockdown: Red PI fluorescence (Ex = 535 nm, Em = 617 nm) indicates anoikis‐induced cell death; scale bar: 100 μm. (V–X) Cellular spatial colocalisation and fluorescence intensity analyses of ITGA5 (green) with PI3K (magenta) and TrkB (yellow) after ITGA5 knockdown ( n = 3); scale bar: 10μm. Data were presented as mean ± SEM. n = 3 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ITGA5 (TargetMol, TMPJ‐00810) was covalently immobilised on a CM5 sensor chip (Cytiva, BR‐1005‐30) using the standard 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide amine coupling chemistry.

Techniques: Knockdown, Western Blot, Analysis, Expressing, Fluorescence, Immunofluorescence

Journal: Cell Proliferation

Article Title: ITGA5 as a Dual Regulator of Epithelial‐Mesenchymal Transition and Epithelial Cell Anoikis Resistance: Functional Validation and Drug Prediction

doi: 10.1111/cpr.70190

Figure Lengend Snippet: Res and M200 pharmacologically inhibited ITGA5 and ameliorated TGF‐ β 1–induced EMT in bronchial epithelium cells. (A and B) Cell viability of HBE135‐E6E7 cells following 48 h treatment with varying concentrations of Res or M200, as measured by CCK‐8 assay. (C–J) Res and M200 inhibited ITGA5 and improved the expression of EMT markers (Fibronectin, N‐cadherin, Vimentin, ZO‐1, E‐cadherin, and Occludin) proteins. Data were presented as mean ± SEM. n = 3 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ITGA5 (TargetMol, TMPJ‐00810) was covalently immobilised on a CM5 sensor chip (Cytiva, BR‐1005‐30) using the standard 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide amine coupling chemistry.

Techniques: CCK-8 Assay, Expressing, Western Blot

Journal: Cell Proliferation

Article Title: ITGA5 as a Dual Regulator of Epithelial‐Mesenchymal Transition and Epithelial Cell Anoikis Resistance: Functional Validation and Drug Prediction

doi: 10.1111/cpr.70190

Figure Lengend Snippet: Res and M200 inhibited ITGA5, suppressed PI3K/Akt pathway activation, and alleviated anoikis resistance. (A–D) Western blot analysis of PI3K/Akt signalling axis proteins in cells treated with Res or M200. (E–K) Western blot analysis of anoikis resistance related proteins (XIAP, MCL‐1, TrkB, Caspase 3, Cleaved‐caspase 3, Caspase 9 and Cleaved‐caspase 9) in cells treated with Res or M200. (L and M) Effects of Res and M200 on anoikis: Green calcein‐AM fluorescence (Ex = 485 nm, Em = 516 nm) indicates viable cells independent of adhesion, red PI fluorescence (Ex = 535 nm, Em = 617 nm) marked anoikis‐induced cell death, scale bar: 100 μm. (N–P) Spatial colocalisation and fluorescence intensity analyses of ITGA5 (green) with PI3K (magenta) and TrkB (yellow); scale bar: 10 μm. Data were presented as mean ± SEM. n = 3 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ITGA5 (TargetMol, TMPJ‐00810) was covalently immobilised on a CM5 sensor chip (Cytiva, BR‐1005‐30) using the standard 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide amine coupling chemistry.

Techniques: Activation Assay, Western Blot, Analysis, Fluorescence, Immunofluorescence

Journal: Cell Proliferation

Article Title: ITGA5 as a Dual Regulator of Epithelial‐Mesenchymal Transition and Epithelial Cell Anoikis Resistance: Functional Validation and Drug Prediction

doi: 10.1111/cpr.70190

Figure Lengend Snippet: Elevated ITGA5 expression in asthma mice lungs accompanied by upregulation of the PI3K/Akt pathway and anoikis resistance marker TrkB. (A–F) Western blot analysis of ITGA5, PI3K/Akt signalling–related proteins and anoikis resistance–associated proteins (XIAP and MCL‐1) in lung tissues of asthma mice. n = 3 for each group. (G–H) Immunohistochemical analysis of ITGA5 localisation and expression in lung tissues of asthma mice. n = 6 for each group. Scale bars: 200 and 50 μm. (I–J) Immunohistochemical analysis of TrkB localisation and expression in lung tissues of asthma mice. n = 6 for each group. Scale bars: 200 and 50 μm. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ITGA5 (TargetMol, TMPJ‐00810) was covalently immobilised on a CM5 sensor chip (Cytiva, BR‐1005‐30) using the standard 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide amine coupling chemistry.

Techniques: Expressing, Marker, Western Blot, Analysis, Immunohistochemical staining, Immunohistochemistry

Journal: Cell Proliferation

Article Title: ITGA5 as a Dual Regulator of Epithelial‐Mesenchymal Transition and Epithelial Cell Anoikis Resistance: Functional Validation and Drug Prediction

doi: 10.1111/cpr.70190

Figure Lengend Snippet: Res and M200 inhibited ITGA5 in vivo and reversed EMT in the lungs of asthma mice. (A–C) Immunofluorescence analysis of the colocalisation and expression of ITGA5, PI3K, and TrkB with the airway epithelial marker EpCAM in the lungs of asthma mice after treatment with Res and M200. n = 6 for each group. Scale bars: 50 and 20 μm. (D–J) Western blot analysis of the protein levels of EMT‐related markers (Fibronectin, N‐cadherin, Vimentin, ZO‐1, E‐cadherin, and Occludin) in lung tissues from asthma mice. n = 3 for each group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ITGA5 (TargetMol, TMPJ‐00810) was covalently immobilised on a CM5 sensor chip (Cytiva, BR‐1005‐30) using the standard 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide amine coupling chemistry.

Techniques: In Vivo, Immunofluorescence, Analysis, Expressing, Marker, Western Blot

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: 13-Methylpalmatine alleviates bleomycin-induced pulmonary fibrosis by suppressing the ITGA5/TGF-β/Smad signaling pathway.

doi: 10.1016/j.phymed.2025.156545

Figure Lengend Snippet: Fig. 5. Transcriptomics analysis, molecular docking, molecular dynamics simulation, and SPR analysis. A Volcano map of the 147 intersecting genes. B PPI network. C Kegg pathway. D Schematic diagram of molecular docking of 13-Me-PLT with ITGA5. E RMSD of ITGA5 with 13-Me-PLT. F Spacing of ITGA5 and 13-Me-PLT binding sites (Dock site-ligand). G Interaction of ITGA5 with 13-Me-PLT. H SPR analysis of ITGA5 and 13- Me-PLT.

Article Snippet: The CM7 chip was selected and ITGA5 protein (50μg, shanghai, MCE) was diluted to 50 μg/ml using sodium acetate with a pH value of 4.0 and the protein was coupled to the chip surface (10,988 RU).

Techniques: Transcriptomics, Binding Assay

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: 13-Methylpalmatine alleviates bleomycin-induced pulmonary fibrosis by suppressing the ITGA5/TGF-β/Smad signaling pathway.

doi: 10.1016/j.phymed.2025.156545

Figure Lengend Snippet: Fig. 6. Effects of 13-Me-PLT on BLM-induced ITGA5 and TGF-β/Smad signaling pathways in mice and TGF-β/Smad signaling pathways in Ang II-stimulated MRC5 cells. A, B Immunohistochemical staining of ITGA5 in lung tissue and relative staining intensity analysis. Scale bar = 50 μm; n = 3. C, D mRNA levels of ITGA5 and TGF-β1 in lung tissue (n = 5–6). E-H Representative blotting and quantitative analysis of protein levels of ITGA5, TGF-β1 and p-Smad3 in lung tissue (n = 6). I, J Immu nofluorescence staining and relative fluorescence intensity analysis of ITGA5 in lung tissue. Scale bar = 100 μm; n = 3. K, l mRNA levels of ITGA5 and TGF-β1 in cells of each group (n = 4–5). M-O Quantitative analysis of protein levels of ITGA5, TGF-β1 and p-Smad3 in cells of each group (n = 4). P, Q Relative fluorescence intensity analysis of ITGA5 in cells of each group (n = 3). Data are expressed as SEM ± mean. **p < 0.01, ***p < 0.001 vs. Control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. BLM or AngII.

Article Snippet: The CM7 chip was selected and ITGA5 protein (50μg, shanghai, MCE) was diluted to 50 μg/ml using sodium acetate with a pH value of 4.0 and the protein was coupled to the chip surface (10,988 RU).

Techniques: Protein-Protein interactions, Immunohistochemical staining, Staining, Fluorescence, Control

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: 13-Methylpalmatine alleviates bleomycin-induced pulmonary fibrosis by suppressing the ITGA5/TGF-β/Smad signaling pathway.

doi: 10.1016/j.phymed.2025.156545

Figure Lengend Snippet: Fig. 7. Effects of silencing ITGA5 on fibrosis-related factors and the TGF-β/Smad signaling pathway in each group of cells. A-E mRNA levels of FN1, Collagen I, α-SMA, ITGA5, and TGF-β1 in cells of each group (n = 4). F-H Representative blotting and quantitative analysis of FN1 and Collagen I protein levels in cells of each group (n = 4). I-l Representative blotting and quantitative analysis of protein levels of ITGA5, TGF-β1 and p-Smad3 in each group of cells (n = 4). M, N Immunofluorescence staining and quantitative analysis of mean fluorescence intensity of α-SMA in each group of cells (n = 3). Scale bar = 100 μm. O, P Immunofluorescence staining and quantitative analysis of mean fluorescence intensity of ITGA5 in each group of cells (n = 3), scale bar = 100 μm. Q, R Transwell detection of cell migration numbers in each group of cells and statistical analysis of migration data. Scale bar = 75 μm; n = 3. Data are expressed as SEM ± mean. **p < 0.01,***p < 0.001 vs. Control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. AngII.

Article Snippet: The CM7 chip was selected and ITGA5 protein (50μg, shanghai, MCE) was diluted to 50 μg/ml using sodium acetate with a pH value of 4.0 and the protein was coupled to the chip surface (10,988 RU).

Techniques: Immunofluorescence, Staining, Fluorescence, Migration, Control

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: 13-Methylpalmatine alleviates bleomycin-induced pulmonary fibrosis by suppressing the ITGA5/TGF-β/Smad signaling pathway.

doi: 10.1016/j.phymed.2025.156545

Figure Lengend Snippet: Fig. 8. Effect of 13-Me-PLT on fibrosis-related indices and TGF-β/Smad signaling pathway in various groups of cells after overexpression of ITGA5. A-E mRNA levels of FN1, Collagen I, α-SMA, ITGA5, and TGF-β1 in cells of each group (n = 4). F-H Representative blotting and quantitative analysis of FN1 and Collagen I protein levels in cells of each group (n = 4). I-l Representative blotting and quantitative analysis of protein levels of ITGA5, TGF-β1 and p-smad3 in each group of cells (n = 4). M, N Immunofluorescence staining and quantitative analysis of mean fluorescence intensity of α-SMA in each group of cells (n = 3). Scale bar = 100 μm. O, P Immunofluorescence staining and quantitative analysis of mean fluorescence intensity of α-SMA in each group of cells (n = 3). Scale bar = 100 μm. Q, R Transwell detection of cell migration numbers in each group of cells and statistical analysis of migration data (n = 3). Scale bar = 75 μm. Data are expressed as SEM ± mean. **p < 0.01,***p < 0.001 vs. Control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. P-ITGA5.

Article Snippet: The CM7 chip was selected and ITGA5 protein (50μg, shanghai, MCE) was diluted to 50 μg/ml using sodium acetate with a pH value of 4.0 and the protein was coupled to the chip surface (10,988 RU).

Techniques: Over Expression, Immunofluorescence, Staining, Fluorescence, Migration, Control

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: 13-Methylpalmatine alleviates bleomycin-induced pulmonary fibrosis by suppressing the ITGA5/TGF-β/Smad signaling pathway.

doi: 10.1016/j.phymed.2025.156545

Figure Lengend Snippet: Fig. 9. Schematic representation of possible targets and molecular mechanisms of 13-Me-PLT for the treatment of IPF. 13-Me-PLT ameliorates pulmonary fibrosis by inhibiting ITGA5, suppressing activation of the TGF-β/Smad signaling pathway, and decreasing phosphorylation of samd3, which reduces the deposition of ECM (collagens, fibronectin, α-SMA, etc.). 13-Me-PLT: 13-methylpalmatine; ITGA5: integrin alpha-5; ECM : extracellular matrix; TGF-β1: transforming growth factor-β1; TβR-I: Transforming Growth Factor Beta-Receptor Type I; TβR-II: Transforming Growth Factor Beta-Receptor Type II.

Article Snippet: The CM7 chip was selected and ITGA5 protein (50μg, shanghai, MCE) was diluted to 50 μg/ml using sodium acetate with a pH value of 4.0 and the protein was coupled to the chip surface (10,988 RU).

Techniques: Activation Assay, Phospho-proteomics

Journal: Acta Pharmaceutica Sinica. B

Article Title: Enhanced radiotheranostic targeting of integrin α 5 β 1 with PEGylation-enabled peptide multidisplay platform (PEGibody): A strategy for prolonged tumor retention with fast blood clearance

doi: 10.1016/j.apsb.2024.07.006

Figure Lengend Snippet: In vitro binding affinity and stability studies of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303. (A) SPR sensorgrams demonstrating the binding affinity of QM-2301, QM-2302, and QM-2303 for human integrin α 5 β 1 in a concentration-dependent manner. (B) The equilibrium dissociation constant ( Κ D ) of each peptide was calculated based on SPR measurements. The K D values of each precursor are shown. (C, D) Schematic diagram of the binding patterns of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302 (C), and [ 64 Cu]QM-2303 (D) to integrin α 5 β 1. Monomeric [ 64 Cu]QM-2301, and [ 64 Cu]QM-2302 bind to receptors in a single-network pattern. For [ 64 Cu]QM-2303, the PEGibody-based radiotracer, one PEGibody can bind to more than two integrin α 5 β 1 receptors, exhibiting better binding affinity. (E, F) The expression of integrin α 5 β 1 in B16F10 cells was analyzed by flow cytometry (E) and Western blotting (F). For flow cytometry assays, an anti-integrin α 5 + β 1 antibody was used. For Western blot assays, integrin α 5 (∼150 kDa) and integrin β 1 (∼138 kDa) were examined using two antibodies. (G) The stability of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 after coincubation with mouse serum within 1 h, as indicated by radio-HPLC. (H) I n vitro cell uptake of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 (750 KBq/mL) when incubated with B16F10 cells for different time period. (I) IC 50 of [ 64 Cu]QM-2301, [ 64 Cu]QM-2302, and [ 64 Cu]QM-2303 when inhibited with antibodies at different concentrations. ∗ P < 0.05, ∗∗ P < 0.01. All the quantitative experiments were performed independently at least three times (data are the mean ± SD, n = 3).

Article Snippet: Recombinant human integrin α 5 β 1 (alpha 5 beta 1, HY-P77718, MCE, NJ, USA) was immobilized on a CM5 sensor chip using a standard amine coupling kit at a temperature of 25 °C.

Techniques: In Vitro, Binding Assay, Concentration Assay, Expressing, Flow Cytometry, Western Blot, Incubation

Journal: bioRxiv

Article Title: Spatial Multi-Omics Reveals Extracellular Matrix Remodeling and VSMC Phenotypic Switching in Moyamoya Disease

doi: 10.64898/2026.04.27.721225

Figure Lengend Snippet: (A) Representative hematoxylin-eosin (HE) staining images of MMD and CTRL arteries, showing characteristic medial thinning and intimal hyperplasia, and based on this, a spatial omics ROI analysis was designed. (B-C) Volcano plots and corresponding heatmaps of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) between the intima and media of MMD arteries. FN1 , ITGA5 , and FAK are significantly upregulated in the intima. (D) Violin plot quantifying the Intima/Media Area Ratio in MMD versus CTRL groups (****p < 0.0001). (E) Representative IF staining of ITGA5 (green) demonstrating its robust accumulation in the thickened intima of MMD compared to CTRL. (F-H) Scratch wound-healing assay and quantification of relative migration ability in VSMCs treated with 10 μg/mL FN1, showing that FN1-induced hyper-migration is significantly abrogated by ITGA5 knockdown (shRNA-ITGA5) or FAK inhibition (VS6063).

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: anti-alpha Smooth Muscle Actin (alpha-SMA) (ab5694, Abcam; 1:200 dilution), anti-Integrin alpha 5 (ITGA5) (A19069, ABclonal; 1:200 dilution).

Techniques: Staining, Wound Healing Assay, Migration, Knockdown, shRNA, Inhibition

Journal: bioRxiv

Article Title: Spatial Multi-Omics Reveals Extracellular Matrix Remodeling and VSMC Phenotypic Switching in Moyamoya Disease

doi: 10.64898/2026.04.27.721225

Figure Lengend Snippet: (A) Workflow for the generation of induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) of MMD patients and healthy controls (HC), followed by directed differentiation into VSMCs. (B-C) Representative images and quantification of Transwell migration assays. MMD-iPSC-SMCs exhibit significantly enhanced migration compared to HC-iPSC-SMCs, which is effectively reversed by shRNA-ITGA5 or VS6063 treatment. (D-F) Western blot analysis and quantification of total FAK and phosphorylated FAK (p-FAK at Tyr397) in the established iPSC-SMC models. The elevated p-FAK/FAK ratio in MMD-iPSC-SMCs is substantially reduced by ITGA5 silencing or direct FAK inhibition.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: anti-alpha Smooth Muscle Actin (alpha-SMA) (ab5694, Abcam; 1:200 dilution), anti-Integrin alpha 5 (ITGA5) (A19069, ABclonal; 1:200 dilution).

Techniques: Migration, shRNA, Western Blot, Inhibition

Journal: bioRxiv

Article Title: Spatial Multi-Omics Reveals Extracellular Matrix Remodeling and VSMC Phenotypic Switching in Moyamoya Disease

doi: 10.64898/2026.04.27.721225

Figure Lengend Snippet: Blocking the ITGA5 pathway may reduce the pathological phenotypic transformation of MMD-iPSC-SMC. (A) Representative Western blot images showing the expression levels of SMC contractile markers, synthetic/matrix markers, and ITGA5 in HC-iPSC-SMCs and MMD-iPSC-SMCs under various treatments. (B-D) Quantification of contractile markers (MYH11, CNN1, TAGLN). Expression is significantly loss in MMD-iPSC-SMCs but rescued upon treatment with shRNA-ITGA5 or VS6063. (E-J) Quantification of synthetic and extracellular matrix-related proteins (FN1, COL1A1, COL3A1, OGN, EFEMP1) and ITGA5. The abnormal upregulation of these markers in MMD-iPSC-SMCs is suppressed by targeting the ITGA5-FAK axis, indicating a reversal from the synthetic back to the contractile phenotype.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: anti-alpha Smooth Muscle Actin (alpha-SMA) (ab5694, Abcam; 1:200 dilution), anti-Integrin alpha 5 (ITGA5) (A19069, ABclonal; 1:200 dilution).

Techniques: Blocking Assay, Transformation Assay, Western Blot, Expressing, shRNA

Journal: bioRxiv

Article Title: Spatial Multi-Omics Reveals Extracellular Matrix Remodeling and VSMC Phenotypic Switching in Moyamoya Disease

doi: 10.64898/2026.04.27.721225

Figure Lengend Snippet: (A) Representative spatial metabolomics mapping (m/z signals) showing the heterogeneous distribution of specific metabolites across different arterial segments. (B) 3D UMAP projection of spatial metabolic segments. (C-E) 2D Principal Component Analysis (PCA) plots distinguishing the distinct molecular signatures between MMD-MID-SMC (media) and MMD-IN-SMC (intima). (F-G) Multi-omics correlation heatmaps illustrating the strong spatial co-expression and inverse correlations between specific intimal genes/proteins (e.g., g.ITGA5 , p.Fibronectin ) and the abundance of localized metabolites. (H) Radar chart highlighting the top depleted compounds in the MMD intima compared to the media, revealing a profound exhaustion of Acyl-CoA derivatives (e.g., trans-Crotonyl CoA, 3-aminobutyryl-CoA).

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: anti-alpha Smooth Muscle Actin (alpha-SMA) (ab5694, Abcam; 1:200 dilution), anti-Integrin alpha 5 (ITGA5) (A19069, ABclonal; 1:200 dilution).

Techniques: Biomarker Discovery, Expressing

Journal: Folia morphologica

Article Title: Integrin subunit alpha 5 maintains mitochondrial function in ox-LDL-induced cardiac microvascular endothelial cells via activating the PI3K/AKT signaling pathway.

doi: 10.5603/fm.95500

Figure Lengend Snippet: Figure 1. Impacts of ITGA5 on CMEC viability and apoptosis. A, B. ITGA5 mRNA and

Article Snippet: Primary antibodies against ITGA5 (A6209), Bcl-2 (A0208), Bax (A12009), and GAPDH (A19056) were obtained from ABclonal.

Techniques:

Journal: Folia morphologica

Article Title: Integrin subunit alpha 5 maintains mitochondrial function in ox-LDL-induced cardiac microvascular endothelial cells via activating the PI3K/AKT signaling pathway.

doi: 10.5603/fm.95500

Figure Lengend Snippet: Figure 2. Impacts of ITGA5 on CMEC dysfunction and oxidative stress. A. the angiogenesis

Article Snippet: Primary antibodies against ITGA5 (A6209), Bcl-2 (A0208), Bax (A12009), and GAPDH (A19056) were obtained from ABclonal.

Techniques:

Journal: European journal of cell biology

Article Title: Regulation of integrin α5β1-mediated Staphylococcus aureus cellular invasion by the septin cytoskeleton.

doi: 10.1016/j.ejcb.2023.151359

Figure Lengend Snippet: Fig. 2. Septins are associated with integrin α5β1-mediated internalisation of S. aureus. (A) HeLa cells were infected with mCherry-S. aureus (blue) for 15 min in DMEM either non-supplemented, supplemented with 10% FBS or supplemented with 10 μg/ml purified bovine plasma fibronectin (Fn). After fixation, cells were stained for SEPT7 (green) and active integrin α5β1 (ITG α5β1, red). Scale bars represent 10 µm (main image) and 2 µm (inset). (B) Quantification of (A). Percentage of bacteria localizing with SEPT7 and/or active integrin α5β1 (ITG α5β1) in presence of DMEM + 10% FBS (+ 10% FBS) or DMEM + 10 μg/ml purified fibronectin (+ 10 μg/ml Fn). The graph represents mean percentage ± SEM of n = 682 (DMEM + 10% FBS) and n = 724 (DMEM + 10 μg/ml purified fibronectin) bacteria from three independent experiments. ns: non-significant by two-tailed Student’s t-test. (C) Septins are associated with bacteria recruiting phosphorylated FAK (pFAK- Y397). HeLa cells were infected with mCherry-S. aureus (blue) for 15 min in DMEM + 10% FBS. After fixation, cells were stained for SEPT7 (green), pFAK-Y397 (red), and S. aureus (blue). Scale bars are 10 µm (main image) and 1 µm (inset).

Article Snippet: Template cDNA was subjected to PCR using primers for gene encoding for integrin a5 (#HP205940, Origene), integrin b1 (#HP232479, Origene) and FAK (#HP208715, Origene).

Techniques: Infection, Purification, Clinical Proteomics, Staining, Bacteria, Two Tailed Test

Journal: European journal of cell biology

Article Title: Regulation of integrin α5β1-mediated Staphylococcus aureus cellular invasion by the septin cytoskeleton.

doi: 10.1016/j.ejcb.2023.151359

Figure Lengend Snippet: Fig. 3. FnBPA is sufficient to trigger septin recruitment to invading beads. (A) HeLa cells stably producing GFP-SEPT6 were challenged with BSA-coated or GST- DuD4-coated beads of 1 µm diameter for 15 min in DMEM + 10% FBS. After fixation, cells were stained for GST (blue) and active integrin α5β1 (ITG α5β1, red). “Merge” indicates the merged channels excluding DIC. DIC microscopy is shown to visualize the beads. Scale bar represents 2 µm. (B) HeLa cells were challenged with GST-DuD4 coated beads of 1 µm diameter for 15 min in DMEM + 10% FBS. Quantification of the mean percentage of beads localizing with SEPT7 and/or active integrin α5β1 (ITG α5β1) from (A). n = 647 beads from three independent experiments.

Article Snippet: Template cDNA was subjected to PCR using primers for gene encoding for integrin a5 (#HP205940, Origene), integrin b1 (#HP232479, Origene) and FAK (#HP208715, Origene).

Techniques: Stable Transfection, Staining, Microscopy

Journal: European journal of cell biology

Article Title: Regulation of integrin α5β1-mediated Staphylococcus aureus cellular invasion by the septin cytoskeleton.

doi: 10.1016/j.ejcb.2023.151359

Figure Lengend Snippet: Fig. 4. SEPT2 depletion impairs cellular invasion by S. aureus. (A) HeLa cells were transfected with the indicated siRNAs for 72 h. Post-siRNA transfection, cells were infected with S. aureus for 30 min in DMEM+10% FBS for gentamicin-lysostaphin protection assays then lysed and plated on TCS agar plates. The graph represents the CFU normalized to the control siRNA treated samples. The CFU ± SEM from 6 independent experiments is presented. * , p < 0.05; * *, p < 0.01; ns: non- significant by one-way Anova and Dunnett’s post-test. (B) HeLa cells were transfected with the indicated siRNAs for 72 h. Post-siRNA transfection, cells were infected with S. aureus for 15 min in DMEM +10% FBS. After thorough washing steps (no gentamicin-lysostaphin incubation step) cells were fixed and stained for SEPT7 and F-actin. The graph represents mean percentage ±SEM normalized to cells treated with control siRNA and SEM of bacteria associated with F-actin from n = 828 (CTRL siRNA) or n = 1080 (SEPT2 siRNA) bacteria in three independent experiments. * *, p < 0.01 by two-tailed Student’s t-test. (C) Number of bacteria per cell ± SEM from the same set of images analysed for Fig. 4B. Each dot represents a single cell (n = 60 cells from three independent experiments). * , p < 0.05 two- tailed by Student’s t-test. (D) Flow cytometry analysis of integrin α5 and β1 surface expression of HeLa cells treated for 72 h with the indicated siRNAs. The graph represents the mean fluorescence intensity (MFI) normalized to cells treated with control siRNA ± SEM from 3 independent experiments. Data is normalised to cells treated with control siRNA. * , p < 0.05; ns: non-significant by two-tailed Student’s t-test. (E) Western blot for siRNA depletion experiments corresponding to data presented in Fig. 4B-D. Whole-cell lysates were from control siRNA or SEPT2 siRNA treated HeLa cells. Integrin α5 (ITG α5), integrin β1 (ITG β1), FAK (FAK) and SEPT2 (SEPT2, to show depletion efficiency) protein levels were detected. GAPDH protein level was used as a loading control. (F) Densitometry-based quantification of integrin α5 (ITG α5), integrin β1 (ITG β1), FAK and SEPT2 signal from (E). All bands were first normalized to the signal of the loading control GAPDH. Graph represents the fold change of the indicated protein normalised to control siRNA treated cells (CTRL) ± SEM from 3 independent experiments. * , p < 0.05, ns: non- significant by two-tailed Student’s t-test.

Article Snippet: Template cDNA was subjected to PCR using primers for gene encoding for integrin a5 (#HP205940, Origene), integrin b1 (#HP232479, Origene) and FAK (#HP208715, Origene).

Techniques: Transfection, Infection, Control, Incubation, Staining, Bacteria, Two Tailed Test, Flow Cytometry, Expressing, Fluorescence, Western Blot

Journal: Nature Communications

Article Title: The CCL20–integrin α5β1 interaction enhances TGF-β/Smad signaling to promote fibroblast activation in pulmonary fibrosis

doi: 10.1038/s41467-025-64211-6

Figure Lengend Snippet: a List of the 10 proteins that interact with CCL20 identified by MS analyses. b Volcano plot of enriched proteins that interact with CCL20 (n = 3 technical replicates per group). Significantly altered proteins are shown in red (false discovery rate (FDR) < 0.05, log 2 FC > 1) and blue (FDR < 0.05, log 2 FC < −1). c Identification of integrin α5 and integrin β1 from CCL20 immunoprecipitates in fibroblasts by MS analysis. d Surface plasmon resonance (SPR) analysis of the kinetic interaction between CCL20 and integrin α5β1. CoIP analysis of the interaction between CCL20 and integrin α5 ( e ) or integrin β1 ( f ) in HEK293T cells. IF images showing the colocalization of CCL20 with integrin α5 ( g ) or integrin β1 ( h ) in MRC-5 cells. Scale bars, 10 μm. i Expression of Col1a1, Col3a1, Fibronectin and Acta2 in primary fibroblasts treated with or without ATN-161 in the presence of CCL20. j Immunoblots showing COL1 and α-SMA levels in primary fibroblasts treated with or without ATN-161 in the presence of CCL20. k Migratory ability of primary fibroblasts treated with or without ATN-161 in the presence of CCL20. Scale bars, 100 μm. l Expression of ACTA2 in MRC-5 cells treated with or without ATN-161 in the presence of CCL20. m Immunoblots showing the levels of COL1 and α-SMA in MRC-5 cells treated with or without ATN-161 in the presence of CCL20. n Migratory ability of MRC-5 cells treated with or without ATN-161 in the presence of CCL20. Scale bars, 100 μm. The data are presented as the means ± SEM. For ( i–n ), n = 3 technical replicates per group. The data in i–n were analyzed by one-way ANOVA. *p < 0.05 ; **p < 0.01; ***p < 0.001. Source data are provided as a file.

Article Snippet: Myc-DDK-tagged integrin α5 was obtained from Origene, Myc-tagged integrin β1 and GFP-tagged human CCL20 were obtained from YouBio (Changsha, China), and HA-tagged CCR6 was purchased from Sino Biological, Inc. (Beijing, China).

Techniques: SPR Assay, Expressing, Western Blot

Journal: Nature Communications

Article Title: The CCL20–integrin α5β1 interaction enhances TGF-β/Smad signaling to promote fibroblast activation in pulmonary fibrosis

doi: 10.1038/s41467-025-64211-6

Figure Lengend Snippet: a CoIP analysis of the interaction between LAP and integrin α5. b Ratio of active TGF-β to total TGF-β in the culture medium of CCL20-treated MRC-5 cells, as determined by ELISA (n = 3 technical replicates per group). c TGF-β-reporter luciferase activity in HEK293T cells in response to the cell supernatant of fibroblasts treated with or without CCL20 (n = 4 technical replicates per group). d Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in primary fibroblasts following CCL20 stimulation (n = 3 technical replicates per group). e Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in MRC-5 cells following CCL20 stimulation (n = 3 technical replicates per group). f IF staining for phosphorylated Smad3 in primary fibroblasts stimulated with CCL20. Scale bars, 20 μm. g IF staining for phosphorylated Smad3 in MRC-5 cells stimulated with CCL20. Scale bars, 20 μm. h Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in primary fibroblasts treated with or without ATN-161 in the presence of CCL20 (n = 3 technical replicates per group). i IF staining for phosphorylated Smad3 in primary fibroblasts treated with or without ATN-161 in the presence of CCL20. Scale bars, 20 μm. j Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in MRC-5 cells treated with or without ATN-161 in the presence of CCL20 (n = 3 technical replicates per group). k IF staining for phosphorylated Smad3 in MRC-5 cells treated with or without ATN-161 in the presence of CCL20. Scale bars, 20 μm. l IF staining for α-SMA in primary fibroblasts treated with or without SIS3 in the presence of CCL20. Scale bars, 20 μm. m IF staining for α-SMA in MRC-5 cells treated with or without SIS3 in the presence of CCL20. Scale bars, 20 μm. n Expression of Acta2 in primary fibroblasts treated with or without SIS3 in the presence of CCL20 (n = 3 technical replicates per group). o Expression of ACTA2 in MRC-5 cells treated with or without SIS3 in the presence of CCL20 (n = 3 technical replicates per group). p Schematic diagram of the method used to evaluate the therapeutic effects of ATN-161 on the PF model. q Hydroxyproline levels in lung tissues from the indicated mice after BLM exposure (n = 7 samples for BLM + PBS, BLM + PBS + ATN161, BLM + CCL20 + ATN161, n = 6 samples for BLM + CCL20 + PBS). r Masson’s trichrome staining of lung tissues from the indicated mice after BLM exposure. Scale bar, 100 μm. s The expression levels of Fibronectin and Acta2 in lung tissues from the indicated mice after BLM exposure (n = 6 mice per group). t Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in primary lung fibroblasts isolated from the indicated mice (n = 3 samples per group). The data are presented as the means ± SEM. The data in b–e , s , t were analyzed via two-tailed Student’s t -test. The data in h–j , n–o , q were analyzed via one-way ANOVA. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Source data are provided as a file.

Article Snippet: Myc-DDK-tagged integrin α5 was obtained from Origene, Myc-tagged integrin β1 and GFP-tagged human CCL20 were obtained from YouBio (Changsha, China), and HA-tagged CCR6 was purchased from Sino Biological, Inc. (Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Western Blot, Staining, Expressing, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: The CCL20–integrin α5β1 interaction enhances TGF-β/Smad signaling to promote fibroblast activation in pulmonary fibrosis

doi: 10.1038/s41467-025-64211-6

Figure Lengend Snippet: a SPR analysis of the kinetic interaction of Pep-CCL20 and integrin α5β1. b IP analysis of the interaction between CCL20 and integrin α5 or integrin β1 in the presence of Pep-CCL20. c TGF-β-reporter luciferase activity in HEK293T cells exposed to the supernatant of fibroblasts subjected to the indicated treatments. d Expression of ACTA2 in primary fibroblasts treated with or without Pep-CCL20 in response to CCL20 stimulation. e Immunoblot showing the levels of COL1 in primary fibroblasts treated with or without Pep-CCL20 in response to CCL20 stimulation. f Migration analysis of primary fibroblasts treated with or without Pep-CCL20 in response to CCL20 stimulation. Scale bars, 100 μm. g Expression of ACTA2 in MRC-5 cells treated with or without Pep-CCL20 in response to CCL20 stimulation. h Immunoblot showing the levels of COL1 in MRC-5 cells treated with or without Pep-CCL20 in response to CCL20 stimulation. i Migration analysis of fibroblasts treated with or without Pep-CCL20 in response to CCL20 stimulation. Scale bars, 100 μm. j Schematic diagram of the method for evaluating the therapeutic effects of Pep-CCL20 on the PF model. Masson’s trichrome staining ( k ), hydroxyproline content ( l ) (n = 6 samples per group), protein and mRNA expression of genes associated with fibrosis ( m , n ), and IHC staining for phosphorylated Smad3 ( o ) in lung tissues from PBS- or Pep-CCL20-treated PF mice (n = 6 samples per group). Scale bars, 100 μm ( k ) and 50 μm ( m, o ). The data are presented as the means ± SEM. For ( c–i ), n = 3 technical replicates per group. For ( m–o ), n = 3 samples per group. The data in m , o were analyzed via two-tailed Student’s t -test. The data in c–i , l were analyzed by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001. Source data are provided as a file.

Article Snippet: Myc-DDK-tagged integrin α5 was obtained from Origene, Myc-tagged integrin β1 and GFP-tagged human CCL20 were obtained from YouBio (Changsha, China), and HA-tagged CCR6 was purchased from Sino Biological, Inc. (Beijing, China).

Techniques: Luciferase, Activity Assay, Expressing, Western Blot, Migration, Staining, Immunohistochemistry, Two Tailed Test