Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 4. Stimulation of IVD cells with HTRA1-generated fibronectin fragments. A, concentrated protein supernatants (15 g) from IVD cells treated for 24 h without or with HTRA1mac (5 g/ml) or HTRA1macSA (5 g/ml) were subjected to immunoblotting using antibody Mab1935 specific for the fibronectincarboxyl-terminalheparin-bindingdomain(Cterminus)orMab1936specificforthefibronectinamino-terminalfibrin-andheparin-bindingdomain (N terminus). Fibronectin fragments containing the amino-terminal fibrin- and heparin-binding domain are identified by the closed arrowhead. B, purified human plasma-derived fibronectin (Fn) was incubated with HTRA1mac or HTRA1macSA at equimolar concentrations in TBS, pH 8.5, for 16 h at 37 °C, and samples were loaded onto a 4–15% gradient gel and stained with Coomassie Blue. Fibronectin and recombinant HTRA1 alone were also loaded and served as controls. C, an equimolar concentration of human plasma-derived fibronectin and HTRA1mac were incubated for 16 h, and fibronectin fragments were visualized by Western blot analysis using the antibodies described in A. D, equimolar concentrations of fibronectin (20 g) and HTRA1mac (5 g) were incubated for 16 h, and fibronectin fragments were purified by affinity chromatography. IVD cells were incubated with purified HTRA1-digested fibronectin (FnHTRA1mac) for 24 h, and expression levels of MMP1, MMP3, and MMP13 mRNA were determined by qRT-PCR and the -fold change as compared with untreatedcontrolswasdeterminedusingthe2CTmethod.Additionalcultureswereincubatedwitheitheraffinity-purifiedTris-bufferedsaline,pH7.6(TBS), fibronectin (Fn), or HTRA1 (HTRA1mac) or left untreated (Control). Data are representative of two separate experiments performed using IVD cells from two patients. Shown are results of triplicate determinations S.D. *, p 0.01, as determined by one-way ANOVA.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Generated, Western Blot, Binding Assay, Purification, Clinical Proteomics, Derivative Assay, Incubation, Staining, Recombinant, Concentration Assay, Affinity Chromatography, Expressing, Quantitative RT-PCR, Control

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 5. Detection of fibronectin fragments in degenerated IVD tissue. A, fibronectin (FN) mRNA levels in intact IVD tissue samples from patients (n 36) with varying degrees of IVD degeneration were determined by qRT-PCR and presented as 2CT S.E. (error bars). B, correlation study between FN and HTRA1 mRNA levels (2CT) in patient IVD tissue samples (n 36). R2, square of correlation coefficient; p 0.01 as determined from Pearson’s correlation coefficient. C, protein extracts from patient IVD tissues (n 12) were loaded onto a 12% SDS-polyacrylamide gel, and immunoblotting was performed using a monoclonal antibody (Mab1936) specific for the amino-terminal fibrin- and heparin-binding domain. D, the PVDF membrane used in C was stained with Coomassie Blue in order to confirm equal protein loading. Lane 1, HTRA1-digested human plasma-derived fibronectin; lanes 2–4, non-degenerated (ND) discs; lanes 5–7, mildly degenerated discs; lanes 8–10, moderately degenerated discs; lanes 11–13, severely degenerated discs.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Quantitative RT-PCR, Western Blot, Binding Assay, Membrane, Staining, Clinical Proteomics, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

doi: 10.1074/jbc.m112.341032

Figure Lengend Snippet: FIGURE 6. A theoretical model for the role of HTRA1 in IVD degeneration. Based on our findings, we propose that HTRA1 accumulates in IVD tissue undergoing degeneration and stimulates MMP production by resident cells in a predominantly protease-dependent manner, via activation of the MEK pathway. Furthermore, we suggest that the stimulatory effects of HTRA1 on IVD cells are mediated indirectly through its ability to generate fibronectin fragments, although other routes of cellular activation cannot be ruled out. IDD, intervertebral disc degeneration.

Article Snippet: Materials—Human fibronectin and rabbit IgG were purchased from R & D Systems (Abingdon, UK).

Techniques: Activation Assay

Journal: Journal of functional biomaterials

Article Title: Differential Effects of Extracellular Matrix Glycoproteins Fibronectin and Laminin-5 on Dental Pulp Stem Cell Phenotypes and Responsiveness.

doi: 10.3390/jfb14020091

Figure Lengend Snippet: Figure 3. DPSC growth assay with fibronectin (FN). Five (n = 5) DPSC isolates exhibited statistically significant changes in growth under FN–induced assay conditions, including rDT DPSC isolates dpsc-5653 (+19.68%), dpsc-9765 (+21.32%), dpsc-3882 (+20.45%); iDT DPSC isolate 8604 (+19.94%); and sDT DPSC isolate: dpsc-11418 (+27.86%), p = 0.0000033. (** denotes statistical significance).

Article Snippet: Experimental wells were treated with 10 ng/mL of recombinant human laminin-5 (NBP256854PEP) or fibronectin (NBP261633PEP) from Novus Biologicals (Littleton, CO, USA).

Techniques: Growth Assay

Journal: Journal of functional biomaterials

Article Title: Differential Effects of Extracellular Matrix Glycoproteins Fibronectin and Laminin-5 on Dental Pulp Stem Cell Phenotypes and Responsiveness.

doi: 10.3390/jfb14020091

Figure Lengend Snippet: Figure 5. DPSC growth assay with ECM of combined fibronectin (FN) and laminin-5 (LN5). Five (n = 5) DPSC isolates exhibited statistically significant changes in growth under the combination of FN/LN5-induced assay conditions, including rDT DPSC dpsc-5653 (+24.20%), dpsc-9765 (+37.40%), dpsc-3882 (+19.95%); one iDT DPSC isolate, dpsc-8604 (+47.03%); and one sDT DPSC isolate, dpsc- 11418 (+30.15%), p = 0.0032. (** denotes statistical significance).

Article Snippet: Experimental wells were treated with 10 ng/mL of recombinant human laminin-5 (NBP256854PEP) or fibronectin (NBP261633PEP) from Novus Biologicals (Littleton, CO, USA).

Techniques: Growth 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: 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