Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Structure of TF and β1-integrin protein constructs. ( A ) Depictions of the sections of the TF protein present in the TED (residues 1–219), LED (residues 106–219) and UED (residues 1–110) protein constructs, produced using the crystal structure of the extracellular domain of TF (1TFH). ( B ) β3-integrin (4G1E) was used as a proxy for β1-integrin, as the crystal structure of β1-integrin has not been published. In β3-integrin, the EGF4 spans residues 563–603 and the βTD residues 604–695. The corresponding domains within β1-integrin were estimated by homology comparison, with the EGF4 domain estimated to span residues 572–610, and βTD was localised to residues 611–728.

Article Snippet: The cells were incubated with mouse anti-HA-tag antibody (1:50 v / v ; C29F4; Cell Signalling) paired with either rabbit polyclonal anti-TF antibody (1:50 v / v ; ab104513; Abcam, Cambridge, UK) or rabbit polyclonal anti-β1-integrin antibody (1:50 v / v ; GTX112971; GeneTex) overnight at 4 °C.

Techniques: Construct, Produced, Comparison

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Association of TF extracellular domain peptide constructs with cellular β1-integrin in HDBEC. HDBEC (5 × 10 4 ) were transfected to express TED, LED or UED constructs or control peptides. The interactions between the expressed peptides and β1-integrin were assessed by PLA using mouse anti-HA-tag (C29F4) and rabbit anti-β1-integrin antibodies. Positive controls were prepared using mouse anti-TF (HTF1) and rabbit anti-TF (FL295) antibodies, as well as mouse anti-TF (HTF1) and rabbit anti-β1-integrin antibodies. A negative control was prepared using the mouse anti-HA-tag antibody (C29F4) paired with a rabbit IgG isotype control antibody. ( A ) The cells were examined by fluorescence microscopy at ×40 magnification. ( B ) The number of red fluorescent interaction events and blue cell nuclei in each field of view was quantified using ImageJ.

Article Snippet: The cells were incubated with mouse anti-HA-tag antibody (1:50 v / v ; C29F4; Cell Signalling) paired with either rabbit polyclonal anti-TF antibody (1:50 v / v ; ab104513; Abcam, Cambridge, UK) or rabbit polyclonal anti-β1-integrin antibody (1:50 v / v ; GTX112971; GeneTex) overnight at 4 °C.

Techniques: Construct, Transfection, Control, Negative Control, Fluorescence, Microscopy

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Association of TF-derived peptides with cellular β1-integrin in MDA-MB-231 cells. MDA-MB-231 cells (10 4 ) were transfected to express the TED, LED or UED constructs or control peptide. The interactions between the expressed constructs and β1-integrin were assessed by PLA using mouse anti-HA-tag (C29F4) and polyclonal rabbit anti-β1-integrin antibodies. ( A ) The cells were examined by fluorescence microscopy at ×40 magnification. ( B ) The number of red fluorescent interaction events and blue cell nuclei in each field of view was quantified using ImageJ. MDA-MB-231 cells (1.5 × 10 5 ) were transfected to express TED, LED or UED constructs or control peptide for 48 h and were then lysed. The recombinant protein constructs were immunoprecipitated from the lysates using the anti-FLAG-tag (4 µg; C29F4) antibody and protein A-magnetic beads. A sample of lysate was also incubated with the protein A-beads. ( C ) Western blot analysis of samples co-precipitated along with the TED, LED or UED constructs was carried out using a polyclonal anti-β1-integrin antibody. Images represent 3 separate experiments. ( D ) The amounts of β1-integrin precipitated from the samples were measured using ImageJ.

Article Snippet: The cells were incubated with mouse anti-HA-tag antibody (1:50 v / v ; C29F4; Cell Signalling) paired with either rabbit polyclonal anti-TF antibody (1:50 v / v ; ab104513; Abcam, Cambridge, UK) or rabbit polyclonal anti-β1-integrin antibody (1:50 v / v ; GTX112971; GeneTex) overnight at 4 °C.

Techniques: Derivative Assay, Transfection, Construct, Control, Fluorescence, Microscopy, Recombinant, Immunoprecipitation, FLAG-tag, Magnetic Beads, Incubation, Western Blot

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Association of EGF4-βTD peptide construct with cellular TF. MDA-MB-231 cells (10 4 ) were transfected to express the EGF4-βTD or EGF4 constructs or control peptide. The interactions between the expressed constructs and TF were assessed by PLA using mouse anti-HA-tag (C29F4) and polyclonal rabbit anti-TF antibodies. ( A ) The cells were examined by fluorescence microscopy at ×40 magnification. ( B ) The number of red fluorescent interaction events and blue cell nuclei in each field of view was quantified using ImageJ. MDA-MB-231 cells (1.5 × 10 5 ) were transfected to express the EGF4-βTD construct or control peptide for 48 h and were then lysed. The recombinant protein constructs were immunoprecipitated from the lysates using the anti-FLAG-tag (4 µg; C29F4) antibody and protein A-magnetic beads. A sample of lysate was also incubated with the protein A-beads. ( C ) Western blot analysis of samples precipitated along with the EGF4-βTD construct was carried out using an anti-TF antibody (HTF-1). Images represent 3 separate experiments. ( D ) The amounts of TF precipitated from the samples were measured using ImageJ.

Article Snippet: The cells were incubated with mouse anti-HA-tag antibody (1:50 v / v ; C29F4; Cell Signalling) paired with either rabbit polyclonal anti-TF antibody (1:50 v / v ; ab104513; Abcam, Cambridge, UK) or rabbit polyclonal anti-β1-integrin antibody (1:50 v / v ; GTX112971; GeneTex) overnight at 4 °C.

Techniques: Construct, Transfection, Control, Fluorescence, Microscopy, Recombinant, Immunoprecipitation, FLAG-tag, Magnetic Beads, Incubation, Western Blot

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Analysis of the availability of the EGF4-βTD domain following expression of TF peptides. ( A ) MDA-MB-231 cells (10 4 ) and ( B ) HDBEC (5 × 10 4 ) were transfected to express TED, LED, UED or control peptides. The cells were probed with an antibody against residues 579–799 of β1-integrin (EGF4 and βTD domains). The cells were then probed with HRP-conjugated anti-rabbit IgG antibody and developed using TMB-one solution HRP substrate. The amount of bound antibody was quantified by measuring the absorptions at 450 nm using a plate reader. Values were normalised to the number of cells in each well, as determined by crystal violet assay measurements.

Article Snippet: The cells were incubated with mouse anti-HA-tag antibody (1:50 v / v ; C29F4; Cell Signalling) paired with either rabbit polyclonal anti-TF antibody (1:50 v / v ; ab104513; Abcam, Cambridge, UK) or rabbit polyclonal anti-β1-integrin antibody (1:50 v / v ; GTX112971; GeneTex) overnight at 4 °C.

Techniques: Expressing, Transfection, Control, Crystal Violet Assay

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: The influence of TF extracellular domain peptide constructs on the conformation of cellular β1-integrin. ( A , B ) MDA-MB-231 cells (10 4 ) and ( C , D ) HDBEC (5 × 10 4 ) were transfected to express TED, LED, UED or control peptides. The cells were probed with antibodies that specifically recognised the ( A , C ) active/open (9EG7) or ( B , D ) inactive/closed (AIIB2) conformation of β1-integrin. The cells were then probed with HRP-conjugated anti-rat IgG or anti-rabbit IgG antibodies and developed using TMB-one solution HRP substrate. The amount of bound antibody was quantified by measuring the absorptions at 450 nm using a plate reader. Values were normalised to the number of cells in each well, as determined by crystal violet assay measurements.

Article Snippet: The cells were incubated with mouse anti-HA-tag antibody (1:50 v / v ; C29F4; Cell Signalling) paired with either rabbit polyclonal anti-TF antibody (1:50 v / v ; ab104513; Abcam, Cambridge, UK) or rabbit polyclonal anti-β1-integrin antibody (1:50 v / v ; GTX112971; GeneTex) overnight at 4 °C.

Techniques: Construct, Transfection, Control, Crystal Violet Assay

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Comparison of the influence of protein constructs in TF + MDA-MB-231 and TF − HDBEC.

Article Snippet: The cells were incubated with mouse anti-HA-tag antibody (1:50 v / v ; C29F4; Cell Signalling) paired with either rabbit polyclonal anti-TF antibody (1:50 v / v ; ab104513; Abcam, Cambridge, UK) or rabbit polyclonal anti-β1-integrin antibody (1:50 v / v ; GTX112971; GeneTex) overnight at 4 °C.

Techniques: Comparison, Construct, Phospho-proteomics, Expressing

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: The influence of an inhibitory anti-β1-integrin antibody on the changes in proliferative signalling caused by TF extracellular domain peptide constructs. MDA-MB-231 cells (1.5 × 10 5 ) were transfected to express UED or the control peptide. The cells were pre-incubated with an inhibitory anti-β1-integrin antibody (AIIB2; 10 μg/mL) for 16 h prior to collection. ( A ) Cells were lysed with Laemmli buffer and western blot analysis was carried out using antibodies against total ERK1/2, phosphorylated ERK1/2 and GAPDH. Images represent 4 separate experiments. ( B ) Band intensities were quantified using ImageJ and the ratio of pERK/tERK was calculated. ( C ) Cells were lysed, and the mRNA extracted and converted to cDNA using cell-2-cDNA kit. The expression of cyclin D1 and β-actin mRNA were measured using RT-PCR and the relative cyclin D1 expression levels calculated using the 2 −ΔΔCq method.

Article Snippet: The cells were incubated with mouse anti-HA-tag antibody (1:50 v / v ; C29F4; Cell Signalling) paired with either rabbit polyclonal anti-TF antibody (1:50 v / v ; ab104513; Abcam, Cambridge, UK) or rabbit polyclonal anti-β1-integrin antibody (1:50 v / v ; GTX112971; GeneTex) overnight at 4 °C.

Techniques: Construct, Transfection, Control, Incubation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Proposed model for the interaction between TF and β1-integrin. ( A ) The proposed positioning for the interacting domains during complex formation between TF (green) and β1-integrin (purple). In the model, the upper region of TF extracellular domain interacts with the EGF4 domain of β1-integrin, and the lower region with the βTD domain. ( B ) The structural loop (blue) within the βTD (cyan) of a β3-integrin interacts with residues in the head group of the integrin and may hold the integrin in a closed configuration.

Article Snippet: The cells were incubated with mouse anti-HA-tag antibody (1:50 v / v ; C29F4; Cell Signalling) paired with either rabbit polyclonal anti-TF antibody (1:50 v / v ; ab104513; Abcam, Cambridge, UK) or rabbit polyclonal anti-β1-integrin antibody (1:50 v / v ; GTX112971; GeneTex) overnight at 4 °C.

Techniques:

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Structure of TF and β1-integrin protein constructs. ( A ) Depictions of the sections of the TF protein present in the TED (residues 1–219), LED (residues 106–219) and UED (residues 1–110) protein constructs, produced using the crystal structure of the extracellular domain of TF (1TFH). ( B ) β3-integrin (4G1E) was used as a proxy for β1-integrin, as the crystal structure of β1-integrin has not been published. In β3-integrin, the EGF4 spans residues 563–603 and the βTD residues 604–695. The corresponding domains within β1-integrin were estimated by homology comparison, with the EGF4 domain estimated to span residues 572–610, and βTD was localised to residues 611–728.

Article Snippet: The wells were washed three times with PBST and then incubated with the rabbit polyclonal anti-β1-integrin antibody specific for the EGF4-βTD domains (1:100 v / v in PBST; PA1-37318; ThermoFisher, Loughborough, UK) for 90 min at room temperature.

Techniques: Construct, Produced, Comparison

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Association of EGF4-βTD peptide construct with cellular TF. MDA-MB-231 cells (10 4 ) were transfected to express the EGF4-βTD or EGF4 constructs or control peptide. The interactions between the expressed constructs and TF were assessed by PLA using mouse anti-HA-tag (C29F4) and polyclonal rabbit anti-TF antibodies. ( A ) The cells were examined by fluorescence microscopy at ×40 magnification. ( B ) The number of red fluorescent interaction events and blue cell nuclei in each field of view was quantified using ImageJ. MDA-MB-231 cells (1.5 × 10 5 ) were transfected to express the EGF4-βTD construct or control peptide for 48 h and were then lysed. The recombinant protein constructs were immunoprecipitated from the lysates using the anti-FLAG-tag (4 µg; C29F4) antibody and protein A-magnetic beads. A sample of lysate was also incubated with the protein A-beads. ( C ) Western blot analysis of samples precipitated along with the EGF4-βTD construct was carried out using an anti-TF antibody (HTF-1). Images represent 3 separate experiments. ( D ) The amounts of TF precipitated from the samples were measured using ImageJ.

Article Snippet: The wells were washed three times with PBST and then incubated with the rabbit polyclonal anti-β1-integrin antibody specific for the EGF4-βTD domains (1:100 v / v in PBST; PA1-37318; ThermoFisher, Loughborough, UK) for 90 min at room temperature.

Techniques: Construct, Transfection, Control, Fluorescence, Microscopy, Recombinant, Immunoprecipitation, FLAG-tag, Magnetic Beads, Incubation, Western Blot

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Analysis of the availability of the EGF4-βTD domain following expression of TF peptides. ( A ) MDA-MB-231 cells (10 4 ) and ( B ) HDBEC (5 × 10 4 ) were transfected to express TED, LED, UED or control peptides. The cells were probed with an antibody against residues 579–799 of β1-integrin (EGF4 and βTD domains). The cells were then probed with HRP-conjugated anti-rabbit IgG antibody and developed using TMB-one solution HRP substrate. The amount of bound antibody was quantified by measuring the absorptions at 450 nm using a plate reader. Values were normalised to the number of cells in each well, as determined by crystal violet assay measurements.

Article Snippet: The wells were washed three times with PBST and then incubated with the rabbit polyclonal anti-β1-integrin antibody specific for the EGF4-βTD domains (1:100 v / v in PBST; PA1-37318; ThermoFisher, Loughborough, UK) for 90 min at room temperature.

Techniques: Expressing, Transfection, Control, Crystal Violet Assay

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: The influence of TF extracellular domain peptide constructs on proliferative signalling in MDA-MB-231 cells. MDA-MB-231 cells (1.5 × 10 5 ) were transfected to express TED, LED, UED, EGF4-βTD or the control peptide. ( A ) Cells were lysed with Laemmli buffer, and Western blot analysis was carried out using antibodies against total ERK1/2, phosphorylated ERK1/2 and GAPDH. Images represent 3 separate experiments. ( B ) Band intensities were quantified using ImageJ, and the ratio of pERK/tERK was calculated. ( C ) Cells were lysed, and the mRNA was extracted and converted to cDNA using cell-2-cDNA kit. The expression of cyclin D1 and β-actin mRNA were measured using RT-PCR and the relative cyclin D1 expression levels calculated using the 2 −ΔΔCq method. ( D ) MDA-MB-231 cells (5 × 10 4 ) were transfected to express TED, LED, UED, EGF4-βTD or control peptide. The cells were incubated for 72 h, and the number of cells was determined using the crystal violet assay.

Article Snippet: The wells were washed three times with PBST and then incubated with the rabbit polyclonal anti-β1-integrin antibody specific for the EGF4-βTD domains (1:100 v / v in PBST; PA1-37318; ThermoFisher, Loughborough, UK) for 90 min at room temperature.

Techniques: Construct, Transfection, Control, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Crystal Violet Assay

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Comparison of the influence of protein constructs in TF + MDA-MB-231 and TF − HDBEC.

Article Snippet: The wells were washed three times with PBST and then incubated with the rabbit polyclonal anti-β1-integrin antibody specific for the EGF4-βTD domains (1:100 v / v in PBST; PA1-37318; ThermoFisher, Loughborough, UK) for 90 min at room temperature.

Techniques: Comparison, Construct, Phospho-proteomics, Expressing

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: The influence of TF extracellular domain peptide constructs on proliferative signalling in HDBEC. HDBEC (5 × 10 5 ) were transfected to express TED, LED, UED, EGF4-βTD or the control peptide. ( A ) Cells were lysed with Laemmli buffer, and Western blot analysis was carried out using antibodies against total ERK1/2, phosphorylated ERK1/2 and GAPDH. Images represent 2 separate experiments. ( B ) Band intensities were quantified using ImageJ and the ratio of pERK/tERK was calculated. ( C ) Cells were lysed, and the mRNA extracted and converted to cDNA using cell-2-cDNA kit. The expression of cyclin D1 and β-actin mRNA were measured using RT-PCR and the relative cyclin D1 expression levels calculated using the 2 −ΔΔCq method. ( D ) HDBEC (5 × 10 4 ) were transfected to express TED, LED, UED, EGF4-βTD or control peptide. The cells were incubated for 72 h and the number of cells were determined using the crystal violet assay.

Article Snippet: The wells were washed three times with PBST and then incubated with the rabbit polyclonal anti-β1-integrin antibody specific for the EGF4-βTD domains (1:100 v / v in PBST; PA1-37318; ThermoFisher, Loughborough, UK) for 90 min at room temperature.

Techniques: Construct, Transfection, Control, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Crystal Violet Assay

Journal: Cancers

Article Title: Identification of the Interacting Domains Between Tissue Factor and β1-Integrin and the Signalling Properties of the Two Fibronectin-like Domains of Tissue Factor

doi: 10.3390/cancers17040644

Figure Lengend Snippet: Proposed model for the interaction between TF and β1-integrin. ( A ) The proposed positioning for the interacting domains during complex formation between TF (green) and β1-integrin (purple). In the model, the upper region of TF extracellular domain interacts with the EGF4 domain of β1-integrin, and the lower region with the βTD domain. ( B ) The structural loop (blue) within the βTD (cyan) of a β3-integrin interacts with residues in the head group of the integrin and may hold the integrin in a closed configuration.

Article Snippet: The wells were washed three times with PBST and then incubated with the rabbit polyclonal anti-β1-integrin antibody specific for the EGF4-βTD domains (1:100 v / v in PBST; PA1-37318; ThermoFisher, Loughborough, UK) for 90 min at room temperature.

Techniques:

Journal: International Journal of Implant Dentistry

Article Title: Effects of the application of low-temperature atmospheric plasma on titanium implants on wound healing in peri-implant connective tissue in rats

doi: 10.1186/s40729-024-00524-3

Figure Lengend Snippet: Connective tissue attachment. a Expression of adhesion-related genes during wound healing. Relative gene expression levels were quantified based on Day 3 of Cont. ** p < 0.01, * p < 0.05. b Immunohistochemistry of integrin α2, α5, and β1 during wound healing of peri-implant connective tissue. High-magnification histological evaluation during wound healing in peri-implant connective tissue is shown (bars: 20 μm). The regions of interest (ROIs) for integrin localization were set at the peri-implant connective tissue around the apical part of the peri-implant epithelium (shown with H&E staining, bar: 200 μm). Immunoreaction for each integrin was observed in the peri-implant connective tissue

Article Snippet: For primary antibody reaction, sections were incubated with the following primary antibodies for 2 h at room temperature: anti-integrin α2 rabbit polyclonal antibody (1:200; St John’s Laboratory Ltd., London, UK), anti-integrin α5 rabbit polyclonal antibody (1:200; St John’s Laboratory Ltd.), and anti-integrin β1 rabbit polyclonal antibody (1:200; Proteintech Group Inc., Chicago, IL, USA).

Techniques: Expressing, Gene Expression, Immunohistochemistry, Staining