human dermal blood endothelial cells  (PromoCell)

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: Primary human dermal blood endothelial cells (HDBEC) were obtained from PromoCell (Heidelberg, Germany) and cultured in MV media containing 5% ( v / v ) FCS and growth supplements (PromoCell).

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: 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: Primary human dermal blood endothelial cells (HDBEC) were obtained from PromoCell (Heidelberg, Germany) and cultured in MV media containing 5% ( v / v ) FCS and growth supplements (PromoCell).

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: Primary human dermal blood endothelial cells (HDBEC) were obtained from PromoCell (Heidelberg, Germany) and cultured in MV media containing 5% ( v / v ) FCS and growth supplements (PromoCell).

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: Primary human dermal blood endothelial cells (HDBEC) were obtained from PromoCell (Heidelberg, Germany) and cultured in MV media containing 5% ( v / v ) FCS and growth supplements (PromoCell).

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: Primary human dermal blood endothelial cells (HDBEC) were obtained from PromoCell (Heidelberg, Germany) and cultured in MV media containing 5% ( v / v ) FCS and growth supplements (PromoCell).

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