primary human dermal blood microvascular endothelial cells endothelial cells hdbec  (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, 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

Journal: Toxins

Article Title: Evaluation of Signaling Pathways Profiling in Human Dermal Endothelial Cells Treated by Snake Venom Cysteine-Rich Secretory Proteins (svCRiSPs) from North American Snakes Using Reverse Phase Protein Array (RPPA)

doi: 10.3390/toxins13090613

Figure Lengend Snippet: The effect of Css-CRiSP and App-CRiSP on signaling pathways in HDBEC and HDLEC cells. HDBEC and HDLEC cells were treated for 30 min with Css-CRiSP and App-CRiSP (1 µM) or PBS (control) and protein lysates were analyzed by RPPA. ( a ) Heat map of protein expression profiles based on RPPA. Values are expressed as log2 fold difference from the control (PBS) samples. Green color represents high protein expression, and red indicates low protein expression. Fold change of the selected proteins in HDBECs ( b ) and HDLECs ( c ) compared to the PBS control. Data are presented as mean ± SD from two independent experiments (n = 2). Statistical significance determined by Student’s t -test (* p < 0.05).

Article Snippet: Primary HDBEC and HDLEC cells were obtained from PromoCell (PromoCell GmbH, Heidelberg, Germany).

Techniques: Expressing

Journal: Cancers

Article Title: De-Palmitoylation of Tissue Factor Regulates Its Activity, Phosphorylation and Cellular Functions

doi: 10.3390/cancers13153837

Figure Lengend Snippet: Analysis of TF-tGFP procoagulant activity and fVIIa-HRP binding in the presence and absence of TF palmitoylation. ( A ) HDBEC (5 × 10 4 ) were transfected with PPT-siRNA (0–15 pmol) and the expression of PPT1 and -2 measured by western blot. ( B ) HDBEC were co-transfected with combinations of pCMV-Ac-TF-tGFP and PPT-siRNA or control siRNA, and fXa-generation was measured in resting and PAR2-activated cells. ( n = 5, * = p < 0.05 vs. the respective non-activated sample.) ( C ) HDBEC were co-transfected to express TF-tGFP, with PPT siRNA or control siRNA. Factor Xa-generation was measured before and after PAR2 activation for up to 60 min. ( n = 4, * = p < 0.05 vs. the respective control siRNA sample.) HDBEC were transfected to express ( D ) TF Ser245 -tGFP or ( E ) TF Phe245 -tGFP and fXa-generation were measured as above. ( n = 6, * = p < 0.05 vs. the respective cells expressing TF Wt -tGFP.) ( F ) MDA-MB-231 (5 × 10 4 ) cells expressing endogenous TF were transfected with PPT-siRNA or control siRNA and fXa-generation measured ( n = 3, * = p < 0.05 vs. cells without siRNA).

Article Snippet: Human dermal blood primary endothelial cells (HDBEC) and coronary artery endothelial cells (HCAEC) devoid of endogenous TF were cultured in MV media containing 5% ( v / v ) foetal calf serum (FCS) and growth supplements (PromoCell, Heidelberg, Germany).

Techniques: Activity Assay, Binding Assay, Transfection, Expressing, Western Blot, Activation Assay

Journal: Cancers

Article Title: De-Palmitoylation of Tissue Factor Regulates Its Activity, Phosphorylation and Cellular Functions

doi: 10.3390/cancers13153837

Figure Lengend Snippet: Analysis of Ser253 phosphorylation in the TF variants. HDBEC (2 × 10 5 ) were transfected ( A ) to express TF Wt -tGFP in the presence and absence of PPT-siRNA or ( B ) to express TF Ser245 -tGFP or TF Phe245 -tGFP. Cells were activated with PAR2-AP (20 µM) for 20 min, and TF-tGFP was then immunoprecipitated and analysed by western blot and ( C ) the phosphorylation of Ser253 was quantified. ( n = 3, * = p < 0.05 vs. the respective cells expressing TF Wt -tGFP.) ( D ) HDBEC (2 × 10 5 ) were transfected to express TF Phe241 -tGFP, TF Ser242 -tGFP, TF ShortTM -tGFP or TF Val225 -tGFP, and the phosphorylation of Ser253 was analysed and ( C ) quantified. ( E ) HDBEC (5 × 10 4 ) were transfected with the TF variants as shown, and the concentration of TF released from cells was measured by TF-ELISA. ( n = 4, * = p < 0.05 vs. the respective cells expressing TF Wt -tGFP.) ( F ) HDBEC (5 × 10 4 ) were transfected to express TF Wt -tGFP and pre-incubated with Gö6976 (100 nM) prior to PAR2-activation and fXa-generation measured in resting and activated cells. ( n = 4). ( G ) MDA-MB-231 (5 × 10 4 ) cells expressing endogenous TF were incubated with Gö6976 and fXa-generation measured ( n = 3).

Article Snippet: Human dermal blood primary endothelial cells (HDBEC) and coronary artery endothelial cells (HCAEC) devoid of endogenous TF were cultured in MV media containing 5% ( v / v ) foetal calf serum (FCS) and growth supplements (PromoCell, Heidelberg, Germany).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay