Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: HDBECs (2×10 5 ) were incubated for 24 h with recombinant TF (0, 0.5 and 2 U/ml) or PAR2-AP (SLIGKV; 20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to addition of TF. The cells were harvested after 24 h, into separate aliquots (1×10 5 cells). Total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) Inhibitor of CDK p16 INKa mRNA (n=5), and (B) the relative amounts of p16 INKa protein (n=3) calculated from (C) the western blots of p16 INKa protein (using a goat anti-human antibody) and against GAPDH. TF, tissue factor; PAR2, protease-activated receptor 2.

Article Snippet: In other experiments, the cells were pre-incubated at 37°C for 60 min with AIIB2 antibody (20 μg/ml; cat. no. AIIB2-c; Merck KGaA) to block β1-integrin signalling, or with SAM11 antibody (20 μg/ml; cat. no. sc-13504; Santa Cruz Biotechnology, Inc.) to block PAR2 activation prior to the addition of TF.

Techniques: Incubation, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: HDBECs (2×10 5 ) were incubated for 24 h with recombinant TF (0, 0.5 and 2 U/ml) or PAR2-AP (SLIGKV; 20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Groups of cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to the addition of TF. The cells were harvested after 24 h, into separate aliquots (1×10 5 cells). Total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 mRNA (n=5), and (B) the relative amounts of p21 CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21 CIP1/WAF1 protein, (using a mouse anti-human antibody) and against GAPDH. TF, tissue factor; PAR2, protease-activated receptor 2.

Article Snippet: In other experiments, the cells were pre-incubated at 37°C for 60 min with AIIB2 antibody (20 μg/ml; cat. no. AIIB2-c; Merck KGaA) to block β1-integrin signalling, or with SAM11 antibody (20 μg/ml; cat. no. sc-13504; Santa Cruz Biotechnology, Inc.) to block PAR2 activation prior to the addition of TF.

Techniques: Incubation, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Examination of the influence of TF on cell cycle indicators. (A) Groups of HDBECs (1×10 5 ) were transfected with the pGL3-promoter vector containing the E2F-1 enhancer sequence. The cells were then treated for 24 h with recombinant TF (0, 0.5 and 2 U/ml), combinations of TF (0.5 U/ml) with the shown antibodies, or with PAR2-AP (SLIGKV; 20 µM) and the luciferase activity was measured within 24 h (n=3). (B) Groups of cells (5×10 4 ) were seeded in 96-well plates and treated with recombinant TF (0, 0.5 and 2 U/ml) for 24 h. The cells were then fixed and probed with an anti-phospho-Thr821/826 human retinoblastoma protein antibody (1:1,000 v/v) for 1 h. The samples were then incubated with an HRP-conjugated donkey anti-goat IgG diluted 1:3,000 v/v) for 1 h, developed with a TMB substrate and the absorptions determined at 450 nm (n=3). (C) Groups of cells (1×10 5 ) were incubated for 24 h with TF (0, 0.5 and 2 U/ml) or PAR2-AP (20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Groups of cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to addition of recombinant TF. The cells were harvested after 24 h, total RNA was isolated and the amount of cyclin D1 mRNA determined against that of β-actin (n=4). (D) Groups of cells (1×10 5 ) were treated as aforementioned and cell numbers were determined using crystal violet staining (n=3). TF, tissue factor; PAR2, protease-activated receptor 2; E2F-1; Early region 2 binding factor.

Article Snippet: In other experiments, the cells were pre-incubated at 37°C for 60 min with AIIB2 antibody (20 μg/ml; cat. no. AIIB2-c; Merck KGaA) to block β1-integrin signalling, or with SAM11 antibody (20 μg/ml; cat. no. sc-13504; Santa Cruz Biotechnology, Inc.) to block PAR2 activation prior to the addition of TF.

Techniques: Transfection, Plasmid Preparation, Sequencing, Recombinant, Luciferase, Activity Assay, Incubation, Isolation, Staining, Binding Assay