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: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

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: Immortalised human epithelial cells (human telomerase reverse transcriptase-human pancreatic nestin-expressing ductal cells) were cultured in 25 cm 2 flasks and supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on the indicated weeks, 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=3), and (B) the relative amounts of p16 INKa protein (n=4) calculated from (C) the western blots of p16 INKa protein (using a goat anti-human antibody) and against GAPDH. TF, tissue factor; Wk, week.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Reverse Transcription, Expressing, Cell Culture, 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: Immortalised human epithelial cells (human telomerase reverse transcriptase-human pancreatic nestin-expressing ductal cells) were supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on week 4, gDNA was extracted from the cells (3×10 4 cells) and bisulphite conversion of the gDNA (750 ng) was carried out using the MethylDetector Bisulfite Modification Kit. DNA was also extracted from HT-29 cells (methylated control) and HDBEC (unmethylated control) and processed as aforementioned. The modified DNA (10 ng/reaction) was amplified with methylation-specific, and unmethylated-specific sets primers to the p16 gene promoter region. Each of the nested amplification steps was carried out for 35 cycles at an annealing temperature of 60°C. Aliquots (4 µl) from the outer reactions were then used as the template for the inner PCR reactions using primers specific for methylated and unmethylated DNA. Both amplicons generated bands of 149 bp. A control β-actin sample was also amplified and examined alongside. (A) The products were examined by 2% (w/v) agarose gel electrophoresis and (B) the band intensities determined and the ratios of the methylated:unmethylated DNA calculated (n=3). TF, tissue factor; gDNA, genomic DNA.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Reverse Transcription, Expressing, Recombinant, Modification, Methylation, Control, Amplification, Generated, Agarose Gel Electrophoresis

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: Proposed model for the mechanism by which the level of TF on the cell surface may have differential outcomes on G1/S checkpoint regulation. The presence of TF on the cell surface differentially upregulates the expression of Inhibitor of CDK p16 INKa , CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 and Alternative reading frame p14 ARF , which is dependent on the concentration of TF and the ability of the cell to dissipate excess TF. Therefore, alterations in p21 CIP1/WAF1 are highly effective in the regulation of the cellular response to acute stress. The interplay between these proteins modulates the signal permitting passage through the cell cycle, or alternatively its arrest. Consequently, the concentration of TF may be an ideal gauge for determining the level of cellular damage. However, the adaptive loss of p16 INK4a function may be promoted by prolonged inflammation leading to permissive transition through the G1/S checkpoint, even in the absence of mutational loss of p16 INKa . TF, tissue factor; PAR2, protease-activated receptor 2, Rb, retinoblastoma protein; E2F, early region 2 binding factor; p21 CIP1/WAF1 , CDK interacting protein/wildtype p53-activated fragment; p16 INK4a , inhibitor of CDK; p14 ARF , alternative reading frame.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Expressing, Concentration Assay, Binding Assay