Thrombin

Human PDLSCs were seeded in 24-well plates at a density of 25000 cells/cm² and cultured with CM or osteogenic medium (OM) (CM + 0.1 mM dexamethasone, 2 mM β-glycerophosphate, and 50 μg/mL ascorbic acid; all from Sigma-Aldrich, St. Louis, MO, USA), and experimental groups were treated with PAR₁-selective agonist peptide TFLLR-NH2 (100 nM) [22 (link)] (Tocris Bioscience Inc., Bristol, UK) or thrombin (0.1 U/mL) (Sigma-Aldrich, St. Louis, MO, USA) for distinct experimental periods based on the analysis performed. To confirm whether the thrombin-induced effect was specifically mediated by PAR₁, cultures were pretreated with PAR₁-selective antagonist RWJ 56110 (100 nM) [23 (link)] (Tocris Bioscience Inc., Bristol, UK) for 30 min prior to thrombin stimulation. Culture medium with its specific treatments (PAR₁ agonist, thrombin, and thrombin + PAR₁ antagonist) was changed every two days. Supernatant and cells were collected for further analysis.