Tfllr nh2

An antibody against PAR1 was purchased from BECKMAN COULTER. Anti-E-cadherin, fibronectin, YAP1, phospho-YAP1 (pYAP1), Lats1 and phospo-Lats1 (pLats1) were purchased from Abcam. Anti-ABCG2, -MRP1, and -P-glycoprotein (P-gp) were purchased from GeneTex. Anti- ribophorin II (RPN2) was purchased from OriGene. Anti-GAPDH was from IMGENEX. The selective PAR1 agonist TFLLR-NH₂ and PAR1 antagonist SCH79797 was purchased from Tocris Bioscience. We used TFLLR-NH₂ (EC50: 1.9 μM) at the 20 μM, and SCH79797 (IC50: 70 nM) at the 70 nM. C3 transferase and Y27932 were purchased from Cytoskeleton. We used C3 at the 2 μg/ml and Y27632 at 10 μM. Small interfering RNA (siRNA) directed against PAR1 (5′-AAGGCUACUAUGCCUACUACU-3′) was synthesized by TOYOBO, and siRNA directed against YAP1 (5′-GGUCAGAGAUACUUCUUA-3′) and Snail (5′- CCACAGAAAUGGCCAUGGGAAGGCCUC-3′) were synthesized by Sigma. Control siRNA is a scrambled sequence with no homology in the human genome (Qiagen) is listed as follows: Scrambled, UUCUCCGAACGUGUCACGUdTdT.

Electrodes made from borosilicate capillaries (tip diameter ranging from 1.5–2 μm; BF150-86-7.5, Sutter Instrument, USA) were either filled with TFLLR-NH₂ (10 μM; Tocris Bioscience, UK), ATP (1 mM; Sigma–Aldrich, St. Louis, MO, USA) or adenosine (1 mM; Sigma–Aldrich, St. Louis, MO, USA). Drugs were puff applied at 14–35 Pa by a homemade time-controlled pressure device.

Male C57BL/6 mice were purchased from Hana Biotech (Ansan, Korea). Mice were housed under a 12 h light/dark cycle and allowed ad libitum access to food and water. The animal protocol used in this study was reviewed by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC) as per their ethical procedures and scientific care, and it has been proven (PNU-2017-1477, PNU-2018-1828). Euthanasia is considered when eating less 40% of food and water, unstable breathing, and continuous for humane endpoint. We assessed and monitored the condition of the feeding and postsurgery daily for any signs of decreased activity and a decrease in food intake. We did not observe any signs of these conditions. Mice were orally administered 0.15 mL of each Tongxuewan or Weisheng-tang extract in 1x PBS at the appropriate concentration once per day for 4 days, as well as 1 h prior to focal cerebral ischemia (a total of five treatments). Experimental drugs, including PAR-1 agonist (TFLLR-NH₂: 3 μmol/kg in 40 μL saline, Tocris, Bristol, UK) or control peptide (RLLFT-NH₂: 3 μmol/kg in 40 μL saline, Tocris) [9 (link)], were injected into the tail vein, 30 min prior to ischemic brain injury.

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.