Ozagrel

N^ω-nitro-L-arginine methyl ester (L-NAME), ACh, phenylephrine (PE) and the COX substrate, AA were purchased from Sigma (St Louis, MO). PGI₂, a stable PGI₂ analogue and IP receptor agonist iloprost, the COX-1 selective inhibitor FR122047, which has an IC₅₀ value of 0.03 or 65 μM for COX-1 and COX-2, respectively [39 (link)], the TP receptor antagonist SQ29548, the IP receptor antagonist CAY10441, and the TxA₂ synthase (TXAS) inhibitor ozagrel, which has an IC₅₀ value of 11 nM [40 (link)], were bought from Cayman Chemicals (Ann Arbor, MI). L-NAME, PE, ACh, iloprost, AA, and FR122047 were dissolved in distilled water (purged with N₂ for dissolving AA), while PGI₂ was dissolved in carbonate buffer (50 mM, pH 10.0). SQ29548, CAY10441, and ozagrel were dissolved in DMSO at 2,000-fold working concentration (the final concentration of DMSO was 0.05/100, v/v).
The compositions of physiological salt solution (PSS; pH 7.4 with 95%O₂-5% CO₂) were as follows (in mM): NaCl 123, KCl 4.7, NaHCO₃ 15.5, KH₂PO₄ 1.2, MgCl₂ 1.2, CaCl₂ 1.25, and d-glucose 11.5. The 60 mM K⁺-PSS (K⁺) was prepared by replacing an equal molar of NaCl with KCl.

Huh7 cells were transfected at 70% confluency in DMEM growth media containing 5% FBS with 8xGTIIC-Tead4-luciferase (Addgene), a 624b YAP1 enhancer-luciferase construct with or without the mutations in the first intronic TCF site^{7 (link)}, and renilla-luciferase plasmids (Addgene) using Biolab transfection reagent. After overnight, the cells were treated with 12(S)-HHTrE (50 nM, cat#34590),12-HETE (100 nM, cat#34570), Ozagrel (1 µM, cat#70515), LY255283 (10 µM, cat#70715) obtained from Cayman Chemical Company and the YAP-TEAD binding inhibitor Super-TDU (1-31) TFA (300 nM, MedChemexpress, cat#HY-P1728A) with respective vehicle controls in serum-free medium for 24 h. Protein concentrations were determined in cell lysates and equal amounts of proteins were analyzed for firefly and renilla luciferase activities using Dual-Glo Luciferase Assay Kit (Promega).

Rats implanted with ventral hippocampal electrodes and dorsal hippocampal optodes were used in these studies. Rats were reused for several drug experiments allowing at least two days between drug treatments. Celecoxib, SC-560, ibuprofen, chelerythrine chloride, milrinone, sildenafil, SKA-31, CAY-10526, seratrodast, ozagrel, paxilline, 2-APB, levetiracetam, and topiramate were obtained from Cayman Chemicals (Ann Arbor, MI). Acetaminophen, nifedipine, bumetanide, ethosuximide, phenytoin, and valproic acid were obtained from Sigma-Aldrich. Lamotrigine was obtained from SelleckChem (Houston, TX). Fasudil was obtained from LC laboratories (Woburn, MA) and phenobarbital was obtained from Strathcona Prescription Center (Canada). Lipophilic drugs were dissolved in 100% DMSO, while hydrophilic drugs were dissolved in saline and injected 30 min prior to seizure induction. The seizure duration and severity of hypoxia (area below 10 mmHg) were compared across kindle (seizure without injection), vehicle-, and drug-treated groups using a within-subject ANOVA and follow-up t-test between vehicle- and drug-treated groups.