Glibenclamide

HSAV was produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. (Zhenjiang, China), and was diluted to the suitable concentrations with purified water. Glibenclamide was purchased from MedChemExpress (Shanghai, China) and was dissolved in the normal saline containing 10% DMSO, 40% PEG300 and 5% Tween-80 for rat administration.

To elucidate the role of the endothelium, K⁺ channel, and PI3K/Akt/Rho-kinase pathways in GPS-mediated vasodilation, the aortic rings with intact endothelium were preincubated with nitric oxide synthase (NOS) inhibitor (100 μM L-NAME), cyclooxygenase (COX) inhibitor (100 μM indomethacin), and soluble guanylyl cyclase (sGC) inhibitor (100 μM methylene blue), respectively, for 30 min, while the aortic rings without endothelium were preincubated with different K⁺ channel blockers of tetraethylammonium chloride (TEA, 10 mM), 4-aminopyridine (4-AP, 1 mM), BaCl₂ (1 mM), glibenclamide (0.01 mM), L-type Ca²⁺ channel inhibitor (100 μM verapamil), PI3K/Akt inhibitor (10 μM LY294002), and Rho-kinase inhibitor (10 μM Y27632) for 30 min (all inhibitors were obtained from MedChem Express, NJ, USA). After achieving a plateau of PE-induced contracted tension, GPS was added in a cumulative manner (20, 80, and 160 μΜ) for 20 min, and the concentration-response curves were recorded.

Cells were seeded at appropriate densities/100 µL/well in 96-well plates in 8 replicates. Detailed densities are listed in Supplementary Table S1. Cells were treated with inhibitors, chemotherapeutics, or in combinational approaches simultaneously at the indicated concentrations for 72 h if not stated otherwise. Cell viability was measured indirectly by fluorescence detection with a Spark^® multimode microplate reader (Tecan, Männedorf, Switzerland) after 2 h of Resazurin treatment (30 µg/mL, R12204; Thermo Fisher Scientific, Waltham, MA, USA). Experiments were performed in triplicates. Graphpad Prism software v8.4.3 (RRID:SCR_002798) was used for the calculation of IC₅₀ values and SynergyFinder (RRID:SCR_019318) was used for the modulation and calculation of synergy scores [26 (link)]. In combinational analyses with inhibitors, stable and non-lethal concentrations (approximately 10% reduction in cell viability or published concentrations) were used: 4 µM GSK126 (HY-13470; MedChemExpress, Sollentuna, Sweden), 4 µM EPZ6438 (HY-13803; MedChemExpress, Sollentuna, Sweden), 125 nM DZNep (Cay-13828; Biomol, Hamburg, Germany), 1 µM Fatostatin (HY-14452; MedChemExpress, Sollentuna, Sweden), or 30 µM Glibenclamide (HY-15206; MedChemExpress, Sollentuna, Sweden) [27 (link)].