Natural product library

Media and reagents for cell culture were from Gibco. The L1 mimetic compounds 6,7-dichloro-1,5-dihydroimidazo (2,1-b) quinazolin-2(3H)-one (anagrelide; CAS 68475-42-3), 5-fluoro-1H-pyrimidin-2-one (2-hydroxy-5-fluoropyrimidine; CAS 2022-78-8), and (8R,9S,13S,14S,17R)-17-ethynyl-3-methoxy-13-methyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-17-ol (mestranol; CAS 72-33-3) were from Sigma-Aldrich (St. Louis, MO, USA). Ortho-phenylenediamine dihydrochloride (OPD, CAS 615-28-1), calcein-AM (CAS 148504-34-1), and propidium iodide (CAS 25535-16-4) were from Thermo Fisher Scientific (Waltham, MA, USA). The NIH Clinical Collection libraries 1 and 2 were from Evotec (San Francisco, CA, USA) and the Natural Product Library was from Selleckchem (Houston, TX, USA). The rat anti-mouse L1 monoclonal antibody 324 (CAS MAB5272) was from Sigma-Aldrich, and secondary donkey anti-rat IgG antibodies coupled to horseradish peroxidase (HRP) were from Jackson ImmunoResearch (West Grove, PA, USA). Recombinant mouse L1CAM-Fc (L1-Fc) chimera (CAS 5674-NC-050) was from R&D Systems (Minneapolis, MN, USA). Human IgG1-Fc tag free protein (CAS FCC-H5214) was from ACROBiosystems (Newark, DE, USA). L1CAM (clone UJ127; catalog# sc-533386) was from Santa Cruz (Dallas, TX, USA). GAPDH (Cat# 60004-1-Ig was from Proteintech (Rosemont, IL, USA)).

Natural Product Library and OA were purchased from Selleck (Houston, United States). H9c2 cells (10⁴ cells per well) were seeded on 96-well plates and cultured in the incubator (95% air, 5% CO₂) at 37°C for 24 h. Subsequently, 2,661 natural compounds were added into each well at a final concentration of 10 μM at the initiation of OGD/R. At the end of the experiment, the cell viability was detected by Cell Counting Kit-8 (CCK-8, Biosharp, China) according to the manufacturer’s instructions and the screened candidate compound, which was OA. To determine the optimal concentration of OA administration, H9c2 cells were treated by different concentrations, which were 5 μM, 10 μM, 100 μM, and 1,000 μM, respectively. Finally, 10 μM was used as the optimal concentration for subsequent experiments. The cells were randomly assigned either into the Control group, the OGD/R group, or the OGD/R + OA group. The latter group represents those cells that were treated with 10 µM OA and subjected to OGD/R procedure.

The Marine Natural Product Library containing 103 compounds was obtained from the Natural Product Library (Cat #L1400; Selleck, Shanghai, China) by searching the Dictionary of Marine Natural Products in the ChemNetBase website (https://dmnp.chemnetbase.com/faces/chemical/ChemicalSearch.xhtml) and applied for preliminary screens against T. gondii. Estradiol benzoate (Cat #S4110; Selleck) and octyl gallate (Cat #S9338; Selleck) were also obtained from Selleck. These compounds were dissolved with dimethyl sulfoxide (DMSO) into a 10 mM solution and stored at −20°C. Pyrimethamine (Cat # 46706, Sigma-Aldrich, Shanghai, China) was used as a reference drug. All information of compounds is listed in Supplementary Table S1.