Cay10526

NR (ChromaDex, Los Angeles, CA, USA) was used in cell culture at 0.5 mM for 16–120 h before subsequent assays. COX-2 inhibitor Celecoxib (Cayman, Ann Arbor, MI, USA) or mPTGES-1 inhibitor Cay10526 (Cayman) was used in cell culture at 5 µM or 10 µM for 48 h, respectively. ON-TARGETplus siRNA for knocking down gene expression of SIRT3 and non-targeting control siRNA were purchased from Horizon Discovery (St. Louis, MO, USA). siRNA against human SIRT3 and control siRNA were incubated with a mixture of nucleofection solution (Human Monocyte Nucleofector Kit)(Lonza, Morristown, NJ, USA) and primary human monocytes and placed in nucleofection cuvettes subjected to program Y-010 for the Nucleofector 2b Device (Lonza). RPMI medium (500 µL) was immediately added into cuvettes after nucleofections. Cells were then plated onto a 6-well plate or 10 cm dish and incubated at 37 °C under 5% CO₂ for 4–5 days for macrophage differentiation followed by M1 macrophage polarization. Plasmid transfection using an empty vector (pcDNA3.1) or SIRT1-, or SIRT3-expression constructs was conducted following the same protocol as siRNA transfection.

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.

Cell viability was determined by MTT conversion assay. The cells were seeded in triplicate in 96 well-plates and incubated with increasing concentrations (between 5 and 100 μM) of compounds 1c, 2c and CAY10526 (Cayman Chemical Company), or DMSO 0.1% (v/v) for the 24, 48, or 72 h in DMEM. Following the treatment, 20 μL of MTT solution (5 mg/mL in PBS) [3-4,5-dimethyldiazol-2-yl]-2,5-diphenyl tetrazolium bromide reagent (Sigma-Aldrich), was added, and the cells were incubated for an additional 3 h at 37°C. The formazan crystals thus formed were dissolved in 100 μL of buffer containing 50% (v/v) N,N-dimethylformamide, 20% SDS (sodium dodecyl sulfate) (pH 4.5). The IC₅₀ values were defined as the compound concentration resulting in 50% inhibition of cell survival compared to control cells treated with DMSO. The absorbance was measured at 570 nm with a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States).