riment. Fig 1. The chemical structure of CYNA. Cynandione A’s Anti-Ischemic Stroke Effects Materials and Methods Experimental tests and validation Reagents. Dulbecco’s Modified Eagle’s Medium and fetal bovine serum were purchased from Gibco. Antibodies for KHSRP, HMGB1, and -actin were obtained from Cell Signaling Technology. Cells cultures and drug treatments. The high differentiated rat pheochromocytoma tumor cell line PC12 was maintained in DMEM containing 10% heat-inactivated equine serum and 5% FBS supplemented with 100 U/mL penicillin and 100 g/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37C. For neuronal cultures, primary cultures of mouse cerebellar granule neurons were prepared from 6- to 8-day-old postnatal mice. Briefly, cerebella were dissected after decapitation and cleaned free of meninges, and then treated with 0.025% w/v trypsin solution for 30 min at 37C. A trypsin inhibitor was then added to block the enzyme, and 0.05% w/v DNase was added to break DNAs from dead cells. After a series of trituration and mild centrifugation steps, the cells were plating in DMEM and 10% FBS supplemented with KCI to a final concentration of 30 mM. Cells were plated onto 24-well dishes containing poly-L-lysine-coated coverslips at a density of 6 105 per well for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19776382 7 days. CYNA was dissolved in dimethyl sulfoxide and were freshly prepared each time before use. For neuroprotection assay, cells were treated with 1, 10 and 100 M CYNA for 6 h before exposure to 5 mM glutamate for PC12 cells or 100 M glutamate for cerebellar granule neurons and then maintained for 24 h. At the end of the treatment period, protein lysates were prepared and western blot analysis was performed. The study protocol was approved by the local institutional review board at the authors’ affiliated institutions and animal experiments were carried out in accordance with the established PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19777101 institutional guidelines for animal care and use at the Second Military Medical University. Cell viability assays. Cell viability was measured by the 2–3-5–2H-tetrazolium monosodium salt method using Cell Counting Kit-8 according to the manufacturer’s instructions. Absorbance was read at 450 nm by a BioTek Synergy 2 plate Crenolanib web reader. Western blotting. Cells were collected by centrifugation at 1000 g for 3 min. The cell pellets were then washed with PBS, resuspended in lysis buffer containing 150 mM NaCl, 50 mM Tris, 0.02% NaN3, 0.01% PMSF, 0.2% aprotinin, 1% tritonX-100 supplemented with protease inhibitor cocktail, and centrifuged at 12,000 g for 10 min. The concentration of total proteins was determined by a BCA kit. 30 g proteins per lane was electrophoresed on 10% SDS polyacrylamide gels after boiling for 5 min and transferred to nitrocellulose membranes. Nonspecific reactivity was blocked by 5% nonfat milk prepared in TBST at room temperature for 1 h. The membranes were incubated with antibodies diluted according to the manufacturers’ instructions. Blots were washed three times in TBST, followed by incubation with the appropriate horseradish peroxidase-linked secondary antibodies for 1 h at room 
temperature. The proteins in the blots were visualized using the ECL plus system. Statistical Analysis. Data were analyzed using GraphPad Prism software version 5. Multiple comparisons were compared by one-way ANOVA analysis of variance followed by Tukey post hoc test. The quantitative data were 3 / 15 Cynandione A’s Anti-Ischemic Stroke Effects reported as means SEM from at least
