Chelerythrine chloride

Cells were grown in Dulbecco’s minimal essential medium (Gibco) with penicillin/streptomycin and 10% fetal bovine serum in a humidified atmosphere of 10% CO₂ at 37 °C. Experiments were performed with HeLa M cells, a sub-line of HeLa. HCT116 cells were obtained from Dr. B. Vogelstein^{61 (link)}, HT-1080 fibroblast cells were from Dr. G.R Stark and wild type and gsk3−/− mouse embryo fibroblast (MEF) were obtained from Dr. Jim Woodgett^{62 (link)}. Cells were synchronized by treating with 2 mM thymidine (Sigma Aldrich) for 20–21 hours for HeLa cells and 24 hours for HCT116 cells, followed by release for 9–10 hours before adding respective drugs.
Taxol (100 nM or 1 μM) (Cayman Chemical), Epothilone (20 nM) (Sigma-Aldrich) or Nocodazole (2 μM) were added to arrest cells in mitosis, after thymidine release where mentioned. The proteasome inhibitor MG132 (Cayman chemical) was used at a concentration of 20 μM and kinase inhibitors were used at previously determined effective concentrations: 2.5 μM ZM447439 (AstraZeneca), 2 μM Reversine (Calbiochem), 2.8 μM LY204002 (Cayman Chemicals), 30 μM SB415286 (Cayman Chemicals), 400 nM WNTc59 (Cayman Chemicals), RO 318220 (Cayman Chemicals), 60 mM Lithium Chloride, 14 μM RO3306 (Cayman Chemicals) and 10 μM Chelerythrine Chloride (Cayman Chemicals). Finally, cell selection was accomplished using puromycin at 2 μg/μL (Invitrogen).

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.

Seven-week-old female C57BL/6 mice were purchased from Charles River and allowed to habituate to the facility for 7 days, followed by 3 days of handling before the start of the experiment. Mice were randomly assigned into groups, with all groups represented in each cage. Depending on the treatment group, lithium chloride (Sigma-Aldrich, 12.8 mg/kg in 0.9% saline), chelerythrine chloride (Cayman Chemical, 2 mg/kg in 0.4% DMSO in saline), or the appropriate vehicle was administered intraperitoneally 1 h before injection of vehicle (20% 50:50 Cremophor EL: ethanol, 80% saline) or paclitaxel (Cayman Chemical, 20 mg/kg in 20% 50:50 Cremophor EL: ethanol, 80% saline) to induce cognitive impairment. Each mouse received a total of 4 pairs of injections over 8 days. During and after injections, mice were weighed daily and checked for general health and any sign of pain or distress.