S1076

The following compounds were used under indicated conditions: Ro-3306 (CDK1 inhibitor, 9 μM, 20 h, S7747; Selleck Chemicals, Houston, TX); anisomycin (500 nM, 24 h, sc-3524; Santa Cruz Biotechnology, Dallas, TX, USA); thapsigargin (100 nM, 24 h, 586005; Sigma-Aldrich); metformin (1 mM, 24 h, 136-18662; FUJIFILM Wako Pure Chemical Corporation); SIN-1 (1 mM, 3 h, ab141525; Abcam); TNF-α (50 ng/ml, 24 h, 300-01 A; PeproTech, Rocky Hill, NJ); cisplatin (2 μM, 24 h, S1166; Selleck Chemicals); doxorubicin (2 μM, 24 h, ab120629; Abcam); SP600125 (JNK inhibitor, 5 μM, 36 h, S1460; Selleck Chemicals); SB203580 (p38 inhibitor, 5 μM, 36 h, S1076; Selleck Chemicals).

Human hepatocarcinoma HepG2 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, 11965-092, Gibco, Grand island, NY, USA) supplemented with 10% fetal bovine serum (FBS, 10270-106, Gibco, Grand island, NY, USA) and 1% penicillin-streptomycin (15140148, Gibco, Grand island, NY, USA) in a humidified incubator containing 5% CO₂ and 95% air at 37°C. The cells were serially subcultured at 1 : 3 split ratio.
HT was prepared in dimethylsulfoxide (DMSO, D2650, Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 100 mM. HepG2 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well in 1 mL DMEM containing 10% FBS for 24 h for attachment. In the experiments with pharmacological inhibitor, the medium was replaced with fresh medium with solvent vehicle (DMSO) or diverse concentrations (10, 25, and 50 μM) of HT or selective inhibitor of p38, SB203580 (SB, 20 μM, S1076, Selleckchem, Houston, USA) for 12 h. An equal amount of DMSO, which the proportion is 1/1000, was always tested as the negative control to assure that the solvent vehicle has no influence.

Wall-climbing 3rd instar larvae were subjected to an electric shock to induce seizure, with or without previous feeding of drug, as described previously (Marley and Baines, 2011 (link)). For drug feeding studies, eggs were laid on food containing drug and larvae were raised until wall-climbing 3rd instar. Concentration of the drugs used are as follows and the most effective concentration shown in Table 2 is underlined: SB203580 (2.6 and 13 µM, S1076, Selleckchem), Losmapimod (2.6, 13 and 26 µM, S7215, Selleckchem), sodium fluoride (1.2 and 2.4 mM, S7920, Sigma-Aldrich), gemcitabine (3.3, 16.5 and 165 µM, G6423, Sigma-Aldrich), Metformin (1.2, 2.4 and 3.6 mM, PHR1084, Sigma-Aldrich), bestatin (81 and 162 µM, J61106.MC, Alfa Aesar), WP1066 (140 and 281 µM, 573097, Merck), valproic acid (0.6, 1.2 and 2.4 mM, P4543, Sigma-Aldrich) and phenytoin (1.6 mM, D4505, Sigma-Aldrich). In response to electroshock, larvae undergo a transitory paralysis during which they tonically contract and, occasionally, spasm (see (Marley and Baines, 2011 (link)) for details on seizure behavior). Recovery time reported represents the average time for larvae to resume normal crawling behaviour and at least 30 larvae were tested for each chemical inhibitor treatment.