Sodium valproate

For the competitive displacement assay, rat synaptosomal membrane extract (0.2 mg protein/tube) was incubated for 120 minutes at 4°C with 20 nM [³H]E2730 and the following ASMs: carbamazepine (FUJIFILM Wako Pure Chemical Corporation; 100 μM); lamotrigine (Sigma‐Aldrich, MO; 300 μM); diazepam (FUJIFILM Wako Pure Chemical Corporation; 10 μM); gabapentin (Tokyo Chemical Industry; 300 μM); ethosuximide (Tokyo Chemical Industry; 3000 μM); perampanel (Eisai Co., Ltd.; 30 μM), retigabine (LKT Laboratories; 100 μM); levetiracetam (Tokyo Chemical Industry; 1000 μM); acetazolamide (Sigma‐Aldrich; 300 μM); sodium valproate (FUJIFILM Wako Pure Chemical Corporation; 3000 μM); zonisamide (FUJIFILM Wako Pure Chemical Corporation; 300 μM); and topiramate (Sigma‐Aldrich; 300 μM). Unlabeled E2730 (0.1–10 μM) was also assessed to confirm a representative displacement. Non‐specific binding was defined as the residual binding observed in the presence of 1 mM unlabeled E2730. Radioactivity of the synaptosome‐bound radioligand was measured in the same way as described above.

MTS assay using CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI, USA), containing a tetrazolium compound [(3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES) was adopted as a colorimetric method for determining the number of viable cells in the cell proliferation or cytotoxicity assays. The absorbance at 490nm was recorded using a Multiskan FC Microplate Photometer (Thermo Scientific).
For the proliferation assay, BAP1 knocked-down GBC cells were seeded on 96-well plates and measured with the MTS assay on days 0, 1, 3, 5, and 7. The data were presented as relative increases of the average intensity compared to the control group.
For drug sensitivity, BAP1 knocked-down cells were incubated on 96-well plates for 3 days after administration of the drugs, gemcitabine (GEM; Wako, Osaka, Japan), fluorouracil (5-FU; Wako), cisplatin (CDDP; Wako), sodium valproate (Wako), 5-azacytidine (Wako), and bortezomib (Wako). Cell viability was measured by MTS assay and the obtained data were analyzed by calculating the area under the curve (AUC) of each group using GraphPad Prism (ver.7.0, GraphPad Software, San Diego, CA, USA).

Drugs used were lamotrigine (Toronto Research Chemicals Inc., Canada), carbamazepine, phenytoin, sodium valproate (Wako Pure Chemical Industries, Ltd., Osaka, Japan), oxcarbazepine, gabapentin, topiramate, and bupivacaine hydrochloride (Tokyo Chemical Industries, Co. Ltd., Tokyo, Japan). Levobupivacaine hydrochloride was kindly gifted by Maruishi Pharmaceutical Co. Ltd. (Osaka, Japan). All of drugs (except for gabapentin, topiramate, and sodium valproate which were directly dissolved in Ringer's solution) were first dissolved in dimethyl sulfoxide (DMSO) as a stock solution and then diluted to the desired concentrations in Ringer's solution immediately before use, where the concentration of DMSO was less than 2%. Drugs at concentrations larger than 10 mM were not tested, because a change in osmotic pressure may affect CAPs. The pH of Ringer's solution containing drugs was adjusted to 7.0 with NaOH.