Mibefradil dihydrochloride hydrate

For work involving parasites, chlorpheniramine maleate salt, chlorpromazine hydrochloride, desipramine hydrochloride, promethazine hydrochloride, verapamil hydrochloride and CQ diphosphate (all from Sigma-Aldrich) were dissolved in PBS to a working concentration of 1 mM. LynxTag-CQ_GREEN (BioLynx Technologies, Singapore; hereafter abbreviated to ‘CQ_GREEN’) was dissolved in DMSO to the same concentration. All compounds were stored at −20°C and protected from light. For microsome uptake assays, methiothepin mesylate salt, metergoline, loperamide hydrochloride, octoclothepin maleate salt, mibefradil dihydrochloride hydrate, L703,606 oxalate salt hydrate, and chlorprothixene hydrochloride (all from Sigma-Aldrich) were dissolved in DMSO to 10 mM and stored at 4°C. verapamil hydrochloride, adenosine triphosphate (ATP), and CQ diphosphate (all from Sigma-Aldrich) were dissolved in water to 7.5 mM, 50 mM and 0.1 M respectively and stored at −20°C. Tritiated CQ (³H-CQ; from Moravek Biochemicals and Radiochemicals) was diluted in water to 5.32 µM and stored at −20°C; specific activity was 4.7 Ci/mmol.

Metabolic activity of cells as a marker of cell viability and proliferation was assessed by WST-1 Cell Proliferation Assay Kit (10,008,883, Cayman Chemical, MICH, USA) according to the manufacture’s recommendation. Three MB cell lines, D341, CHLA-01 and CHLA-01R, were used in this assay. Briefly, cells were seeded at a density of 30,000 cells/well in 96-well plates. Twenty-four hours later, treatment was added as shown in the “Results” section. Assay reaction was assessed 24 or 72 h after treatment. For that, 10 μL of WST-1 reagent was added to each well for 4 h, and absorption was measured using a plate reader (Multiskan Go, Thermo Fisher Scientific, Scoresby, VIC, Australia). Drugs used, including mibefradil dihydrochloride hydrate (M5441), NNC55-0396 hydrate (N0287), verapamil hydrochloride (V4629), nifedipine (N7634), vincristine (V0400000) and lomustine (L5918), were purchased from Sigma-Aldrich.

Buffers including MES, HEPES, and Tris were obtained from VWR Life Science as well as ethylenediaminetetraacetic acid (EDTA). Nifedipine and choline chloride were from Beantown Chemical. Pluronic F-127 and mibefradil dihydrochloride hydrate were from Sigma Aldrich. Verapamil hydrochloride, glybenclamide, 4-Aminopyridine (4-AP), amiloride hydrochloride dehydrate, NaOH, HCl, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and DMSO were purchased from AdipoGen, InvivoGen, Acros Organics, ENZO, Fisher Scientific, EMD, Alfa Aesar, and Macron Fine Chemicals, respectively. KCl, CaCl₂ × 2H₂O, MgCl₂ × 6H₂O, and MgSO₄ × 7H₂O were purchased from VWR Analytical, Ward’s Science, EMD Millipore Corp, and Ward’s Science, respectively.