Lomustine

Meclofenamate and lomustine were purchased from Sigma. For preparation of 100-mM stock solutions, Meclofenamate was dissolved in dimethyl sulfoxide (DMSO) and lomustine in ethanol. Both drugs were aliquoted and stored at −20 °C. Final DMSO and ethanol concentrations did not exceed 0.1%.

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.

Cisplatin ((SP-4-2)-diamminedichloridoplatinum(II); CIS), lomustine (N-(2-Chloroethyl)-N’-cyclohexyl-N-nitrosourea; CCNU), and temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide; TMZ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Imidazolyl ethanamide pentandioic acid (5-[2-(1H-imidazol-5-yl)ethylamino]-5-oxopentanoic acid; IEPA; for chemical structure see Figure 9) was kindly provided by MyeloTherapeutics (Berlin, Germany). Stock solutions were prepared as follows: 30 mM CIS (molecular weight (MW) 300 g/mol) in 33% dimethyl sulfoxide (DMSO); 100 mM CCNU (MW 249.69 g/mol) in 5% ethanol; 100 mM TMZ (MW 194 g/mol) in 10% DMSO; and 100 mM IEPA (MW 225.25 g/mol) in phosphate-buffered saline (PBS; Lonza, Basel, Switzerland). Aliquots were stored at −20 °C and further working solutions were prepared with assay-specific culture medium (see cell culture and labeling of CD34⁺ cells) or PBS. Appropriate solvent controls were implemented.