Salinomycin

XAV939, an inhibitor of TNKS activity, Salinomycin, an inhibitor of the Wnt pathway that decreases the phosphorylation of low-density lipoprotein receptor-related protein 6, and ABT-888, PARP1 and PARP2 inhibitor, were obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in water or DMSO. A stock of 10 mM was stored at −80 °C. The 50% inhibitory concentration (IC₅₀) of XAV939 for TNKS1 and 2 is 10 and 4 nM, respectively and for PARP1and 2–2.2 and 0.1 μM. Therefore, XAV939 was used at concentrations of 0.1 and 1 μM, which are specific for inhibition of TNKS1 and 2. The IC50 of ABT88 towards PARP1 and TNK1 is 5.2 nM, and 15 μM; therefore, it was used at 0.1 μM. Cycloheximide (Sigma-Aldrich) was dissolved in DMSO and used at 100 μg/mL. Ganciclovir (Sigma-Aldrich) was dissolved in PBS and used at 5 μM.

Celecoxib (Pfizer, Täby, Sweden), dimethyl-PGE₂ (dmPGE₂), cyclophosphamide (given as the active metabolite 4-hydroxycyclophosphamide), temozolomide, doxycycline, XAV-939, salinomycin (all from Sigma-Aldrich, Solna, Sweden), O6-BG, Wnt-C59 and LGK974 (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in dimethyl sulfoxide (Sigma-Aldrich). Cisplatin, irinotecan, doxorubicin and vincristine were purchased from the local pharmacy (Apoteket AB, Sweden) and diluted according to manufactures instructions. G007-LK was a kind gift from Dr Krauss, University of Oslo, Norway14 (link)27 (link). The mTOR inhibitors rapamycin (Sirolimus, LC Laboratories, Woodburn, MA, USA) and CCI-779 (TemSirolimus, a kind gift from Wyeth Pew River, NY, USA) were dissolved in 99.5% ethanol. LiCl was dissolved in H₂O. All inhibitors/activators were further diluted in OptiMEM (Gibco BRL, Sundbyberg, Sweden) to the desired in vitro concentration. temozolomide for the in vivo studies was supplied by the local pharmacy (Apoteket AB). For in vivo use of Celecoxib and temozolomide in the D283 xenograft study, the stock was prepared as a suspension in a vehicle fluid consisting of 0.5% methylcellulose (w/v; Sigma-Aldrich) and 0.1% Tween 80 (v/v; Sigma-Aldrich) in sterile water. For the LS174T xenograft study, temozolomide was dissolved in NaCl and doxycycline in water.

Stock solutions were prepared, sterile-filtered, and kept frozen at − 20 °C until needed. Doxorubicin (Sigma-Aldrich) was prepared in saline (2 mg/ml) and salinomycin (Sigma-Aldrich) was prepared in DMSO (5 mM). Aliquots of stock solution were diluted as needed in saline (for Doxorubicin) or DMEM (for salinomycin) for use in experiments.

Cells (150 000 cells/well) were treated with docetaxel (20 nm) (Sigma‐Aldrich) or salinomycin (0.1 μm) (Sigma‐Aldrich) prior to assessment of viability and apoptosis.

Three immunotoxins, HB21PE40, HA22 and SS1P were produced recombinantly in E. coli as described previously [38 (link)]. Nilotinib and everolimus were purchased from Selleck Chemicals LLC (Houston, TX), dissolved in dimethyl sulphoxide (DMSO) at 10 mmol/L stock concentration, and stored frozen at −80°C. Salinomycin was from Sigma-Aldrich (St. Louis, MO) and was handled similarly.

Parental GBM cells and iGSCs were seeded at 5000 cells per well onto 96-well plates and cultured in medium containing increasing concentrations of salinomycin (Sigma-Aldrich) for 2 days or temozolomide (Sigma-Aldrich) for 4 days. Untreated cells were used as controls. Four wells were used for each concentration group. After treatment, viability of cells was measured with the MTT assay 20 (link).