Dimethyl sulfoxide (dmso)

NALM6 cells and the RS4;11 TP53 wild-type BCP-ALL cell line were obtained from the JCRB Cell Bank (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA), respectively. These cells were cultured in RPMI-1640 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) containing 10% heat-inactivated FBS (Thermo Fisher, Waltham, MA, USA), in a humidified incubator at 37 °C under 5% CO₂.
The following chemicals were purchased: actinomycin D, daunorubicin, dimethyl sulfoxide (DMSO), methotrexate, prednisolone, vincristine, cytarabine, L-asparaginase, 6-mercaptopurine, and HDM201 (Cayman Chemical, Ann Arbor, MI, USA; Fujifilm Wako Pure Chemical Corporation; MedChemExpress, Monmouth Junction, NJ, USA; ProSpec, Ness-Ziona, Israel; TCI, Tokyo, Japan).

Cellular viability was measured with CellTiter-Glo (CTG) assays (Promega) according to manufacturer’s instructions. To address the effect of EGFR inhibition on cell viability in the validation experiments, cells were plated on 96-well plates at 5000 cells per well. Cells were treated with 1.6–1000 ng/ml of gefitinib in 0.01% DMSO (Cayman Chemicals) for 72 h starting at the moment of plating. DMSO-treated cells were used as controls.

A172, LN18, U87MG, and U373 GBM cell lines and human embryonic kidney 293 T cells were obtained from American Tissue Type Culture Collection (USA) and cultured in DMEM medium (Gibco, USA) with %10 fetal bovine serum (Gibco, USA) and %1 penicillin-streptomycin (Gibco, USA). MGG152, GBM8-TR, and GBM8-TS cells were kindly gifted by Dr. Hiroaki Wakimoto (Massachusetts General Hospital, Boston, MA)^{19 (link)}. GBM8-TR cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. GBM8-TS cells were cultured in neurobasal medium (Gibco) supplemented with 3 mmol/L of L-Glutamine (Mediatech), B27 (Invitrogen/GIBCO), 2 μg/mL of heparin (StemCell Technologies), 20 ng/mL of human EGF (R&D Systems), and 20 ng/mL of human FGF-2 (fibroblast growth factor; PeproTech). All cells were grown in 37 °C, 5% CO₂ in a humidified incubator. TRAIL was commercially supplied (Enzo Life Sciences, US) or produced from 293T cells as described^{29 (link)}. Stock of MS-275 (Cayman Chemicals, USA) was prepared in DMSO. ZVAD-FmK and ZVA-FMK were prepared in DMSO (Promega, USA).

Wildtype and mutant mice were treated weekly, three times with Sugen5416 (20 mg/kg in DMSO, s.c., Cayman Chemical) and normobaric, 10% O₂. Mice were weighed at the end of the exposure period (Figure S1). Right ventricle pressures were measured as previously described (Breen et al., 2017 (link)). The right ventricular chamber was accessed by way of Sastry et al. (2006 (link)) with a small pressure‐conductance transducer. Briefly, mice were anesthetized and an incision was made on the midline of the neck to allow the right jugular vein to be exposed by blunt dissection. A distal tie (6–0 silk suture) on the vein was made to serve as a retractor while a loose silk knot was placed at the proximal end that was used to occlude the vein temporally. A small incision was made between the two sutures and a 1.4F pressure‐conductance transducer (Millar SPR‐839) was inserted and advanced into the vessel and to the RV cavity. The catheter was secured by the proximal suture once the RV pressure waveform was identified. Right ventricular pressures (RVPs) were recorded, converted digitally via an analog‐digital converter (emka Technologies, IOX 1.8), and stored on a computer for analyses. Upon completion of data collection, the mouse was euthanized by an overdose of isoflurane (5%).

SK2 (MW = 292.1059), IUPAC name: 6-n-butoxy-10-nitro-12,13-dioxa-11-azatricyclo [7.3.1.0^2,7]trideca-2,4,6,10-tetraene, was prepared with >95% purity, as previously described [25 (link)], dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) for all experiments.
N-acetylcysteine (NAC) (Sigma-Aldrich, St. Louis, MO, USA) [26 (link),27 (link)] and MitoTEMPO [28 (link)] (Cayman Chemical, Ann Arbor, MI, USA), the inhibitors of reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX), were dissolved in 1 x PBS and DMSO for stock preparation, respectively. Z-VAD-FMK (ZVAD) [29 (link)] (Selleckchem.com; Houston, TX, USA), a panapoptosis inhibitor, was dissolved in DMSO for stock preparation.