Temozolomide

GBM1 cells were a kind gift from Dr. Yu Shi at Institute of Pathology and Southwest Cancer Center, Southwest Hospital and were characterized previously (Zhou et al., 2018 ). GBM1 cells were cultured in Neurobasal A medium (Gibco, 2585380) containing B27 supplement (1:50, Invitrogen, Cat#2175161), GlutaMAX (1:100, Gibco, Cat#35-50-061), Sodium pyruvate (1:100, Gibco Cat#R25-0000-Cl), MEM NEAA (1:100, Gibco, Cat#11140-050), 20 ng/mL bFGF (Gibco, Cat#PHG0266) and 1% penicillin/streptomycin. The GSC11 cells were cultured in DMEM/F12, supplemented with B27, 20 ng/mL EGF (Gibco, Cat#PHG0311L), 20 ng/mL bFGF, 4 μg/mL heparin (Sigma, H3149-500KU-9) and 1% penicillin/streptomycin. Normal human astrocyte cells (NHA) were a kind gift from Dr. Xiaozhong Peng at Institute of Medical Biology, Chinese Academy of Medical Sciences (Hu et al., 2019 (link)). NHA cells were cultured in commercial astrocyte medium (Cat#1801, ScienCell) supplemented with 1% astrocyte growth factor (ScienCell) and 2% FBS (ScienCell). All cells were cultured at 37°C in a 5% CO₂ humidified environment. Ivacaftor and temozolomide were purchased from MedChemExpress (Ivacaftor, Cat#HY-13017), (temozolomide, Cat#HY-17364/CS-0943).

U251 cells and SHG-44 cells (Shanghai Institutes for Biological Sciences, China Academy of Science, Shanghai) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (BioInd, Kibbutz Beit Haemek, Israel), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in 5% CO₂.
On the basis of observations in a preliminary study, U251 cells and SHG-44 cells were treated with 100 μΜ temozolomide (MedChemExpress, MCE, New Jersey, USA) for 24 h to cause cell injury. The cells were pretreated with 100 μM TRPA1 agonist (PF-4840154, MCE) or 100 μM TRPA1 antagonist (HC030031, MCE) for 1 h and then cultured with vehicle (culture medium) or TMZ for another 24 h.