Tak 243

Human normal astrocyte cell line (HA 1800) and human GBM cell lines (LN229 and U251) were cultured in DMEM supplemented with 10% FBS solution at 37°C in 5% CO₂. The GSC cell line was cultured in Neurobasal™ Medium containing basic fibroblast growth factor, EGF, B27 supplement, heparin, L-glutamine, and N2 supplement to form a GSC-rich neurosphere culture. Cleaved caspase-3 (#9661, 1:1,000), 53BP1 (#4937, 1:200), γ-H2AX (#9718, 1:500 for WB, 1:200 for IF and 1:100 for IHC), Rad51 (#8875, 1:500) and β-actin (#8457, 1:1,000) primary antibodies were purchased from Cell Signaling Technology (CST, MA, United States). TAK-243 from CSNpharm (CSNpharm, Chicago, IL, United States) were dissolved in DMSO to create a 10 mmol/L solution, which was diluted to different concentrations in DMEM medium before use.

Ubiquitin (#3933), UBA1 (#4891), p21 (#2947), Cyclin D1 (#2922), CDC2 (#9116), c-Jun (#9165), cleaved PARP (#9532), LC3B (#3868), PERK (#5683), p-PERK (#3179), CHOP (#2895), ATF4 (#11815), IRE1α (#3294), eIF2α (#5324), p-eIF2α (#9721), XBP1s (#27901), GRP78 (#3177), cleaved caspase-3 (#9661), p-JNK (#9251), and β-actin (#9562) primary antibodies were purchased from Cell Signaling Technology (CST, MA, USA). Antibody against Ki-67 (RM-9106) was purchased from Thermo Fisher (Waltham, MA, USA). ATF6 (sc-166659 and JNK1 (sc-1648) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-IRE1α (ab243665) and p62 (ab56416) were purchased from Abcam (Burlingame, CA, USA). UBA1 inhibitor TAK-243 was obtained from CSNpharm (CSNpharm, Chicago, IL, USA). TAK-243 was dissolved in DMSO to create a 10 mmol/L solution, which was diluted to different concentrations in DMEM medium before use.