Gsk2606414

24S-OHC [52 (link)] was dissolved in EtOH (Wako, Osaka, Japan). F12511 was the generous gift of Kowa (Aichi, Japan). Thapsigargin and CHX were purchased from Wako (Osaka, Japan). GSK2606414 and ISRIB were from Cayman Chemical (Ann Arbor, MI, USA). GCN2iB was from MedChemExpress (Monmouth Junction, NJ, USA). Thapsigargin, GSK2606414, CHX, and ISRIB were dissolved in dimethyl sulfoxide (DMSO; Wako). The following antibodies were from commercial sources: anti-PERK (Cat# 3192), anti-phospho-eIF2α (Cat# 3398), anti-eIF2α (Cat# 5324), and anti-GCN2 (Cat# 3302) were from Cell Signaling (Danvers, MA, USA); anti-β-actin (Cat# A5441) was from Sigma-Aldrich (St. Louis, MO, USA); anti-TIA1 (Cat# 12133-2-AP), anti-ATF4 (Cat# 10835-1-AP), and anti-TDP-43 (Cat# 12782-2-AP) were all from Proteintech (Chicago, IL, USA); anti-G3BP1 (Cat# 611126) was from BD Biosciences (Franklin Lakes, NJ, USA); anti-phospho-GCN2 (Cat# ab75836) was from Abcam (Cambridge, UK); and anti-puromycin (Cat# MABE343) was from Merck Millipore (Burlington, MA, USA); All other chemicals, of analytical grade, were obtained from Sigma-Aldrich or Wako.

Tunicamycin, cisplatin, GSK2606414, and 4μ8C were obtained from Wako. Cells were incubated in DMEM + 10% FBS + antibiotics in 10-cm normal plates at a density of 5×10⁶ cells/plate for monolayer cells or in DMEM/F-12 + 0.4% BSA + 20 ng/mL EGF + 10 ng/mL FGF in 10-cm ultra-low attachment plates at the same density for sphere-forming cells. After a 3-hour incubation, cells were treated with or without TM 0.03 μM/cisplatin 20 μM, GSK2606414 1.5 μM/4μ8C 7 μM for 72 hours.