ER Stress Induction in HFFF2 and HEK293FT Cells

HFFF2 (Sigma‐Aldrich, St. Louis, MO, USA) and HEK293FT (Invitrogen, Carlsbad, CA, USA) cells were cultured in the Dulbecco's Modified Eagle's Medium (DMEM; Corning, Glendale, AZ, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U·mL⁻¹ penicillin, 100 μg·mL⁻¹ streptomycin, and 0.292 mg·mL⁻¹l‐glutamine (HyClone, Marlborough, MA, USA). Cells were maintained at 37 °C in a humidified environment with 5% CO₂ and 95% air. For ER stress induction, cells were split into six‐well plates, at 300 000 cells/well, and cultured for 24 h. Then, cells were treated with either tunicamycin (5 μg·mL⁻¹; Sigma) or thapsigargin (3 μm; Tocris, Minneapolis, MN, USA) with or without the addition of ISRIB (500 nm; Sigma) for 5 h, immediately followed by RNA extraction.