Investigating Unfolded Protein Response in MEFs

Mouse embryo fibroblasts (MEFs), including Eif2α^A/A, Eif2α^S/S, Perk^+/+, Perk^−/−, Ire1α^−/−, Ire1α^+/+, Atf6α^−/−, and Atf6α^+/+ were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% of penicillin/streptomycin as described earlier [29 (link)]. MEFs were incubated at 37 °C in humidified 5% CO₂ incubator for maintenance. To induce heat stress, MEFs were incubated at 42 °C in humidified 5% CO₂ incubators for indicated times. Tunicamycin (Tm, Sigma, St. Louis, MO, USA, T7765), thapsigargin (Tg, Sigma, St. Louis, MO, USA, T9033), tauroursodeoxycholic acid (TUDCA, Calbiochem, St. Louis, MO, USA, 580549), cycloheximide (Sigma, St. Louis, MO, USA, C6255), homoharringtonine (Sigma, St. Louis, MO, USA, SML1091), guanabenz (GA, Sigma, St. Louis, MO, USA, G110) ISRIB (Sigma, St. Louis, MO, USA, SML0843), GSK2606414 (Tocris, Bristol, England, 5107), and 4μ8c (Cayman, Ann Arbor, MI, USA, 22110) were utilized as described in the text.