The gene expression program activated under conditions of eIF2a phosphorylation, the ISR, promotes amino acid sufficiency and an anti-oxidant state (Harding et al., 2002; Harding et al., 2003). Cells with defective activation of this pathway have varying levels of impairment in these functions. For example the eIF2a^S51A/S51A cells generated in the Kaufman lab were noted to require supplementation by non-essential amino acids (Scheuner et al., 2001), whereas cells lacking ATF4, an important effector of the ISR, are completely dependent on such supplementation and also require supplemental reducing substances in the media (Harding et al., 2003). A similar, milder phenotype is also present in PERK-/- cells (Harding et al., 2003) and can be imposed on certain cells by blocking the ISR through over-expression of an eIF2a phosphatase, GADD34 (Novoa et al., 2001; Lu, et al., 2004).

Therefore the ISR^def cells arevery sensitive to media depletion; one lapse in feeding or just an old batch of glutamine will be their undoing. We recommend frequent media changes (never let the media get yellow) and supplementing the media of cells suspected of being ISR^def with b-mercaptoethanol (55mcM final concentration, gibco cat # 21985023) and 1X MEM Non-essential amino acids (Gibco cat# 11140050).

References:

• Harding, H., Zhang, Y., Zeng, H., Novoa, I., Lu, P., Calfon, M., Sadri, N., Yun, C., Popko, B., Paules, R., Stojdl, D., Bell, J., Hettmann, T., Leiden, J., and Ron, D. (2003). An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 11, 619-633.
• Harding, H. P., Calfon, M., Urano, F., Novoa, I., and Ron, D. (2002). Transcriptional and translational control in the mammalian unfolded protein response. Annu Rev Cell Dev Biol 18, 575-599.
• Lu P.D., Jousse C., Marciniak S.J., Zhang Y., Novoa I., Scheuner D., Kaufman R.J., Ron D. and Harding H.P. 2004. Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2. Embo J. 23:169-179.
• Novoa, I., Zeng, H., Harding, H., and Ron, D. (2001). Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2a. J Cell Biol 153, 1011-1022.
• Scheuner, D., Song, B., McEwen, E., Gillespie, P., Saunders, T., Bonner-Weir, S., and Kaufman, R. J. (2001). Translational control is required for the unfolded protein response and in-vivo glucose homeostasis. Mol Cell 7, 1165-1176.