Fig. 6. Stress-mediated phosphorylation of EIF2S1 induces resistance to the folate-based antimetabolites methotrexate and pemetrexed.

Survival of LN-308 cells (a) and A459 cells (b) after 24 h of treatment with the indicated concentrations of the FDA-approved chemotherapeutic reagents in the absence (black lines, vehicle control) or presence of tunicamycin (red lines). c Survival of stably transfected HEK293 cells before (black lines, vehicle control) and after induction of expression of a phosphomimetic mutant of EIF2S1 (S51D) (red lines). Cells were treated for 24 h with the indicated concentrations of FDA-approved chemotherapeutic reagents before scoring survival. d Survival of LN-308 cells in the presence of MTX after activation of the kinase HRI. Left panel: representative western blots of ATF4 and beta-tubulin protein levels after stimulation with the HRI activator BTdCPU, or the control compound NCPdCPU. Treatment with DMSO or TM served as control. Right panel: Cell survival in the presence of the indicated concentrations of MTX of control-treated LN-308 cells (black), TM-treated (red), or BTdCPU-treated LN-308 cells (blue). Data are represented as mean ± SD of three (a, c, and d) or five (b) biologically independent experiments. Representative western blots of at least three biologically independent replicates are depicted; pictures of the uncropped membranes are provided in the supplementary information.