Figure 12. ISR-deficient Eif2s1S51A (eIF2αS51A) cells retain their responsiveness to Sephin1.
(A) Schematic representation of procedure used to create dual reporter (CHOP::GFP, XBP1s::Turquoise) Eif2s1S51A (eIF2αS51A) CHO-K1 cells using CRISPR-Cas9 system. (B) Immunoblot of CHO-K1 cell lysates using anti- eIF2αP (upper panel), anti- eIF2α (middle panel) and anti-BiP (lower panel) antibodies. Two-fold more cell lysate was loaded onto lanes 5 and 6 to compensate for the lower eIF2α content of the haploid mutant Eif2s1S51A cells. (C) Two-dimensional plot and histograms of the fluorescent signal of the CHOP::GFP and XBP1s::Turquoise reporters in the Eif2s1S51A CHO-K1 cells. Where indicated, the cells were exposed to a low concentration of tunicamycin (0.2 µg/mL; 20 hr) alone or together with Sephin1 (50 µM). The mean ± CV (coefficient of variation) of the fluorescence intensity of the two reporters in each of the two clones is displayed in the bar diagram. Shown is representative experiment of two independent experiments performed. Note the blunted expression of CHOP::GFP wrought by the ISR-defect imposed by the Eif2s1S51A mutation.