Fig. 3

Stress-induced RAN translation elevation is mediated through eIF2α phosphorylation. a Diagram of the integrated stress response (ISR) and the actions of small-molecular inhibitors of ISR. b Schematic timeline of stress stimuli and inhibitor treatments, and induction of luciferase reporters. c G3BP immunofluorescence of reporter cells treated with arsenite and MG132, without or with pretreatment of ISRIB and PERKi. Stress granules were detected with G3BP antibody. d Quantification of stress granule numbers per cell in c. More than 100 cells were counted in each condition. ***P < 0.0005, two-tailed t-test. e Fold change of relative RAN translation vs. AUG translation in bicistronic reporters under arsenite (left) and MG132 (right) stimuli, without or with pretreatment of ISRIB or PERKi compounds targeting the eIF2α pathway. NLuc signals were normalized to FLuc. **P < 0.005, ***P < 0.0005, two-tailed t-test. f Schematic timeline of cDNA transfection and inhibitor treatment (as in h), and induction of luciferase reporters. g Relative expression of C9R-NLuc vs. AUG-FLuc reporters upon transfection of eIF2α wild-type or S51D mutant were compared with negative control. NLuc signals were normalized to FLuc in each sample. *P < 0.05, **P < 0.005, two-tailed t-test. h Fold change of bicistronic RAN translation reporter expression upon eIF2α-S51D transfection, without or with ISRIB treatment. *P < 0.05, **P < 0.005, two-tailed t-test. Data are mean ± SEM from three biological replicates