Fig. 6.

Translation of intronic repeats does not require 5′-cap and can be upregulated by stress. a HeLa reporter cells were transfected with non-targeting siRNA or siRNA against cap-binding protein eIF4E. Immunoblotting of eIF4E showed the knockdown efficiency. β-actin was blotted as internal control. b Expression of AUG-FLuc translation (left) and C9R-NLuc RAN translation (right) reporters in presence of non-targeting siRNA or siRNA against eIF4E. **P < 0.005, ***P < 0.0005, two-tailed t-test. c Bicistronic splicing reporter cells were treated with mTOR inhibitor Torin 1 for 24 h. Immunoblotting of phospho-4E-BP1 using antibodies recognizing different phosphorylation sites. β-actin was blotted as internal control. d Expression of AUG-FLuc and C9R-NLuc in bicistronic splicing reporter cells with the treatment of mTOR inhibitor Torin 1. **P < 0.005, ***P < 0.0005, two-tailed t-test. e Fold change of relative RAN translation vs. AUG translation of the bicistronic splicing reporters under arsenite (left) and MG132 (right) stimuli, without or with pretreatment of ISRIB or PERKi. NLuc signals were normalized to FLuc in each sample. *P < 0.05, **P < 0.005, ***P < 0.0001, two-tailed t-test. f Fold change of RAN translation upon eIF2α-S51D expression, without or with the treatment of ISRIB. *P < 0.05, **P < 0.005, two-tailed t-test. g Diagram for a feedforward loop with C9ORF72 (GGGGCC)_n RAN translation being enhanced by initial repeat-mediated toxicity through eIF2α phosphorylation pathway. Data are mean ± SEM from three biological replicates