Fig. 6. Knockdown of eIF2D suppresses DPR expression in NSC-34 and HEK293 cells.

Monocistronic or bicistronic constructs were cotransfected along with either control or anti-eIF2D shRNA and cultured for 48 h. a Schematic diagram showing C9-HA construct. AG promoter: CMV enhanced chicken β-actin promoter. HA is in poly-GA frame. FLAG and Myc are in poly-GR and poly-GP frames, respectively, but are not schematically depicted. BGH bovine growth hormone. b A representative western blot of NSC-34 lysates to detect poly-GA, eIF2D, and β-actin. c, d Quantification of eIF2D (c) and poly-GA (d) expression from the western blots. e–f Quantification of poly-GP (e) and poly-GR (f) by ELISA in NSC-34 cells. The experiments were repeated four times. mean ± s.e.m. Turkey’s multiple comparisons test was used to calculate P-values. One-way ANOVA with Tukey’s multiple comparisons test was performed. g Schematic diagram showing the bicistronic constructs. CMV cytomegalovirus promoter; fLuc firefly luciferase; nLuc nanoluciferase; BGH bovine growth hormone. h The cell lysates were processed for western blotting and immunostained with poly-GA, fLuc, eIF2D, and α-tubulin antibodies. i, j, n Quantification of eIF2D (i), poly-GA(j), and fLuc (n) on western blots. The experiments were repeated three times. mean ± s.e.m. Two-tailed unpaired t-test was performed. k The levels of luciferase activity were assessed by dual-luciferase assays. The experiments were repeated three times. mean ± s.e.m. l, m Poly-GP and poly-GR detected with ELISA in HEK293 cells transfected with a ΔC9 or C9 bicistronic construct. The experiments were repeated three times. mean ± s.e.m. Two-tailed unpaired t-test was performed.