GCN2 knockdown does not interfere with transgene reactivation in HepG2 cells.

(A, B) Downregulation of GCN2 protein by RNAi, shown by immunoblotting (A) and quantification (B) of protein extracts from HepG2-OA1 cells, transfected with control or anti-GCN2 siRNAs, and incubated with anti-GCN2 antibody. Arrow, GCN2 specific band; asterisk, non-specific signal detected by the Ab. Ponceau staining was used as loading control. Data are expressed as fold change vs. control siRNA (siRNA CTRL = 1). (C) Relative GCN2 mRNA abundance in HepG2-OA1 cells transfected with control or anti-GCN2 siRNAs. Mean ± SEM of 3 independent experiments. Data are expressed as fold change vs. control siRNA (siRNA CTRL = 1). ***P<0.001 (paired two-tailed Student’s t-test vs. control). (D) Relative transgene (OA1) mRNA abundance in HepG2-OA1 cells transfected with control or anti-GCN2 siRNAs and incubated for 6 h with L-Histidinol (HisOH, GCN2 activator; 4 mM). Mean ± SEM of 3 independent experiments. Data are expressed as fold change vs. untreated control (w/o HisOH = 1). *P<0.05, ***P<0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). (E) Schematic representation of the experiment.