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

(A) Immunoblotting of protein extracts from the HepG2-OA1 parental cell line and GCN2-KO clones 185#27, E23, F22, F27, immunodecorated with anti-GCN2 antibody. Clone 185#27 results from the first round of selection, and was used to generate clones E23, F22, F27. Arrow, GCN2 specific band. For GCN2 protein quantification, Ponceau staining was used as loading control and data are expressed as fold change vs. parental cell line (= 1). (B, C) Relative transgene (OA1) mRNA abundance in HepG2-OA1 cells and GCN2-KO clones, cultured in Met/Cys (B) or His (C) deprived medium for 24 h, compared to full medium. Mean ± SD of 3 technical replicates from 1 experiment. Data are expressed as fold change vs. control (full medium = 1). Since independent clones may display variable reactivation responses (e.g. due to different levels of transgene expression in basal conditions), the results are not shown as means of the three clones, but as separate replicates. The greater reactivation of the transgene in the HepG2 clones (particularly upon Met/Cys deprivation) depends on the lower expression level of the transgene in basal conditions, probably secondary to subculturing, resulting in a higher starved vs. control ratio.