shRNA-triggered TGA is associated with recruitment of Ago2 and enrichment of gene activation marker into the CMV region. (A) Western blot of Ago2 protein after knockdown using siRNAs against Ago1, Ago2, the combination of both, or an irrelevant siRNA. (B) Quantitative RT-PCR to detect TGA response in normal C5 clones (white bars) or C5 clones with depleted levels of endogenous Ago2 (black bars). Anti-Ago2 siRNAs and plasmids expressing TGA-inducing shRNAs were cotransfected into cells. The RNA samples were extracted 4 d post-transfection for analysis. (C) ChIP analysis for the enrichment of Ago2 at the CMV promoter region after shRNA-mediated gene activation in the C5 clone. Plasmids expressing 3xHA-Ago2 driven by the ubiquitin promoter were cotransfected with shRNA-(0)AS, shRNA-(-4)AS, and an irrelevant shRNA (ctrl-shRNA) into cells. ChIP results were represented as the fold of enrichment relative to an irrelevant shRNA. Statistical analysis in this study was performed using a two-tailed t-test. (D) ChIP analysis for H3K4me2 at the CMV region after shRNA-mediated gene activation in C5 clone.
