Efficiency of myc-Ago2 phospho-mutants in slicer-dependent and slicer-independent silencing assays. (A) Schematic representation of dsGFP-miR21 reporter used to assay slicer-dependent silencing activity. (B) Ago2−/− mouse embryonic fibroblast cells were transiently electroporated with plasmids expressing wild-type or mutant Ago2 alongside dsGFP-miR21 (or dsGFP-nsc as a control), and pDsRed-C1-monomer. GFP expression was determined by flow cytometry and normalized against DsRed expression. The bar graph represents the expression of dsGFP-miR21 relative to dsGFP-nsc for each Ago2 construct. (C) Schematic representation of dsGFP-Nonslicer reporter used to assay slicer-independent silencing activity. (D) Ago2−/− mouse embryonic fibroblast cells were transiently electroporated with plasmids expressing wild-type or mutant Ago2 alongside dsGFP-NonSlicer (or dsGFP-nsc as a control), and pDsRed-C1-monomer. Cells were then transfected with Dicer-substrate siRNA. GFP expression was determined by flow cytometry and normalized against DsRed expression. The bar graph represents the expression of dsGFP-NonSlicer relative to dsGFP-nsc for each Ago2 construct. Paired Student's two-tailed t-test was used to compare amounts of dsGFP expression in cells cotransfected with mutant Ago2 constructs to those cotransfected with wild-type Ago2, (*) P < 0.05; (**) P < 0.01. Error bars indicate SE.
