FIG. 7.

Silencing by miRNAs or dsRNAs does not require microscopic P bodies. (A) The indicated proteins were depleted from S2 cells (kd, knockdown). Depleted cells were cotransfected with plasmids expressing miRNA reporters (Nerfin or CG10011), plasmids expressing miR-9b or miR-12 or the corresponding empty vector. R-Luc served as a transfection control. F-Luc activity and the corresponding mRNA levels were measured and normalized to those of the Renilla control. Normalized F-Luc activities and mRNA levels in cells transfected with the empty vector were set to 100% for each knockdown (not shown). Error bars represent standard deviations from three independent experiments. (B) S2 cells expressing adh mRNA were treated with a dsRNA targeting a central region of the adh open reading frame or with GFP dsRNA as a control. The levels of adh mRNA were quantitated and normalized to those of a CAT mRNA transfection control. These values were set to 100 in cells treated with GFP dsRNA. The left panel shows results for S2 cells expressing adh mRNA that were treated with adh or GFP dsRNAs. These cells were then treated with dsRNAs targeting the proteins indicated. The levels of adh mRNA were quantitated and normalized to those of a transfection control (CAT mRNA). For each knockdown, the normalized values obtained for the cells in the presence of the adh dsRNA were divided by those obtained for cells treated with GFP dsRNA to compensate for unspecific effects of the depletions. Error bars represent standard deviations from three independent experiments. (C) Depletions shown in panel B resulted in P-body dispersion as shown by fluorescent staining of cells with anti-Tral antibodies. Numbers indicate the fraction of cells exhibiting a staining identical to that shown in the representative panel in the three independent knockdowns performed per protein (at least 100 cells were counted per knockdown). Scale bar, 5 μm.