Fig. 4.

The localization of Ago2 at SGs, but not at PBs, depends on the presence of short RNAs. (a) Dicer+/+ (Upper) and Dicer−/− (Lower) cells transiently transfected with EGFP-Ago2 were fixed after 24 h and immunolabeled with PB marker Dcp1a. In both cases, EGFP-Ago2 colocalized with PBs (arrows). (b) Upon addition of 1 μM hippuristanol (HIPP), transiently expressed EGFP-Ago2 still colocalized with PB marker Dcp1a in both Dicer+/+ (Top, arrows) and Dicer−/− (Middle and Bottom) cells. However, EGFP-Ago2 no longer colocalized with SG marker TIA1 (arrowheads) in Dicer−/− cells. Cotransfection of 100 nM of miRNA let7a in the form of siRNA duplex resulted in the localization of EGFP-Ago2 at SGs in Dicer−/− cells (Bottom). (c) The intensities of EGFP-Ago2 at SGs were compared with the cytoplasm in each case, and a histogram was plotted with the percentage of SGs as the y axis, for each relative intensity with an interval of 0.05 difference on the x axis. Correlated with the image data in b, the intensities of EGFP-Ago2 at SGs relative to the cytoplasm were centered at ≈1.0 in Dicer−/− cells, suggesting that there is no enrichment of EGFP-Ago2 at SGs in the absence of short RNAs. (Scale bars, 5 μm.)