Supporting Information

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Fig. 5. Quantitation of Ago2 at PBs and SGs. The intensities of EGFP-Ago2 at PBs and PBs+SGs were compared with the total intensities in the cytoplasm in the presence of DMSO and 1mM hippuristanol (HIPP), respectively, in fixed HeLa^EGFP-Ago2 cells (first row). The corresponding volumes of PBs and PBs+SGs relative to the cytoplasm volumes are shown in the second row (n = 5).

Fig. 6. The localization of let-7a repressed mRNAs at PBs and SGs upon inhibition of translation initiation. HeLa cells stably expressing EGFP-Ago2 were transfected with a Renilla luciferase construct with three bulged let7a binding sites (RL-3xBulge) [Pillai RS, Bhattacharyya SN, Artus CG, Zoller T, Cougot N, Basyuk E, Bertrand E, Filipowicz W (2005) Science 309:1573-1576] and fixed after 24 hours for in situ hybridization. The let7a repressed mRNAs colocalized with EGFP-Ago2 at PBs (arrows, as reported in Pillai et al.) (a) and colocalized at PBs and SGs (arrowheads) after 250 mM arsenite treatment for 30 min (b). (Scale bars, 5 mm.)

Fig. 7. Quantitative dynamics of Ago2 at PBs and SGs. (a) Selected time-lapse micrographs of single live HeLa cell stably expressing EGFP-Ago2 (same cell as in Fig. 2a) upon addition of 1 mM hippuristanol (HIPP). Arrowheads in Insets indicate the progression of SG formation, and see also Movie 1 for the complete time-lapse series. (b) The intensities of individual PBs were followed every 30 s, which appeared to remain relatively unchanged over 20 min either upon addition of DMSO (blue series) or 1 mM HIPP (orange series). (c) FRAP analyses of EGFP-Ago2 at single PBs showed that the recovery kinetics of EGFP-Ago2 at PBs remained relatively unchanged in the absence (orange, n = 5) or presence (purple, n = 5) of 250 mM arsenite. (d) FRAP analyses of EGFP-Dcp1a (n = 6) showed that another PB component, Dcp1a, is relatively mobile as compared with Ago2 (c). (e) PAGFP-Ago2 (Upper) and PAGFP-Dcp1a (Lower) were followed after photoactivation at single PBs over 13 min (same cells as in Fig. 2d; broken circles indicate the photoactivation spots). Arrows and broken arrows indicate the photoactivated PBs and neighboring PBs, respectively. (Scale bars, 5 mm.)

Fig. 8. Activity of quenched TAMRA-labeled siCXCR4. (a) To minimize the fluorescence emitted in the double-stranded forms of siRNA, we devised a labeling scheme such that the fluorescence from the siRNA duplex was quenched when antisense strands conjugated with tetramethylrhodamine (TAMRA) at the 3' end were hybridized with sense strands conjugated with Black Hole Quencher-2 at the 5' end. (b) Fluorescence emission profiles of unquenched (red) and quenched TAMRA siRNAs (blue) against endogenous gene CXCR4 (siCXCR4) were measured at the excitation wavelength of 559 nm. Emission profiles of TAMRA-labeled siRNAs were obtained using a Tecan Xfluor4 microplate reader. (c) HeLa cells were transfected with 10 nM siCXCR4, firefly luciferase control, and different Renilla luciferase constructs to measure the quenched TAMRA and unmodified siCXCR4 activities as miRNAs and siRNAs. Activities of the Renilla luciferase construct containing no binding site for siCXCR4 (no target), one perfect binding site (siRNA target), and six bulged binding sites (miRNA target) were measured relative to the cotransfected firefly luciferase control and no siRNA control. Quenched TAMRA siCXCR4 (purple) retained 100% siRNA and 86% miRNA activities of the unmodified siCXCR4 (yellow), respectively (n = 12).

Fig. 9. Characterization of Dicer-/- Cells. (a) Short RNA Northern blots probing total RNA extracted from mouse Dicer+/+, Dicer+/-, and Dicer-/- ES cells showed that Dicer-/- cells lacked mature miRNAs but not pre-miRNA hairpins. (b) Dicer-/- cells were still capable of posttranscriptional silencing triggered by short RNAs acting as either siRNAs or miRNAs. Dual luciferase assays showed that Dicer+/+ and Dicer-/- cells responded comparably to either an siRNA that targets the coding region (siRL, Left) or the 3' UTR of a cotransfected Renilla luciferase containing six bulged binding sites of siCXCR4 (Right).

Movie 1. The localization of stably expressed EGFP-Ago2 in HeLa cells upon hippuristanol treatment. The time indicated (mm:ss) is relative to the instantaneous moment of drug addition.

Supporting Text

For quantitating the EGFP-Ago2 signals of the cytoplasm, PBs and SGs reported in Fig. 5, HeLa(EGFP-Ago2) and parental HeLa cells were seeded on the left- and right-hand side of the same coverglass, respectively, and therefore both populations of cells were treated with the same growth, fixation, and mounting conditions. The intensities of the 3D deconvolved images were first subtracted from the background fluorescence resulted from cellular autofluorescence and mounting medium. The amount of subtraction is calculated from the fluorescence intensities of coseeded, parental HeLa cells. The cytoplasmic volumes were then rendered from the background-corrected 3D data sets, and the intensities were calculated by using Imaris 4.0.6 (Bitplane). Similarly, the volumes of PBs and PBs/SGs were determined by an empirical threshold in each image data set, and the intensities were calculated by using 2D polygon finder in the softWORX Imaging Package (Applied Precision Inc.).

For quantitating the siRNA and EGFP-Ago2 signals in Figs. 3 and 4, PBs and SGs were first identified by immunostaining of anti-Dcp1a and anti-TIA1, respectively. Segmentation algorithm is based on a combination of an empirical intensity threshold and object size to create a binary mask that represents structures of interest. Mean fluorescence intensities were then measured in all channels of the original deconvolved images within these areas. Each sample measurement corresponds to a single structure of interest in one optical section. To compare the signal of the structure relative to the cytoplasm, the cytoplasmic value is obtained either by picking randomly non-PB/SG spots of the same size in the same cell by using a home-built Perl script in Fig. 3 or by using the average pixel value of 5 optical sections of a particular cell in Fig. 4. Student's t tests were performed by using the R package (www.r-project.org).

For photokinetics experiments, the microscope was set up with an additional module of two 20-W solid-state 410-nm and 488-nm lasers for photoactivation and photobleaching experiments, respectively. In each case, three to five prebleach images were acquired before the laser-activated event, which is followed by the acquisition of 100-200 images, and the time interval between each acquisition depends on the mobility of the protein of interest. For photobleaching, a diffusion-limited spot was focused to the structure of interest in the cell such that the initial intensity is reduced to 20-50% of its original value. For photoactivation, PBs were first identified by mRFP-Dcp1a, and laser was activated at a single PB for ~1 s such that the increase in intensities for PA-Dcp1a and PA-Ago2 is comparable. The resultant time-lapse images were normalized with total cell intensities and data sets with potential z-drifts were removed from the analysis.

1. Leung AK, Gerlich D, Miller G, Lyon C, Lam YW, Lleres D, Daigle N, Zomerdijk J, Ellenberg J, Lamond AI (2004) J Cell Biol 166:787-800.

2. Pillai RS, Bhattacharyya SN, Artus CG, Zoller T, Cougot N, Basyuk E, Bertrand E, Filipowicz W (2005) Science 309:1573-1576.