4604 Nucleic Acids Research, 2006, Vol. 34, No. 16 Figure 5. NOP132 is required for DDX47 to properly localize to the nucleolus. Localization of DDX47 by anti-DDX47 immunofluorescence in HeLa cells treated with NOP132 siRNAs to reduce expression of NOP132. Control cells were transfected with luciferase siRNA duplex. (a) NOP132 RNAi in HeLa cells (upper panels). Control luciferase RNAi in HeLa cells (lower panels). Confocal images were taken with the Olympus laser-scanning confocal microscope LSM- GB200 system (left panel). DNA was stained with DAPI. Merge images of DDX47 and DNA are shown in the rightmost panels. Fluorescence images were taken with the Zeiss Axiophot microscope (right panels). Scale bars, 10 mm. (b) HeLa cells were treated without (control, lower panels) or with 5 mg/ml actinomycin D for 8 h (upper panels) and then immunostained with anti-DDX47 and stained with DAPI. Left panels, confocal fluorescence microscopy. Right panels, fluorescence microscopy. (c) A schematic model of the change in the localization of DDX47. In the untreated state, DDX47 (in black) is localized to the nucleolus. (d) 293 cells were transiently cotransfected with vectors carrying EGFP-DDX47 and FLAG-NOP132 coiled-coil domain minus a NLS (residues 667– 960; FLAG-Coil). The cells were fixed and stained with anti-FLAG followed by Alexa Fluor 594-goat anti-mouse IgG. Upper and middle panels, fluorescence microscopy; lower panels, confocal fluorescence microscopy. Arrowheads indicate cells in which the EGFP-DDX47 protein distributed evenly in the nucleus. DNA was stained with Hoechst dye. (e) 293 cells were transiently transfected with the NOP132 FLAG-Coil vector. The cells were fixed and stained with FLAG monoclonal antibody and then with Alexa Fluor 594-goat anti-mouse IgG. DNA was stained with Hoechst dye. Fluorescence images were taken with the Zeiss Axiophot microscope. (f) 293 cells were transiently transfected with EGFP-DDX47 vector. DNA was stained with Hoechst dye. Fluorescence images were taken with the Zeiss Axiophot microscope.