Figure 3
From: Identification of DHX9 as a cell cycle regulated nucleolar recruitment factor for CIZ1
CIZ1 colocalises with DHX9 in the nucleolus of cells normal fibroblasts and HeLa cells. (A) HeLa cells were imaged by immunofluorescence confocal microscopy. Upper panel: Total DNA is shown in blue; DHX9, green; CIZ1, Red and merged image. Middle panel: Total DNA shown in blue; DHX9, green; nucleophosmin (B23), Red and merged image. Lower panel: Total DNA shown in blue; UBF, green; CIZ1, Red and merged image. Yellow indicates colocalisation. White bar = 10 µm (B) As for A except human BJ fibroblasts were used. (C) Differential interference contrast light microscopy showing purified nucleoli. Black bar = 10 µm. (D) Western blot analysis of nucleoli from (C) for DHX9, CIZ1, nucleophosmin (B23) and actin. (E) Representative HeLa cell nuclei imaged by immunofluorescence confocal microscopy after synchronisation in G1, S and G2 phase. Percentage of cells in S-phase in each population, as determined by BrdU incorporation, is shown. Total DNA is stained with DAPI (blue), CIZ1 (red) and DHX9 (green). White scale bar = 10 µm. (F) HeLa cells were synchronised in early S-phase with thymidine treatment and CIZ1 and DHX9 visualised by confocal fluorescence microscopy. Representative images were taken as indicated after removal of thymidine. Total DNA is shown in blue; DHX9, green; CIZ1, red and merged image, with yellow showing colocalisation. (G) Flow cytometry profiles of propidium iodide stained DNA in HeLa cells at indicated time points after release from thymidine block. (H) Percentage of BrdU positive S-phase cells determined by pulse labelling at indicated time points after release from thymidine block. Data shows mean ± SD, where 100 nuclei scored for each time point I) Percentage of nuclei showing nucleolar colocalisation for CIZ1/DHX9 at indicated time points. Data from three independent experiments showing mean ± SD, where > 50 nuclei scored for each time point.
