Skip to main content
. 2007 Sep 28;35(19):6571–6587. doi: 10.1093/nar/gkm753

Figure 3.

Figure 3.

Subcellular redistribution and nucleolar accumulation of RDM1α in response to proteotoxic treatment. (A–B) HEK293T cells transiently expressing EGFP-RDM1α were treated with 10 μM MG132 (A) or lactacystin (B) for 4 h, followed by fixation, counterstaining with DRAQ5 (blue) and confocal microscopic analysis. The second image of (B) is identical to the first, except that the confocal settings have been optimized for the analysis of the GFP signal within the nucleolus. (C) HEK293 cells stably expressing RDM1α-EGFP were treated with 10 μM lactacystin for 8 h and living cells were analysed following counterstaining with DRAQ5. (D) HEK293T cell displaying the accumulation of the EGFP-RDM1α protein as nucleolar foci in response to treatment with MG132 (10 μM, 4 h). (E) HeLa cells expressing Flag-RDM1α were treated with 10 μM MG132 for 4 h, followed by fixation and staining of the Flag epitope (red). Nucleolar foci and entangled structures can be seen in the left and right cells shown on this image, respectively. (F–H) Lack of colocalization of stress-induced, nucleolar RDM1α with nucleolin and fibrillarin. (F) HEK293T cells expressing RDM1α-EGFP were treated with 10 μM lactacystin for 8 h, followed by fixation and staining for nucleolin. Nuclei were counterstained with DRAQ5 (blue). (G) HEK293 cells stably expressing RDM1α-EGFP were treated with 25 μM MG132 for 8 h and analysed as described for (F). (H) HeLa cells expressing RDM1α-EGFP and Fibrillarin-RFPCherry were treated with 10 μM MG132 for 8 h followed by fixation, counterstaining with DAPI (blue) and visualization. Signals from EGFP (green) and nucleolin or fibrillarin (red) were merged, revealing distinct distribution patterns. (I) Colocalization of RDM1α with nucleolar RNA in MG132-treated cells. HeLa cells expressing RDM1α-EGFP were treated with MG132 (10 μM MG132, 8 h) and the RNA was visualized with the cell-permeant, RNA-specific dye F22, followed by processing as for (H). In the merged image, RDM1α-EGFP (green) and RNA (red) coincide extensively (yellow) within the nucleoli.