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Fig. 3. Ataxin-1 sub-nuclear localization is dependent on RNA and transcription. Fluorescence micrographs of eGFP-ataxin-1[Q26]- or [Q84]-expressing NIH 3T3 cells. (A) panels A,E: no drug added; all other panels show the effects of the indicated drugs under the following conditions: panel B: 25 µg/ml {alpha}-amanatin for 3 hours at 37°C; panels C,F: a 0.05 µg/ml actinomycin D at 37°C for 3 hours; panel B: 25 µg/ml {alpha}-amanatin for 3 hours at 37°C; panel D: RNase for 1 hour; panel G: 0.5 µm colchicine for 3 hours; panel H, 5% DMSO for 1 hour; panels I, J, MG132 and cycloheximide respectively, for 1 hour. (B) panels A-D: live-cell microscopy of ataxin-1[Q84]-expressing cell over a time course of 2-4 hours of treatment with 25 µg/ml {alpha}-amanatin, showing inclusion dispersal. (See also Movie 6 in supplementary material.) (C) SYTO Green total RNA staining of cells expressing mRFP-ataxin-1[Q84] (panels A-C). Arrows indicate the presence of RNA in nuclear ataxin-1 inclusions in panels A and B. Bar: ~10 µm.