Fig. 3. Genotoxic stress eliminates wild-type p53, Rev NoLSC'p53 and Rev sub-nucleolar association but only wild-type p53 nucleolar localization was ATP dependent. (A) H1299 p53-null cells were transfected with 1 µg of the following plasmids: wild-type p53, Rev NoLSC'p53 and Rev-GFP, and were either left untreated or were treated with daunorubicin (D) for 16 hours. 8 hours before the cells were lysed they were treated with 10 µM MG132 (M). Alternatively, 45 minutes before the cells were lysed they were treated with azide (a) as indicated. Combined treatments with MG132 and azide (Ma) and MG132 and daunorubicin (MD) were also performed, as indicated. Cells were then lysed in electrophoresis sample buffer and were subjected to western blotting. (B) Parallel treatments as in A were used to perform immunofluorescence experiments as described in Fig. 1C and Materials and Methods. (C) To quantify the immunofluorescence results, approximately 200 cells were counted from different fields in two independent experiments, and the results are presented as the percentage of cells with accumulated p53 or Rev NoLSC'p53 in the FC of the nucleolus.