TABLE 1.
Nucleolar localization of the ARF-Mdm2 complex correlates with growth arrest
| Retrovirus | ARF localizationa | Hdm2 localizationa | % BrdU-positive cellsb |
|---|---|---|---|
| Mouse | |||
| CD8 vector control | 96.4 ± 6.7 | ||
| ARF (wild type) | Nucleoli | Nucleoli | 7.6 ± 4.3 |
| ARF Δ1–14 | Nucleoli | Nucleoplasm | 81.2 ± 9.5 |
| ARF Δ15–25 | Nucleoli | Nucleoli, nucleoplasm | 11.6 ± 5.1 |
| ARF Δ26–37 (75%) | Nucleoplasm, cytoplasm | Nucleoplasm | 85.6 ± 8.5 |
| ARF Δ26–37 (25%) | Nucleoli, nucleoplasm, cytoplasm | Nucleoli, nucleoplasm | 16.1 ± 4.2 |
| ARF Δ1–14-Δ26–37 | Nucleoplasm, cytoplasm | Nucleoplasm, cytoplasm | 95.7 ± 6.0 |
| Human | |||
| CD8 vector control | 92.6 ± 4.1 | ||
| ARF (wild type) | Nucleoli | Nucleoli | 9.2 ± 4.0 |
| ARF Δ2–14 (80%) | Nucleoplasm, cytoplasm | Nucleoplasm, cytoplasm | 84.9 ± 7.1 |
| ARF Δ2–14 (20%) | Nucleoli, nucleoplasm, cytoplasm | Nucleoli, nucleoplasm, cytoplasm | 30.5 ± 5.8 |
| ARF Δ26–37 | Nucleoli, nucleoplasm | Nucleoli, nucleoplasm | 16.8 ± 8.3 |
| ARF Δ82–101 | Nucleoli, cytoplasm | Nucleoli, nucleoplasm, cytoplasm | 11.5 ± 5.6 |
| ARF Δ2–14-Δ82–101 | Cytoplasm | Nucleoplasm, cytoplasm | 92.6 ± 3.0 |
NIH 3T3 cells were cotransfected with expression vectors encoding T7-tagged Hdm2 and the indicated ARF mutants. Mouse ARF was detected using antibodies to the p19ARF C terminus, with simultaneous detection of Hdm2 using antibody to the T7 epitope. Human ARF was scored using antibodies to the p14ARF C terminus, with simultaneous detection of Hdm2 using monoclonal antibody 2A10. The relative distribution of staining is summarized, as documented in Fig. 5.
NIH 3T3 cells were infected in parallel with retroviruses encoding the same ARF mutants, but without Hdm2, and scored 24 h postinfection for BrdU incorporation into replicating DNA (24-h pulse, equivalent to one cell cycle). Three independent coverslips were scored, and over 100 cells were counted for each (± standard deviation).