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First published online 12 August 2003
doi: 10.1242/jcs.00714

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Cellular stress and DNA damage invoke temporally distinct Mdm2, p53 and PML complexes and damage-specific nuclear relocalization

Haartman Institute and Molecular and Cancer Biology Program, Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital, PO BOX 63, FIN-00014 Helsinki, Finland

View larger version (60K): [in a new window]   Fig. 1. Mdm2-PML colocalization. (A) SaOS-2 cells were left untreated (control) or were treated with MG132 (10 µM), As2O3 (1 µM) or were radiated with UVC (35 J/m2) and incubated for 6 hours, except with As2O3 for 16 hours. Cells were fixed and stained for endogenous Mdm2 and PML. (B) Localizations of Mdm2 and PML in a nucleolar necklace structure after UV-treatment. The image is a 3.75x magnification from A. (C) Localizations of insoluble Mdm2 and PML. SaOS-2 cells, treated as in A, were permeabilized with 0.5% NP-40, followed by staining for endogenous Mdm2 and PML. (D) Confocal image of Mdm2-PML colocalization. WS1 cells were treated with UVC and were incubated for 6 hours and stained for endogenous Mdm2 (red) and PML (green). The localizations were visualized by confocal microscopy. A layer (0.36 µm) of the merged projection is shown. Cells were visualized by differential interference (DIC) or phase contrast. The overlay for Mdm2 and PML is shown in MERGE as yellow staining. Arrows, colocalization of Mdm2 and PML. Bars, 10 µm.

View larger version (78K): [in a new window]   Fig. 6. Immunofluorescence staining of WS1 cells showing p53 and PML localizations following UV-irradiation. Cells were treated with UVC (35 J/m2) and were incubated for the indicated times.

View larger version (47K): [in a new window]   Fig. 2. PML sequesters Mdm2. p53-/-mdm2-/- cells were transfected with Mdm2 and PML III or PML IV expression vectors as indicated. The cells were stained for Mdm2 (red) and PML (green). Nuclei were visualized with DAPI. The overlay for Mdm2 and PML is shown in MERGE as yellow staining. Bars, 10 µm.

View larger version (35K): [in a new window]   Fig. 3. Mdm2 interacts with PML in vitro. (A) PML isoforms III, IV, IV-3K and PML-RAR were translated in vitro and mixed with equal amounts of in vitro translated wild-type Mdm2. The translation products were precipitated with an Mdm2 antibody mix (IF2, 2A10, SMP14) and blotted against PML (H-238) and Mdm2. Recognition of the different PML forms by the H-238 antibody was verified in separate experiments. (B) Mdm2 C-terminus is required for PML interaction. Mdm2 deletion mutants were translated in vitro and precipitated with in vitro translated PML IV. The products were precipitated with the Mdm2 antibody mix and blotted against the indicated proteins. Deletion constructs 58-89 and 89-222 were analysed in separate experiments together with full-length Mdm2. Note that Mdm2 migrates as three bands because of alternative translation initiation. As a negative control, in vitro translated Mdm2 was omitted from the binding reaction (neg. ctrl).

View larger version (39K): [in a new window]   Fig. 4. UVC-radiation alters PML subcellular levels and its interaction with Mdm2. SaOS-2 cells were treated with MG132 (10 µM) or were radiated with UVC and incubated for 6 hours. (A) Soluble and insoluble PML fractions were analysed by immunoblotting. (B) Cellular lysates were immunoprecipitated with Mdm2 antibodies followed by immunoblotting as indicated.

View larger version (28K): [in a new window]   Fig. 5. Temporal complexes between p53, Mdm2 and PML. WS1 cells were treated with UVC (35 J/m2) and were incubated for the given time. (A) Cellular lysates were precipitated with an p53 antibody mix (DO1, PAb1801, PAb421) or precipitated with a PML antibody (PG-M3). The immunoprecipitates were blotted against p53 (FL-393), Mdm2 (antibody mix of 2A10, IF2, SMP-14) or PML (H-238) as indicated. (B) Insoluble fractions of p53, Mdm2 and PML. Following UVC-treatment, the cells were lysed with NP-40 lysis buffer and the NP-40-insoluble pellet was analysed by immunoblotting as indicated. (C) WS1 cells were extracted in Laemli sample buffer and total levels of p53, Mdm2 and PML were analysed by western blotting.

View larger version (33K): [in a new window]   Fig. 7. PML, Mdm2 and p53 form trimeric complexes. In vitro translated p53 and increasing amounts of Mdm2 or PML were mixed followed by immunoprecipitation with an antibody against p53 (PAb421) or an Mdm2 antibody mix (IF2, 2A10, SMP14), followed by immunoblotting for PML (H-238), p53 (FL-393) and Mdm2 (IF2, 2A10, SMP14) as indicated.