Fig. 5

Mad1 destabilizes p53 by preventing sequestration of MDM2 into nucleoli. a–d In response to DNA damage, upregulated Mad1 replaces MDM2 in nucleoli. a, b MDM2 relocalizes to the nucleolus after DNA damage caused by doxorubicin in control cells (-tet) but not in cells expressing Mad1 in response to tet. HeLa cells stably expressing tet-inducible full length wild type Mad1 and GFP-nucleolin-P2A-3xFLAG-MDM2 were cultured for 24 h in the presence of tet and an additional 8 h with 2 µg/mL doxorubicin. a Immunofluorescence showing colocalization of MDM2 with GFP-nucleolin after DNA damage. Scale bar, 2.5 µm. b Quantification of the percentage of cells (±SD) exhibiting nucleolar MDM2. n > 200 cells from each of three independent experiments. c, d Upregulated Mad1 localizes to nucleoli in response to DNA damage. HeLa cells stably expressing GFP-nucleolin and tet-inducible wild type Mad1 were cultured with tet for 24 h and doxorubicin for another 8 h. c Immunofluorescence showing colocalization between Mad1 and GFP-nucleolin in response to DNA damage. Scale bar, 2.5 µm. d Quantitation of the percentage of cells (±SD) with nucleolar Mad1 staining. n ≥ 200 cells from each of three independent experiments. e, f HeLa cells stably expressing tet-inducible Mad1(SIM mutant)-3xFLAG were incubated with adenovirus expressing nucleolin-mNeonGreen-P2A-mScarlet-MDM2 for 1 h and then cultured with tet for 24 h and doxorubicin for another 8 h. e Immunofluorescence showing MDM2 localization to nucleoli after doxorubicin was unaffected by expression of SIM mutant Mad1. f Quantification of the percentage of cells (±SD) exhibiting nucleolar MDM2 in (c). n > 200 cells from each of three independent experiments. g, h HeLa cells stably expressing tet-inducible SIM mutant Mad1 were incubated with adenovirus expressing mNeonGreen-nucleolin for 1 h and then cultured for 24 h in the presence of tet and an additional 8 h ± 2 µg/mL doxorubicin. g SIM mutant Mad1 does not colocalize with nucleoli. Scale bar, 5 µm. h Rotated views of (g), showing the lack of colocalization between SIM mutant Mad1 and GFP-nucleolin. *p < 0.05. **p < 0.001. ns non-significant using t test. For specific p values, see Source Data file