Fig. 4

Mad1 upregulation destabilizes p53 and impairs cell death in response to DNA damage. a MDA-MB-231 cells stably expressing Mad1-YFP in response to tet were treated ± tet for 24 h and then with the topoisomerase II inhibitor doxorubicin (2 µg/mL) to induce DNA damage for the indicated times. Blot is representative of 3 independent experiments. b The CTD of Mad1 is essential for its role in preventing stabilization of p53. HeLa cells stably expressing full length (FL) Mad1-3xFLAG or Mad1 lacking the CTD (aa 1–596) in a tet-inducible manner were treated with tet for 24 h and then with doxorubicin (2 µg/mL) for the indicated times. Blot is representative of 3 independent experiments. c MCF10A cells were infected with adenoviruses expressing mNeonGreen or Mad1-mNeonGreen for 1 h and then treated with 1 µg/mL doxorubicin to induce DNA damage for the indicated times. Expression of Mad1-mNeonGreen prevents stabilization of p53 and its downstream effector p21. d The SIM in the CTD of Mad1 is necessary for Mad1 to prevent stabilization of p53 and p21. HeLa cells stably expressing tet-inducible wild type Mad1-3xFLAG or Mad1-SIM mutant-3xFLAG were treated with tet for 24 h and then with 2 µg/mL doxorubicin for the indicated number of hours. Wild type Mad1 but not SIM mutant Mad1 prevents stabilization of p53 and p21. Blot is representative of 3 independent experiments. e Quantitation of cell death (±SD) in HeLa cells stably expressing tet-inducible 3xFLAG tagged full length or ΔCTD Mad1 ± 24 h tet, then ±2 µg/mL doxorubicin for 0, 12, and 24 h. Full length Mad1, but not Mad1 lacking the CTD, prevented cell death due to DNA damage. n > 250 cells from each of 3 independent experiments