Figure 6.

Depletion of hMps1 or MDM2 dampens DNA repair and cell survival in cells encountering oxidative stress. (A) Western blots showing downregulation of hMps1 and MDM2 by siRNA in HeLa cells. sc, scramble control. (B) Comet assays demonstrating delayed repair of oxidative DNA damage in hMps1 and MDM2 knockdown cells. HeLa cells transfected with sc, hMps1 or MDM siRNA were treated or not with 0.1 mM H₂O₂ for 15 min and then recovered in drug-free fresh medium for 0, 60 or 90 min. UT, untreated. (C) Quantification of comet tails shown in (B). Mean ± SD from three independent experiments is shown. (D) Downregulation of hMps1 or MDM2 sensitized HeLa cells to H₂O₂ treatment. Colony survival assay was performed using HeLa cells transfected with control (sc), hMps1 or MDM2 siRNA. Cells were untreated or treated with various concentrations of H₂O₂ for 15 min before plating. Shown are results from three independent experiments. (E) Re-expression of WT MDM2 but not the 3A mutant restored DNA repair in MDM2 knockdown cells. Comet assays were performed as in (B) but using the Tet-off HeLa cell lines induced to express siRNA-resistant WT or 3A MDM2 (Supplementary Figure S6A and B). Mean ± SD from three independent experiments is shown. (F and G) Delayed repair of 8-oxoguanine (8-oxoG) lesions in hMps1 and MDM2-downregulated cells. HeLa cells treated as in (A) were fixed and stained with an anti-8-oxoG antibody for immunofluorescence microscopy (F). Quantification of the 8-oxoG fluorescence intensity from three experiments is shown in (G). (H) Expression of the H2B 2KR mutant hampered oxidative DNA damage repair. Repair in HeLa cells stably expressing WT or the 2KR (K120/125R) mutant of Flag-H2B was assessed by the comet assay (Supplementary Figure S6E). Quantification of comet tails from three experiments is shown. Expression of WT and 2KR H2B is shown in Supplementary Figure S6F. (I) H2B ubiquitination acts downstream of hMps1 and MDM2 in oxidative DNA damage repair. hMps1 or MDM2 was first depleted with siRNA in H2B WT and 2KR cells, and comet assays were performed after 0.1 mM H₂O₂ treatment. For each treatment, 150 cells were quantified. The results were analyzed by Prism. (J) The MDM2 3A mutation compromises the ability of MDM2 to suppress mutation caused by oxidative stress. The HPRT mutation assay was conducted using HeLa Tet-off cells expressing the empty vector (V), MDM2 WT, or 3A. * and **P < 0.05 and 0.01, respectively.