Figure 4.

hMps1 and MDM2 regulate ubiquitination of endogenous histone H2B upon oxidative stress. (A) H2B ubiquitination was increased by H₂O₂ treatment. HeLa cells were untreated (UT) or treated with 0.5 mM H₂O₂ and collected at the indicated time points. Chromatin fractions were isolated and analyzed by immunoblotting. (B) H₂O₂ treatment promotes interaction between endogenous hMps1 and MDM2 in HeLa cells. Co-immunoprecipitation was performed using the anti-hMps1 antibody. (C) H2B ubiquitination was diminished in cells depleted of hMps1 and MDM2. HeLa cells transfected with scramble control (SC), sihMps1 or siMDM2 siRNA were incubated for 2 days before treatment with 0.5 mM H₂O₂ for the indicated time. (D) Quantification of results shown in (C) after normalization to total H2B. Mean ± SD from three independent experiments is shown. **P < 0.01. (E) Effects of hMps1 and MDM2 knockdown on H2B ubiquitination verified using a second set of siRNAs. (F) Inducible expression of siRNA resistant WT or 3A mutant of myc-MDM2 in Tet-off HeLa cells. (G) MDM2 WT but not the 3A mutant restores H2B ubiquitination in MDM2-depleted HeLa cells. Cells as in (F) were transfected with MDM2 siRNA then treated or not with 0.5 mM H₂O₂. (H) Complementation with WT but not 3A myc-MDM2 restored H2B ubiquitination in MDM2 knockdown HCT116 cells. Cells were first transfected with control or MDM2 siRNA then the following day with plasmids expressing siRNA-resistant WT or 3A MDM2. Treatment and analysis were as in (G).