Celastrol disrupts the cooperation of Myb with the coactivator p300. (A) QT6 fibroblasts were cotransfected with the Myb-inducible luciferase reporter gene pGL4-5xMRE(GG)-Myc (containing a cluster of high-affinity Myb binding sites), the β-galactosidase expression vector pCMVβ, and an expression vector for v-Myb. The cells were incubated for 12 hours with the indicated concentrations of Celastrol and then analyzed for luciferase and β-galactosidase activities. The columns show the average luciferase activity normalized to the β-galactosidase activity. Thin lines show standard deviations. v-Myb expression was visualized by western blotting (bottom). (B) HD11-C3-GFP1 cells grown for 12 hours in the presence or absence of doxycycline and the indicated concentrations of Celastrol, Trolox, or vitamin (vit.) C were analyzed by fluorescence microscopy. The fluorescence of cells only treated with doxycycline was set to 100%. (C) The positions of cysteine residues in v-Myb are shown schematically at the top. HD11 cells transfected with expression vectors for wild-type (wt) or the CallA mutant of v-Myb were treated for 24 hours with the indicated concentrations of Celastrol and analyzed by northern blotting for expression of the endogenous mim-1 and S17 mRNAs. (D) QT6 fibroblasts were transfected and analyzed as in (A), except that expression vectors for c-Myb and p300 were used. (E) QT6 fibroblasts were transfected with the Gal4-dependent reporter gene pG5E4-38luc, pCMVβ, and expression vectors for Gal4/Myb and KIX-VP19, as indicated. The transfected cells were incubated for 12 hours with or without Celastrol followed by analysis of reporter gene activities. The columns show the average luciferase activity normalized against the β-galactosidase activity. The luciferase activity in the Gal4/Myb and KIX-VP16 transfected cells was set to 100%. Asterisks indicate statistical significance (**P < .01, ***P < .001, Student t test).