Celastrol suppresses the activity of Myb. (A) Schematic illustration of the fluorescence-based reporter cell line HD11-C3-GFP1. The cells carry a stably transfected mim-1 promoter/enhancer GFP reporter gene, an expression vector for the Tet-repressor (Tet-R), and a cytomegalovirus (CMV) promoter-based expression vector for chicken Myb that contains Tet-operator sites close to the transcriptional start site (CMV*). (B) HD11-C3-GFP1 cells grown for 12 hours in the presence or absence of doxycycline (dox) were analyzed by fluorescence microscopy. (C) Structure of Celastrol. (D) The cells were grown for 12 hours in the presence of doxycycline and the indicated concentrations of Celastrol. Columns on the left show the mean GFP fluorescence with standard deviations. Columns on the right show the viability of the cells as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The values were normalized to the fluorescence and viability of cells treated only with doxycycline. Asterisks indicate statistical significance (**P < .01, ***P < .001, Student t test). (E) HD11-C3-GFP1 cells were grown for 12 hours in the presence or absence of doxycycline and Celastrol, as indicated. The cells were then analyzed by northern blotting for expression of mim-1 and ribosomal protein S17 mRNAs (upper) and by western blotting for Myb and β-actin (lower).