Celastrol suppresses the expression of Myb target genes and induces myeloid differentiation and apoptosis in HL60 cells. (A) Effect of Celastrol on human Myb target genes. Real-time PCR analysis of ACTB, c-MYC, ADA, CDC2 and c-KIT expression in HL60 cells treated for 24 hours with or without 0.5 μM Celastrol. Columns show the amount of the indicated mRNAs normalized to the amount of β-actin mRNA. (B) Analysis of c-myc expression at different times of Celastrol treatment. (C) Effect of Celastrol on the differentiation of HL60 cells. Cells were treated for 2 days with the indicated concentrations of Celastrol or ATRA. Columns indicate the percentage of CD11b-positive cells, as determined by flow cytometry. (D) May-Grünwald staining of HL60 cells treated for 2 days with dimethylsulfoxide (DMSO) or 0.5 μM Celastrol. (E) HL60 cells treated for 2 days Celastrol and LBH589 were analyzed as in C. (F) HL60 cells were treated for 24 hours with the indicated concentrations of Celastrol and analyzed for staining with Annexin V. The columns indicate the percentage of apoptotic cells. Asterisks indicate statistical significance (*P < .05, **P < .01, ***P < .001, Student t test).