Effect of ectopic c-Myb expression on Celastrol-induced differentiation apoptosis of HL60 cells. (A) HL60 cells infected by a control lentivirus or a lentivirus encoding c-MybΔ3 were treated for 2 days with 0.5 μM Celastrol. Columns indicate the percentage of CD11b-positive cells. (B) Lentivirally infected HL60 cells were treated for 24 hours with the indicated concentration of Celastrol and analyzed for staining with Annexin V. The columns indicate the percentage of apoptotic cells. Asterisks indicate statistical significance (**P < .01, ***P < .001, Student t test). (C) Western blot analysis of total cell extracts of the cells with antibodies against Myb and β-actin. c-Myb refers to the endogenous protein. (D) QT6 cells were transfected with the Gal4-dependent reporter gene pG5E4-38luc, pCMVβ, and expression vectors for Gal4/Myb and Gal/CREB, as indicated. The transfected cells were incubated for 12 hours with or without Celastrol followed by analysis of reporter gene activities. Gal/CREB transfected cells were additionally incubated with 10 μM forskolin. The columns show the average luciferase activity normalized against the β-galactosidase activity. The luciferase activity in the absence of Celastrol was set to 100%.