Conditional expression of CBFB-SMMHC in U937 leukemic cells. U937 cells were analyzed before (day 0) and 1, 2, 3, 5, and 7 days after withdrawal of tetracycline. (A) Quantitative RT-PCR analysis for CBFB-SMMHC and CEBPAmRNAexpression. Gray bars indicate CEBPAmRNA levels, and black bars indicate induction of CBFB-SMMHC mRNA. Results of 5 independent experiments are expressed as n-fold up/down-regulation and compared to day 0. Mean values and SDs are shown. (B) Western blot analysis of whole-cell lysates harvested at the same time points noted in panel A. The CBFB-SMMHC fusion protein (71-kDa) was detected with a CBFB antibody. TNT is an in vitro-translated CBFB-SMMHC protein from a CBFB-SMMHC expression construct that served as positive control. The membrane was further incubated with antibodies against CEBPA, CEBPE, granulocyte-colony-stimulating factor receptor (G-CSFR) and B-actin. (C) Parental U937 cells with the tetracycline-inducible construct lacking the CBFB-SMMHC cDNA (U937-T) were analyzed by quantitative RT-PCR for CEBPA mRNA expression (top panel) and by Western blot analysis for CEBPA and β-actin protein expression (lower panels). Mean values and SDs are shown. (D) CEBPA-binding activity to a CEBP site present in the G-CSF R promoter was assessed by EMSA using nuclear extracts from time points as indicated after CBFB-SMMHC induction. In vitro-translated CEBPA protein served as a positive control. U937-T indicates the parental U937 cell line lacking the inducible CBFB-SMMHC construct; S, shifted CEBPA protein; SS, supershifted CEBPA complex. Binding of the extracts to an octamer (OCT) consensus site indicates equal loading and integrity of the samples.