Regulation of E-box–dependent promoter/reporter activity during induced differentiation of U-937 cells. (A) Schematic presentation of the reporter constructs. A detailed description of the constructs is given in Materials and Methods. (B) Repression of basal E-box–dependent PrT-CAT activity by a dominant negative c-Myc mutant. 20 × 106 cells of the indicated U-937 clones were electroporated with 20 μg of Pr-T-CAT or GalmE-PrT-CAT with or without 30 μg of pCMV-myc▵BR. CAT activity was determined 16 hours after transfection. CAT or luciferase activities (B through D) were normalized to β-gal activity by cotransfection with 20 μg hubactp/lacZ. (C) PrT-CAT activity during differentiation of U-937 clones. PrT-CAT or GalmE-PrT-CAT were electroporated with or without 5 μg of pCMV-myc as described in (B). Cells from separate electroporations were pooled and divided into aliquots, which were treated with the inducers indicated. (D) m4mintk-Luc activity during differentiation of U-937 clones. The experiment was performed as in (C). As a reference, mintk-Luc, lacking E-boxes and pCMV-Luc, which contains the luciferase gene driven by a CMV-promoter, was used. (E) Kinetics of m4mintk-Luc activity during differentiation of U-937-m112 cells. U-937-m112 is a subclone of U-937-myc-6 containing a stably integrated m4mintk-Luc construct. The cells were induced by TPA, IFN-γ, or the TPA + IFN-γ for the indicated time points and assayed for luciferase activity. The results (C through E) are presented as percentage of untreated U-937-myc-6 cells. (B-E) The data of at least three independent experiments are summarized.
