(A) Effect of mutation of C/EBP site (−55) on lactoferrin promoter activity in U937 cells. The −230-bp lactoferrin promoter luciferase (Lac-Luc) reporter either with or without a mutation at the C/EBP site as well as β-galactosidase plasmid were transfected into U937 cells. After 16 hours, cells were harvested to determine luciferase activity. Relative light units (RLU) were normalized for β-galactosidase activity. Data shown represent mean ± standard deviation of 3 independent experiments. (B) Western blot for C/EBPɛ expression in U937 cells stably integated with either zinc-inducible C/EBPɛ expression vector or empty expression vector (PMT). Addition of 100 μmol/L zinc-sulfate strongly induces C/EBPɛ protein (labeled p32). (C) U937 cells containing either a stably integrated, zinc-inducible C/EBPɛ expression vector (labeled C/EBPɛ) or empty vector (PMT, control) were transiently transfected with either the −87 lactoferrin promoter reporter plasmid (labeled −87 Lac-Luc) or empty vector pGL 3basic. After transfection, half of the cells were treated with 100 μmol/L zinc (▪) either to induce expression of C/EBPɛ or control for a possible nonspecific zinc effect on promoter activity in the case of U937 with integrated empty vector. Half of the cells were not treated with zinc () and used to determine the basal activity of the −87 Lac-Luc plasmid in U937/C/EBPɛ and U937/PMT. Data represent mean and standard deviation of 3 independent experiments.