Down-regulation of ESE-3 during DC development leads to an impaired DC phenotype. CD14⁺ peripheral-blood monocytes were isolated from buffy coat preparations with magnetic beads and transfected with 5 nM siRNA specific for ESE-3 (siRNA ESE3) or firefly luciferase GL2 (siRNA GL2). Mock transfection was carried out with the equivalent amount of water. (A) After 48 hours of culturing the cells in RP10 medium supplemented with GM-CSF and IL-4, the phenotype of the cells was analyzed by FACS staining. Shaded histograms represent staining with the indicated antibody; open histograms, the isotype control. The level of surface expression is indicated as mean fluorescence intensity. (B) LPS was added as a maturation stimulus 24 hours before harvesting the cells. The data shown are representative of 4 independent experiments. (C) Real-time quantitative RT-PCR to analyze the amount of ESE-3 mRNA after siRNA treatment. The amount of mRNA was determined in relation to GAPDH.