Figure 2

NF-κB binding sites are required for E2F1 regulation of ICAM-1 promoter. A. The histograph represent the human ICAM-1 promoter luciferase reporters used in this study. The location of each binding site on the promoter of ICAM-1 is indicated and labeled. The crossed boxes indicate the mutation sites on each construct. The mutated nucleotides corresponding to κB-1-mut and κB-2-mut are underlined. B and C. DU145/sh-Con and DU145/sh-E2F1 cells were co-transfected with duplex siRNA targeting p65 and either ICAM-1 wild type or mutant promoter reporters. The stars indicate the significant differences (P < 0.05) (B). The expression of membrane ICAM-1 was measured by Western blot (C). The data are shown as the mean ± SD of triplicate measurements. D and E. DU145/sh-Con and DU145/sh-E2F1 cells were transiently transfected with either the empty vector or the mutant IκBα expressing plasmids. The phosphorylation of p65 and IκBα and the expression of ICAM-1 in these indicated cells were monitered by Western blot (D). DU145/sh-Con and DU145/sh-E2F1 cells were co-transfected with IκBα expressing plasmids and either the ICAM-1 wild type or mutant promoter reporters for the luciferase activity assay (E). The results are shown as the mean ± SD of triplicate measurements. The stars indicate the significant differences (P < 0.05).