Decreased expression of VCAM-1 in c-Maf^−/− fetal liver macrophage. mRNA expression profiles of erythroblast-macrophage adhesive interaction genes at E13.5 (A) and at E14.5 (B). Total RNA obtained from the Mac-1⁺ fraction (gray bar) and Mac-1⁻ fraction (open bar) of fetal liver cells was used for analyses. VCAM-1 expression was decreased in c-Maf^−/− macrophages at E13.5 and E14.5; n = 7 per group; *P < .05. The expression level of c-Maf^+/+ fetal liver Mac-1 fraction was set to 1.0. All of the data are presented as mean ± SEM (C) Relative mean fluorescent intensity (MFI) values of VCAM-1 and Integrin αV for the c-Maf^+/+ fetal liver Mac-1 fraction (normalized to MFI = 1) and the c-Maf^−/− fetal liver Mac-1 fraction. E13.5 fetal liver cells were stained with FITC-conjugated anti–Mac-1 mAb, APC-conjugated anti–VCAM-1 mAb, and PE-conjugated anti-Integrin αV mAb. Bar graphs represent mean ratio ± SEM. Consistent with real-time RT-PCR analysis, significant differences in VCAM-1 and Integrin αV protein expression were observed; n = 8 per group; *P < .05. (D) Schematic diagram of a luciferase reporter construct with the use of a VCAM-1 0.7-kb promoter (VCAM-1 Luc, top) ligated to a firefly luciferase cassette. Three putative half-MARE sites (5′ −318 bp, −221 bp, and −136 bp) are indicated. A luciferase assay was performed with a VCAM-1 Luc and that with mutations in half-MARE (VCAM-1 mut Luc, bottom) as reporters. (E) The pEFX3-FLAG-cMaf expression vector (c-Maf Vector) was cotransfected with the reporter plasmid into the macrophage cell line J774. The relative luciferase activity shown is derived from averages of 2 independent experiments (shown as mean ± SEM). The luciferase activity seen in J774 cells transfected with the reporter plasmid and with an empty vector was normalized to a value of 1 as the standard (*P < .05).