Figure 4
MYCN directly binds
CCL2 promoter.
(A) CCL2 promoter cDNA constructs with (2.8 kb) and without (2.6 kb) E-box site were fused with a Firefly luciferase cDNA. A pGL3 plasmid containing only Firefly luciferase cDNA was used as a negative control. CHLA-255 or CHLA-255/MYCN cells were transiently cotransfected with the indicated constructs and pRLSV40 plasmid containing Renilla luciferase cDNA as an internal control. The activity of CCL2 promoter was detected in a dual-luciferase assay and expressed as a ratio of Firefly to Renilla luciferase luminescence intensity. (B) Nuclear extract from neuroblastoma cells was analyzed by EMSA with biotinylated E-box probe from CCL2 promoter. Lane 1, free probe; lane 2, nonbiotin (cold) probe plus probe plus CHLA-255/MYCN; lane 3, probe plus CHLA-255/vector; lane 4, probe plus CHLA-255/MYCN; lane 5, probe plus CHLA-255/MYCN plus anti-MYCN mAb; lane 6, probe plus CHLA-255/MYCN plus IgG control. Data are representative of 6 experiments. (C) Nuclear extract from CHLA-255/MYCN was analyzed by EMSA with E-box probe as above (positive control) or with INR probes. Lane 1, free E-box probe; lane 2, E-box probe plus CHLA-255/MYCN; lanes 3, 5, and 7, free probes for INR-1, INR-2, and INR-3, respectively; lanes 4, 6, and 8, CHLA-255/MYCN plus probes for INR-1, INR-2, and INR-3, respectively. Data are representative of 3 experiments.
