Fig. 4

Validation of the N-Myc target sites in the PLAGL2 promoter region. a Schematic diagram showing the five putative N-Myc binding E-boxes (E1-E5) in the PLAGL2 gene promoter region and the amplified regions to construct the luciferase reporters. Three luciferase reporters, including plaP_(E1–5)-Luc (spanning all 5 E-boxes), plaP_(E1–3)-Luc (spanning E1 to E3) and plaP_(E4–5)-Luc (spanning E4 and E5), were generated. b Validation of the target sites by luciferase assay in HEK 293T cells. A MYCN over-expression vector (MYCN(+)) or a control expression vector (MYCN(−)) was co-transfected with the indicated luciferase reporter or a Control reporter into cells. After 2 days, the luciferase activity was measured. *, p < 0.05. c-d Validation of the PLAGL2 target sites by CHIP-PCR assay. c The primers sets used in the assay. Primers 1, 2 and 3 were used to validate the E4-E5, E2-E3 and E1 sites, respectively. d, The CHIP-PCR results performed using the indicated primers sets under the indicated treatments. The relative band intensity shown above each band was normalized as above