Fig. 3
Validation of the PLAGL2 binding site in the MYCN promoter region. a Schematic diagram showing the putative PLAGL2 binding site in the MYCN promoter region and the region to be amplified to construct the luciferase reporter. The PLAGL2 binding site starts at − 378 nt. A segment of DNA (− 480 bp to − 124 bp) containing the predicted target site was amplified and inserted upstream of luciferase gene in pGL3B reporter to construct a wildtype luciferase reporter (mycPWT-Luc). A mutant vector with the binding site mutated was generated to use as a control (mycPMU-Luc, mutated nucleotides are shown in red). b Validation of the target site by luciferase assay in HEK 293T cells. Cells were co-transfected with the indicated luciferase reporters and PLAGL2 over-expression vector (or the Control vector) for 2 days, and luciferase activity was measured. *, p < 0.05. c Validation of the direct binding of PLAGL2 to its target site in the MYCN promoter by CHIP-PCR assay. CHIP were performed in two cell lines, BE(2)-C and KELLY. PCR was performed using the primer sets shown in (a). Shown are the representative PCR results under the indicated treatments. Values shown above the bands are the relative band intensities derived by normalizing the raw band intensities in the IP samples to those in the Input samples of the corresponding cells lines. d The depletion of PLAGL2 protein expression by the siPLAGL2 used in the CHIP-PCR experiment was confirmed by Western blots. Cells were transfected with the siPLAGL2 or control oligo (25 nM) for two days and protein levels were measured and quantified as above