Figure 1.

N-Myc upregulates LYAR expression in neuroblastoma cells by binding to the LYAR gene promoter. (a and b) BE(2)-C and CHP134 cells were transfected with control siRNA, N-Myc siRNA-1 or N-Myc siRNA-2. Seventy-two hours later, RNA and protein were extracted from the cells for RT-PCR (a) and immunoblot (b) analyses of N-Myc and LYAR expression. (c) SHEP21N cells were treated with doxycycline (2 μg/ml) or vehicle control for 72 h. RT-PCR and immunoblot analyses were conducted on N-Myc and LYAR mRNA and protein expression. (d) Schematic representation of the LYAR gene promoter. TSS represented transcription start site. (e) ChIP assays were performed with a control or anti-N-Myc antibody, followed by PCR with primers targeting the negative control region (Amplicon A) or the LYAR gene core promoter containing the Myc responsive element E-Box (Amplicons B) in BE(2)-C and CHP134 cells 24 h after transfection with control siRNA, N-Myc siRNA-1 or N-Myc siRNA-2. Fold enrichment of the LYAR gene promoter was calculated as the difference in PCR cycle thresholds obtained with the anti-N-Myc antibody and with the control antibody. Error bars represented standard errors (S.E.). *P<0.05 and ***P<0.001