SMAD4 and neuroblastoma: To determine whether Smad4 could directly target its binding site, the HPSE promoter luciferase reporter vector and its truncates were transfected into NB cells stably transfected with empty vector (mock) or Smad4. Dual-luciferase assay indicated that −3.5 to −0.7 kb relative to TSS was essential for the negative control of HPSE promoter activity, and mutation of Smad4 binding site within this region resulted in increased HPSE promoter activity in cultured SH-SY5Y and SK-N-SH cells (Fig. 2a).