Studies using the transcriptional inhibitor, Act D, demonstrate that the induction of Ndrg1 is transcriptionally regulated following Fe chelation. MCF-7 breast cancer cells were preincubated for 2 hours at 37°C with either control media or media containing the transcriptional inhibitor, Act D (4 μM). This media were then subsequently removed and replaced with control media (MEM) or media containing either DFO (250 μM), 311 (25 μM), or Act D (4 μM) for an additional 6 hours at 37°C. The mRNA levels for Ndrg1 (A), VEGF-1 (B), and TfR1 (C) were then assessed by using semiquantitative RT-PCR (described in “Materials and methods”). Densitometry was performed, and gene expression was then calculated relative to the β-actin control. Results are a typical experiment from 3 performed.