GADD45A is both required for and the rate-limiting step in activation of AREG and EREG expression. (A) Cells were treated with 10 μM Gemcitabine for 72 hours and expression of AREG and EREG measured by QPCR. (B) Summary of current literature for how GADD45A mediated DNA demethylation is achieved. Transient knockdown of GADD45A was achieved using electroporation and specific siRNA (E). Adenovirus was used to overexpress GADD45A in Panc1-2G6 and Panc1-3D7 cells (H). Changes in AREG and EREG expression were measured by QPCR in Panc1-2G6 (C,F) and Panc1-3D7 (D,G). Data depicted here are representative of at least three different experiments and represent the mean +/− standard deviation. Statistical significance was calculated using a one-tailed t-test in which * denotes P < 0.05 as compared to controls.