Since our data suggest that AREG and EREG DNA demethylation is an active process, we tested the hypothesis that DNA repair is required to maintain their expression downstream of integrin α6β4 signaling. The NER pathway, including the Xeroderma pigmentosum complementation group proteins XPA, XPG, and XPF, has been implicated in active DNA demethylation by DNA repair37, 38. Accordingly, we targeted molecules critical for and specific to the NER pathway and examined their impact on AREG and EREG expression. When knockdown of XPA (Fig. 4A) was achieved, transcription of AREG and EREG in Panc-3D7 (high α6β4; Fig. 4C) remained unaffected. AREG and EREG transcription in Panc-2G6 (low α6β4; Fig. 4B) showed a statistically significant increase when XPA was knocked down, which implies negative regulation. However, due to very low basal expression of AREG and EREG in these cells (cT value >35) it is unlikely to be biologically significant. Using specific shRNAs we knocked down ERCC4 (XPF; Fig. 4D) and ERCC5 (XPG; Fig. 4G) and demonstrated that effective knockdown of NER genes had relatively little or no effect on AREG and EREG expression (Fig. 4E–F,I–J). Taken together these data indicate that NER is not required to maintain AREG or EREG expression.Figure 4NER is not required for expression of AREG and EREG. Using lentiviral transfection stable knockdown of XPA (A), ERCC4 (XPF) (D), and ERCC5 (XPG) (G) was achieved in Panc1-2G6 (low α6β4) and Panc1-3D7 (high α6β4) cells as confirmed by QPCR. AREG and EREG expression was examined following knockdown in cells with both low α6β4 (B,E,H) and high α6β4 (C,F,I) expression. Data depicted 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.