NF-κB regulates Nrf2 overexpression in human AML. (A) AML and control cells were treated with Act D (5 μg/mL) for up to 6 hours. RNA was extracted and examined for Nrf2 mRNA expression by real-time PCR. mRNA expression was normalized to GAPDH mRNA levels. (B) AML and control cells were treated for 6 and 24 hours with U0126 (10μM) and RNA was extracted and examined for Nrf2 mRNA expression by real-time PCR. mRNA expression was normalized to GAPDH mRNA levels. Data presented as percent of control. (C) Schematic representation of the Nrf2 promoter. (D) AML and control cells were treated for 6 and 24 hours with BAY 11-7082 (10μM) and RNA was extracted and examined for Nrf2 mRNA expression by real-time PCR. mRNA expression was normalized to GAPDH mRNA levels. Data are presented as percent of control. *P < .05 between the different treatment groups. (E) Nuclear and cytosolic Western blots from Figure 1A were reprobed for p65 expression. (F) Analysis of p65 and Nrf2 subcellular localization in AML cell nucleus. Data were calculated as a percentage of total localization and is expressed as fold increase compared with healthy CD34⁺ cell levels. Values indicate the mean ± SEM from at least 5 cells per sample (statistical significance *P ≤ .05 compared with an average of both CD34⁺ samples). Relative fold p65 versus fold Nrf2 scatter plot indicates a correlation with a slope of 1.