Silencing Nrf2 enhances chemotherapy-induced apoptosis in AML. THP-1 cells were transduced with Nrf2-targeted miRNA lentiviral constructs. (A) The percentage of gated GFP-positive cells was measured with flow cytometry. (B). RNA was extracted from AML cells transduced with Nrf2-targeted and nonsilencing miRNA control constructs and examined for Nrf2 expression by real-time PCR compared with CD34⁺ control cells (dashed line; C) RNA was extracted from THP-1 cells transduced with Nrf2-targeted and nonsilencing miRNA control constructs and examined for Nrf2 expression by real-time PCR at the indicated times. mRNA expression was normalized to GAPDH mRNA levels. (D) Protein extracts were also obtained and Western blot analysis was conducted for nuclear Nrf2 protein levels. (E) AML cells were transduced with either Nrf2-targeted miRNA or nonsilencing control miRNA construct for 4 days, then treated with 0.5μM cytarabine and 0.2μM daunorubicin for 24 hours. Cell number was assessed by MTS assay. In all panels values indicate the mean ± SEM from 3 independent experiments. *Statistical significance of P < .05 between the different treatment groups. (F) AML cells and control cells were transduced with Nrf2-targeted miRNA and control miRNA constructs and treated with 0.5μM cytarabine and 0.2μM daunorubicin. Cells were washed with PBS and incubated with 10μM of H₂DCFDA for 15 minutes. Cells were then assessed for H₂DCFDA oxidation using flow cytometry. *Statistical significance of P < .05 by 2-way ANOVA analysis. (G) Protein extracts also were obtained, and Western blot analysis was conducted for Nrf2 and p65 protein levels.