KLF2 is essential for and contributes significantly to statin enhancement of tBHQ-induced γ-globin gene expression. K562 cells were transiently transfected with siRNA specific for KLF2. Twenty-four hours later, cells were treated with tBHQ or tBHQ and simvastatin (statin) for 48 hours before RNA isolation. Mock transfection (UT) and control siRNA (siC) were used as controls. (A) qPCR results measuring KLF2 steady-state mRNA levels 24 hours after siRNA transfection presented as the percentage relative to siC. (B) Effect of KLF2 siRNA on 25μM tBHQ-induced γ-globin mRNA levels. Results are expressed as the fold increase over untreated controls to emphasize that KLF2 knock-down had no effect on tBHQ induction of γ-globin. (C) Effect of KLF2 siRNA on statin-enhanced tBHQ-induced γ-globin mRNA levels. Results are expressed as the fold increase over tBHQ alone to emphasize the fold induction-dependent on the addition of statin. Error bars represent ± SEM. *P < .05 compared with siC at 2.5 or 5μM statin (n = 3). (D-E) klf2 overexpression significantly enhances tBHQ-induced γ-globin gene expression. (D) Real-time analysis of mRNA levels and Western blot analysis of nuclear extracts of empty vector (V) used as control and cells stably overexpressing murine klf2. TBP was used as a nuclear loading control. (E) Effect of overexpressing klf2 on tBHQ induction of V and γ-globin mRNA. Cells overexpressing V or klf2 were treated with tBHQ and harvested for RNA at 6, 12, and 24 hours after treatment. Results of real-time PCR are presented as the relative mRNA expression compared with 0-hour controls. Error bars represent ± SEM. *P < .05 (n = 2).