Overexpression of FoxO3 enhances gilz promoter activity and endogenous gilz expression.(A) FoxO3 is transcriptionally active in CTLL-2 cells. CTLL-2 cells were transiently transfected with 10 μg FHRE-Luc and 10 μg pcDNA3 (□) or pcDNA3-FoxO3 (▦) and cultured with or without IL-2 for 8 hours. Results are expressed as percentage of FHRE-Luc activity and represent the mean ± SEM (error bars) of 2 independent experiments performed in duplicate. Expression of FoxO3 was controlled by Western blot (bottom panel). (B) FoxO3 overexpression enhances gilz promoter activity. CTLL-2 cells were transiently transfected with 10 μg of either p-1940, p-1940_FHRE1-mut, p-1940_FHRE3-mut, or p-1940_FHRE1+3-mut luciferase constructs and 10 μg pcDNA3 (□) or pcDNA3-FoxO3 (▦) and cultured for 8 hours in the absence of IL-2. Data are expressed as a percentage of p-1940 activity, with 100% being the activity measured in cells transfected with pcDNA3. Data represent the mean ± SEM (error bars) of 3 independent experiments performed in duplicate. Expression of FoxO3 was controlled by Western blot (bottom panel). (C) Overexpression of FoxO3 enhanced endogenous GILZ expression. CTLL-2 cells were transiently transfected with 10 μg pcDNA3 or pcDNA3-FoxO3. After 16 hours of expression, cells were deprived of IL-2 for 6 hours. Total RNA was extracted and used as a template for RT-PCR using primers specific for gilz or β actin. Gilz expression was quantified by densitometric analysis and normalized to the densitometric value of the β actin (ratio GILZ/β actin).