Figure 7.
Figure 7. GILZ inhibits FoxO3 transcriptional activity and consequently down-regulates its own expression. (A) GILZ inhibits FoxO3 transcriptional activity. CTLL-2 cells were transiently transfected with 5 μg FHRE-Luc reporter construct along with increasing doses (1, 5, and 10 μg) of the expression vector for GILZ and/or FoxO3 as indicated. The total amounts of transfected DNA were kept constant by addition of empty control vector. Data are expressed as a percentage of FHRE-Luc activity and represent the mean ± SEM (error bars) of 3 independent experiments performed in triplicate. Lower panel: Western blot analysis for expression of Myc-GILZ and FoxO3. (B) GILZ inhibits Bim promoter transcriptional activity. CTLL-2 cells were transiently transfected with 5 μg pBim-Luc reporter construct along with increasing doses of the expression vector for GILZ (1, 5, and 10 μg) and/or FoxO3 (1 and 5 μg) as indicated. The total amounts of transfected DNA were kept constant by addition of empty control vector. Data are expressed as a percentage of pBim-Luc activity and represent the mean ± SEM (error bars) of 2 independent experiments performed in triplicate. Lower panel: Western blot analysis for expression of Myc-GILZ and FoxO3. (C) GILZ down-regulates the activity of its own promoter. CTLL-2 cells were transiently transfected with 5 μg p-1940 or p-1940FHRE1+3-mut reporter constructs along with increasing doses (1, 5, and 10 μg) of the expression vector for GILZ. The total amounts of transfected DNA were kept constant by addition of empty control vector. Results are expressed as a percentage of p-1940 activity and represent the mean ± SEM (error bars) of 3 independent experiments performed in triplicate. Lower panel: Western blot analysis of Myc-GILZ expression. (D) gilz transcripts are down-regulated in Myc-GILZ–overexpressing CTLL-2 clones. GILZ-overexpressing clones (4 and 6) and control clones (1 and 2) were deprived of IL-2 and lysed at the indicated period of time. Total RNA was extracted and used as a template for RT-PCR using primers specific for gilz or β actin. Differential detection of endogenous gilz transcript was performed using a primer in the 3′ untranslated region of the gilz mRNA (GILZ 3′ UTR). Endogenous gilz expression was quantified by densitometric analysis and normalized to the densitometric value of β actin (ratio GILZ 3′ UTR/β actin).

GILZ inhibits FoxO3 transcriptional activity and consequently down-regulates its own expression. (A) GILZ inhibits FoxO3 transcriptional activity. CTLL-2 cells were transiently transfected with 5 μg FHRE-Luc reporter construct along with increasing doses (1, 5, and 10 μg) of the expression vector for GILZ and/or FoxO3 as indicated. The total amounts of transfected DNA were kept constant by addition of empty control vector. Data are expressed as a percentage of FHRE-Luc activity and represent the mean ± SEM (error bars) of 3 independent experiments performed in triplicate. Lower panel: Western blot analysis for expression of Myc-GILZ and FoxO3. (B) GILZ inhibits Bim promoter transcriptional activity. CTLL-2 cells were transiently transfected with 5 μg pBim-Luc reporter construct along with increasing doses of the expression vector for GILZ (1, 5, and 10 μg) and/or FoxO3 (1 and 5 μg) as indicated. The total amounts of transfected DNA were kept constant by addition of empty control vector. Data are expressed as a percentage of pBim-Luc activity and represent the mean ± SEM (error bars) of 2 independent experiments performed in triplicate. Lower panel: Western blot analysis for expression of Myc-GILZ and FoxO3. (C) GILZ down-regulates the activity of its own promoter. CTLL-2 cells were transiently transfected with 5 μg p-1940 or p-1940FHRE1+3-mut reporter constructs along with increasing doses (1, 5, and 10 μg) of the expression vector for GILZ. The total amounts of transfected DNA were kept constant by addition of empty control vector. Results are expressed as a percentage of p-1940 activity and represent the mean ± SEM (error bars) of 3 independent experiments performed in triplicate. Lower panel: Western blot analysis of Myc-GILZ expression. (D) gilz transcripts are down-regulated in Myc-GILZ–overexpressing CTLL-2 clones. GILZ-overexpressing clones (4 and 6) and control clones (1 and 2) were deprived of IL-2 and lysed at the indicated period of time. Total RNA was extracted and used as a template for RT-PCR using primers specific for gilz or β actin. Differential detection of endogenous gilz transcript was performed using a primer in the 3′ untranslated region of the gilz mRNA (GILZ 3′ UTR). Endogenous gilz expression was quantified by densitometric analysis and normalized to the densitometric value of β actin (ratio GILZ 3′ UTR/β actin).

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