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. 2002 Dec;22(24):8695–8708. doi: 10.1128/MCB.22.24.8695-8708.2002

FIG. 7.

FIG. 7.

Regulation of tradd gene transcription by FKHR. (A) 293T cells were transfected with either the pGL3-promoter vector containing nothing (Control), an FHRE-containing 582-bp segment of intron 1 of the tradd gene (Intron 1), three copies of a 10-bp FHRE-like domain of IGF-BP1 gene (3× IRS) together with the pcDNA3 vector encoding nothing (Mock), WT-FKHR (WT), AAA-FKHR (AAA), or H215R-FKHR (H215R). The pRL-TK plasmid was also cotransfected as a transfection efficiency control. After 24 h of transfection, luciferase activities were calculated with the dual-luciferase reporter assay system as described in Materials and Methods. (B) 293T cells were transfected with vector pGL3 containing one (×1) or two (×2) copies of the 10-bp potential FKHR-binding sequence in the tradd gene or one mutant copy of the potential FKHR-binding sequence (Mutant) together with vector pcDNA3 encoding nothing (Mock), WT-FKHR (WT), AAA-FKHR (AAA), or H215R-FKHR (H215R). The pRL-TK plasmid was also transfected as a transfection efficiency control. After 24 h of transfection, luciferase activities were calculated with the Dual-luciferase reporter assay system as described in Materials and Methods. (C) HT1080 cells were cotransfected with vector pGL3 containing two copies of the 10-bp potential FKHR-binding sequence in the tradd gene (pGL3-×2) and the pRL-TK plasmid as a transfection efficiency control. The pcDNA3 vector encoding nothing (Mock), WT-FKHR (WT), AAA-FKHR (AAA), or H215R-FKHR (H215R) was also cotransfected with a pUSEamp vector encoding nothing (Mock), Myr-Akt, or DN-Akt. After 24 h of transfection, luciferase activities were calculated with the dual-luciferase reporter assay system as described in Materials and Methods. (D) HT1080 cells were transfected with either two copies of the 10-bp potential FKHR-binding sequence in the tradd gene (×2) or three copies of the 10-bp potential FKHR-binding sequence in IGF-BP1 (3×IRS) together with the pRL-TK plasmid as a transfection efficiency control. After 24 h of transfection, cells were treated with 50 μM LY294002 for 12 h or 10 μg of VP-16 per ml for 36 h. Luciferase activities were calculated with the dual-luciferase reporter assay system as described in Materials and Methods.

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