Fig. 9.
Fig. 9. Activation of Stat1 and Stat3 is required for the. / c-myc gene activation. (A) EPO-induced activation of c-myc promoter via the putative binding site of Stat1 and Stat3. Luciferase reporter constructs containing human c-mycgene promoter and internal control reporter plasmid pRL-TK vector were introduced into UT-7/EPO cells. The cells were starved for 12 hours and then transfected with the reporter constructs. After an additional 12 hours of incubation, the cells were treated with or without EPO (10 U/mL). Finally, 6 hours later, the cells were harvested for dual luciferase assay. The results for EPO-exposed cells are expressed as a fold-induction of the luciferase activity of the same construct in the control condition (without EPO). The values represent the mean ± SD from triplicate experiments. The box indicates the putative binding site of Stat1 and Stat3. (B) The effects of dominant-negative Stat1 and Stat3 on EPO-induced activation of c-myc promoter. Thec-myc reporter constructs −2039/+532 myc were introduced into the UT-7/EPO cells with expression vector pCAGGS-neo or pCAGGS-Stat1F bearing cDNA encoding dominant-negative Stat1, or with pCAGGS-Stat3F bearing the cDNA encoding dominant-negative Stat3. After transfection, the cells were cultured with EPO for 6 hours and then harvested for luciferase assay. The results are expressed as a fold-induction of the luciferase activity of the combination of −2039/+532 myc and pCAGGS vector. The values represent the mean ± SD from triplicate experiments. (C) G-CSF–induced activation ofc-myc promoter in UT-7/EPO cells expressing the chimeric receptor. The transfectant cells were starved for 12 hours and then introduced with the −2039/+532 myc reporter constructs. After an additional 12 hours of incubation, the cells were treated with or without G-CSF (250 ng/mL) for 6 hours and then harvested for luciferase assay. The results for G-CSF–exposed cells are expressed as a fold-induction of the luciferase activity of the same construct in the control condition (without G-CSF). The values represent the mean ± SD from triplicate experiments.

Activation of Stat1 and Stat3 is required for the

c-myc gene activation. (A) EPO-induced activation of c-myc promoter via the putative binding site of Stat1 and Stat3. Luciferase reporter constructs containing human c-mycgene promoter and internal control reporter plasmid pRL-TK vector were introduced into UT-7/EPO cells. The cells were starved for 12 hours and then transfected with the reporter constructs. After an additional 12 hours of incubation, the cells were treated with or without EPO (10 U/mL). Finally, 6 hours later, the cells were harvested for dual luciferase assay. The results for EPO-exposed cells are expressed as a fold-induction of the luciferase activity of the same construct in the control condition (without EPO). The values represent the mean ± SD from triplicate experiments. The box indicates the putative binding site of Stat1 and Stat3. (B) The effects of dominant-negative Stat1 and Stat3 on EPO-induced activation of c-myc promoter. Thec-myc reporter constructs −2039/+532 myc were introduced into the UT-7/EPO cells with expression vector pCAGGS-neo or pCAGGS-Stat1F bearing cDNA encoding dominant-negative Stat1, or with pCAGGS-Stat3F bearing the cDNA encoding dominant-negative Stat3. After transfection, the cells were cultured with EPO for 6 hours and then harvested for luciferase assay. The results are expressed as a fold-induction of the luciferase activity of the combination of −2039/+532 myc and pCAGGS vector. The values represent the mean ± SD from triplicate experiments. (C) G-CSF–induced activation ofc-myc promoter in UT-7/EPO cells expressing the chimeric receptor. The transfectant cells were starved for 12 hours and then introduced with the −2039/+532 myc reporter constructs. After an additional 12 hours of incubation, the cells were treated with or without G-CSF (250 ng/mL) for 6 hours and then harvested for luciferase assay. The results for G-CSF–exposed cells are expressed as a fold-induction of the luciferase activity of the same construct in the control condition (without G-CSF). The values represent the mean ± SD from triplicate experiments.

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