Figure 3
Figure 3. Figure 3. Effect of AML1 on NF-E2 1A promoter activity. (A) Cotransfection of AML1. Plasmids encoding the −3606 bp NF-E2-1A promoter-Luciferase construct or the −1740-bp fragment, a −1439-bp fragment, a −167-bp fragment, or the empty pGL3 vector were cotransfected into γ-2A cells either with an expression vector for AML1 (filled bars) or with an empty control vector (open bars). Luciferase activity was measured 16 hours after transfection and normalized for transfection efficiency by determination of Renilla luciferase activity from a cotransfected vector. Activity of the empty pGL3 vector was set at 1, and fold activity relative to this control is depicted. Bar graphs represent the mean ± SD of 4 independent experiments, each performed in duplicate, ***P < .001 by one-way ANOVA. (B) Cotransfection of AML1 and mutation of AML1 binding sites. Plasmids encoding the wild-type −3606 bp NF-E2-1A promoter-Luciferase construct or vectors in which one or several of the AML1 binding sites (open boxes) were mutated (indicated by crosses in the boxes) were cotransfected into γ-2A cells, either with an expression vector for AML1 (filled bars) or with an empty control vector (open bars). The introduced mutations have previously been shown to inactivate AML1 DNA binding.30 Luciferase activity was measured 16 hours after transfection and normalized for transfection efficiency by determination of Renilla luciferase activity from a cotransfected vector. Activity of the empty pGL3 vector was set at 1, and fold activity relative to this control is depicted. Bar graphs represent the mean ± SD of 4 independent experiments, each performed in duplicate ***P < .001, **P < .01 by one-way ANOVA. (C-D) Effect of mutating the AML1 binding sites on NF-E2 promoter activity in hematopoietic cells. Plasmids encoding the −3606-bp NF-E2-1A promoter-Luciferase construct, the −3606-bp NF-E2-1A construct in which one or several of the AML1 binding sites (open boxes) were mutated (indicated by crosses in the boxes) or the −1740-bp NF-E2 1A fragment were transiently nucleofected into K562 cells (C) or UKE-1 cells (D). At 24 hours after transfection, luciferase activity was determined. Results were normalized for transfection efficiency by cotransfection of a Tk-driven Renilla luciferase reporter gene vector. Mean and SD of 3 independent experiments, each measured in duplicate, are shown. *P < .05 by Kruskal Wallis one-way ANOVA on Ranks.

Figure 3. Effect of AML1 on NF-E2 1A promoter activity. (A) Cotransfection of AML1. Plasmids encoding the −3606 bp NF-E2-1A promoter-Luciferase construct or the −1740-bp fragment, a −1439-bp fragment, a −167-bp fragment, or the empty pGL3 vector were cotransfected into γ-2A cells either with an expression vector for AML1 (filled bars) or with an empty control vector (open bars). Luciferase activity was measured 16 hours after transfection and normalized for transfection efficiency by determination of Renilla luciferase activity from a cotransfected vector. Activity of the empty pGL3 vector was set at 1, and fold activity relative to this control is depicted. Bar graphs represent the mean ± SD of 4 independent experiments, each performed in duplicate, ***P < .001 by one-way ANOVA. (B) Cotransfection of AML1 and mutation of AML1 binding sites. Plasmids encoding the wild-type −3606 bp NF-E2-1A promoter-Luciferase construct or vectors in which one or several of the AML1 binding sites (open boxes) were mutated (indicated by crosses in the boxes) were cotransfected into γ-2A cells, either with an expression vector for AML1 (filled bars) or with an empty control vector (open bars). The introduced mutations have previously been shown to inactivate AML1 DNA binding.30  Luciferase activity was measured 16 hours after transfection and normalized for transfection efficiency by determination of Renilla luciferase activity from a cotransfected vector. Activity of the empty pGL3 vector was set at 1, and fold activity relative to this control is depicted. Bar graphs represent the mean ± SD of 4 independent experiments, each performed in duplicate ***P < .001, **P < .01 by one-way ANOVA. (C-D) Effect of mutating the AML1 binding sites on NF-E2 promoter activity in hematopoietic cells. Plasmids encoding the −3606-bp NF-E2-1A promoter-Luciferase construct, the −3606-bp NF-E2-1A construct in which one or several of the AML1 binding sites (open boxes) were mutated (indicated by crosses in the boxes) or the −1740-bp NF-E2 1A fragment were transiently nucleofected into K562 cells (C) or UKE-1 cells (D). At 24 hours after transfection, luciferase activity was determined. Results were normalized for transfection efficiency by cotransfection of a Tk-driven Renilla luciferase reporter gene vector. Mean and SD of 3 independent experiments, each measured in duplicate, are shown. *P < .05 by Kruskal Wallis one-way ANOVA on Ranks.

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