Figure 4
Figure 4. Promoter activity assay. (A) Luciferase reporter activity was examined with various amounts (nanograms) of pcDNA6-HA-AML1–, pcDNA6-HA-AML1-ETO–, or pcDNA6-HA-AML1-ETO9a–transfected 293T cells. pBAAE35-TK-luc (TC, 50 ng, wild type), pCMV5-CBFb 21.5 (150 ng), and pNull-renilla luciferase (5 ng) were cotransfected. The value of y-axis is a fold change of luciferase activity compared with vector alone. The results are presented as the mean value of 3 independent experiments; error bars represent standard deviation. The almost equal level of protein expression induced from each amount of plasmid DNA was confirmed by immunoblotting with anti-HA antibody (data not shown). (B) Luciferase reporter assay was performed with 293T cells transfected with 50 ng wild-type (TC) or mutant (mC and Tm) of pBAAE35-TK-luc, 150 ng pCMV5-CBFb 21.5, and 5 ng pNull-renilla luciferase and increasing amount of pcDNA6-HA-AML1-ETO expression plasmid. (C) U937T-AML1-ETO cells were stably transfected with wild-type phSeparase-luc (TC) or the constructs including mutated AML1-binding sites (mC, Tm, mm). Two independent cell pools stably transfected with each construct were used in this study. The value of y-axis is a fold change of luciferase activity compared with day 0 of tetracycline (tet) withdrawal. The results are presented as the mean value of 3 independent experiments; error bars represent standard deviation. At day 1 of the tet withdrawal, statistically significant P values compared with mm control (*t test, P < .001 for TC, mC, and Tm) are indicated. At day 2 of the tet withdrawal, statistically significant P value compared with mm control (**t test, P = .005 for TC) is indicated. The expression of AML1-ETO was controlled by tet withdrawal, and protein expression was confirmed by Western blotting using anti-AML1 antibody (◀). Ponceau staining shows the relative amount of protein loading. As a negative control, all of these phSeparase-luc constructs were also stably transfected into parental U937T cells and no change of luciferase activity was detected after tet withdrawal (data not shown).

Promoter activity assay. (A) Luciferase reporter activity was examined with various amounts (nanograms) of pcDNA6-HA-AML1–, pcDNA6-HA-AML1-ETO–, or pcDNA6-HA-AML1-ETO9a–transfected 293T cells. pBAAE35-TK-luc (TC, 50 ng, wild type), pCMV5-CBFb 21.5 (150 ng), and pNull-renilla luciferase (5 ng) were cotransfected. The value of y-axis is a fold change of luciferase activity compared with vector alone. The results are presented as the mean value of 3 independent experiments; error bars represent standard deviation. The almost equal level of protein expression induced from each amount of plasmid DNA was confirmed by immunoblotting with anti-HA antibody (data not shown). (B) Luciferase reporter assay was performed with 293T cells transfected with 50 ng wild-type (TC) or mutant (mC and Tm) of pBAAE35-TK-luc, 150 ng pCMV5-CBFb 21.5, and 5 ng pNull-renilla luciferase and increasing amount of pcDNA6-HA-AML1-ETO expression plasmid. (C) U937T-AML1-ETO cells were stably transfected with wild-type phSeparase-luc (TC) or the constructs including mutated AML1-binding sites (mC, Tm, mm). Two independent cell pools stably transfected with each construct were used in this study. The value of y-axis is a fold change of luciferase activity compared with day 0 of tetracycline (tet) withdrawal. The results are presented as the mean value of 3 independent experiments; error bars represent standard deviation. At day 1 of the tet withdrawal, statistically significant P values compared with mm control (*t test, P < .001 for TC, mC, and Tm) are indicated. At day 2 of the tet withdrawal, statistically significant P value compared with mm control (**t test, P = .005 for TC) is indicated. The expression of AML1-ETO was controlled by tet withdrawal, and protein expression was confirmed by Western blotting using anti-AML1 antibody (◀). Ponceau staining shows the relative amount of protein loading. As a negative control, all of these phSeparase-luc constructs were also stably transfected into parental U937T cells and no change of luciferase activity was detected after tet withdrawal (data not shown).

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