Figure 5
Figure 5. RUNX1-ETO specifically repressed the SFRP1 gene through a consensus RUNX binding site on the SFRP1 promoter. (A) The SFRP1 promoter-luciferase construct −706-Luc was cotransfected with pCMV-RUNX1-ETO, pCMV-CBFβ-MYH11, or pCMV-RUNX1-ETOΔ469 together with pRL-CMV into U937 cells. Results are presented as relative promoter activity by comparing the normalized firefly luciferase activity of the construct cotransfected with the expression plasmids to that cotransfected with empty pCMV. (B top) Confirmation of RUNX1-ETO induction on tetracycline withdrawal in U937T-AE cells by semiquantitative RT-PCR. Amplification of GAPDH cDNA was done as internal control. No RUNX1-ETO induction was observed in the parental U937T cells. (B bottom) The SFRP1 promoter-luciferase construct −706-Luc was cotransfected with pRL-CMV into U937T and U937T-AE cells. Six hours after transfection, the medium was removed, and cells were cultured for another 48 hours in the presence or absence of tetracycline (Tet) before luciferase measurement. Transfection with the same amount of pGL3-Basic and pRL-CMV was done in parallel. Results are presented as relative promoter activity by comparing the normalized firefly luciferase activity of the construct with that of pGL3-Basic in each respective group (+Tet or −Tet). The fold of repression is indicated. (C) SFRP1, SFRP2, and SFRP3 promoter-luciferase constructs were cotransfected with pCMV-RUNX1-ETO and pRL-CMV into HeLa cells. Cotransfection with the same amount of empty pCMV was done in parallel. Results are presented as fold of repression by comparing the normalized firefly luciferase activity of the construct cotransfected with pCMV-RUNX1-ETO with that cotransfected with empty pCMV. All numberings are relative to the first nucleotide of the start codon (+1) of each gene. In all experiments, transfection efficiency was normalized according to the cotransfected pRL-CMV Renilla luciferase activity. (D) A549 cells were transfected with 10 μg of pCMV or pCMV-RUNX1-ETO with the use of Lipofectamine 2000. RUNX1-ETO expression was validated by RT-PCR (top), and SFRP mRNA levels were determined by real-time RT-PCR and normalized with GAPDH (bottom) 48 hours after transfection. All results are expressed as mean ± SE from at least triplicate assays. *P < .05.

RUNX1-ETO specifically repressed the SFRP1 gene through a consensus RUNX binding site on the SFRP1 promoter. (A) The SFRP1 promoter-luciferase construct −706-Luc was cotransfected with pCMV-RUNX1-ETO, pCMV-CBFβ-MYH11, or pCMV-RUNX1-ETOΔ469 together with pRL-CMV into U937 cells. Results are presented as relative promoter activity by comparing the normalized firefly luciferase activity of the construct cotransfected with the expression plasmids to that cotransfected with empty pCMV. (B top) Confirmation of RUNX1-ETO induction on tetracycline withdrawal in U937T-AE cells by semiquantitative RT-PCR. Amplification of GAPDH cDNA was done as internal control. No RUNX1-ETO induction was observed in the parental U937T cells. (B bottom) The SFRP1 promoter-luciferase construct −706-Luc was cotransfected with pRL-CMV into U937T and U937T-AE cells. Six hours after transfection, the medium was removed, and cells were cultured for another 48 hours in the presence or absence of tetracycline (Tet) before luciferase measurement. Transfection with the same amount of pGL3-Basic and pRL-CMV was done in parallel. Results are presented as relative promoter activity by comparing the normalized firefly luciferase activity of the construct with that of pGL3-Basic in each respective group (+Tet or −Tet). The fold of repression is indicated. (C) SFRP1, SFRP2, and SFRP3 promoter-luciferase constructs were cotransfected with pCMV-RUNX1-ETO and pRL-CMV into HeLa cells. Cotransfection with the same amount of empty pCMV was done in parallel. Results are presented as fold of repression by comparing the normalized firefly luciferase activity of the construct cotransfected with pCMV-RUNX1-ETO with that cotransfected with empty pCMV. All numberings are relative to the first nucleotide of the start codon (+1) of each gene. In all experiments, transfection efficiency was normalized according to the cotransfected pRL-CMV Renilla luciferase activity. (D) A549 cells were transfected with 10 μg of pCMV or pCMV-RUNX1-ETO with the use of Lipofectamine 2000. RUNX1-ETO expression was validated by RT-PCR (top), and SFRP mRNA levels were determined by real-time RT-PCR and normalized with GAPDH (bottom) 48 hours after transfection. All results are expressed as mean ± SE from at least triplicate assays. *P < .05.

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