American Society of Hematology

Figure 2

$Figure 2. Effect of RNA splicing on vWF promoter activity in cultured primary human endothelial cells and MEG-01 cells. (A) HUVECs were transiently transfected with a pGL3 construct containing the human vWF promoter spanning the region between −2182 and +246 (which corresponds to the end of the first exon) without an intron (vWFΔInt) or linked 5′ of the first intron from the human vWF gene (vWF2), the second intron from the human β-globin gene (vWFΔIntGIn), the second intron from the mouse DSCR-1 gene (vWFΔIntDIn), or the sixth intron from the hagfish factor X gene (vWFΔIntHIn). Alternatively, cells were transfected with a pGL3 construct in which the first intron of the vWF gene was placed upstream of the −2182 and +246 vWF promoter in the correct orientation (Int-vWFΔInt) or in the reverse direction (RInt-vWFΔInt), with a a pGL3 construct in which the second intron from the human β-globin gene was placed upstream of the −2182 and +246 vWF promoter in the correct orientation (GInt-vWFΔInt) or with a pGL3 construct containing a 2100-bp fragment of the mouse Tie2 promoter linked in the absence or presence of the first intron of human vWF (Tie2P and Tie2P-vWFIn, respectively). (Inset) MEG-01 cells were transiently transfected with a pGL3 construct containing the human vWF promoter spanning the region between −2182 and +246 (which corresponds to the end of the first exon) in the absence or presence of the first intron from the human vWF gene. The results show the mean ± standard deviation of luciferase light units (relative to vWF2) obtained in triplicate from ≥3 independent experiments. *P < .05. n.s., nonsignificant. (B) HUVECs were transfected with vWF2-luc or vWFΔInt-luc constructs. After a 24-hour incubation, total or cytoplasmic (cyto) extract fractions were prepared. RNA was extracted, and luciferase mRNA levels were measured by qPCR. The results show the mean ± standard deviation of luciferase mRNA levels (relative to vWF2) obtained in triplicate from 3 independent experiments. n.s., nonsignificant. (C) HUVECs were transfected with vWF2-luc or vWFΔInt-luc plasmids. After a 24-hour incubation, cells were treated with cyclohexamide (100 μg/mL) and collected at the indicated time points to measure the luciferase activity. Data are presented as percentage relative to 0 hours. n = 3 independent experiments.$

Effect of RNA splicing on vWF promoter activity in cultured primary human endothelial cells and MEG-01 cells. (A) HUVECs were transiently transfected with a pGL3 construct containing the human vWF promoter spanning the region between −2182 and +246 (which corresponds to the end of the first exon) without an intron (vWFΔInt) or linked 5′ of the first intron from the human vWF gene (vWF2), the second intron from the human β-globin gene (vWFΔIntGIn), the second intron from the mouse DSCR-1 gene (vWFΔIntDIn), or the sixth intron from the hagfish factor X gene (vWFΔIntHIn). Alternatively, cells were transfected with a pGL3 construct in which the first intron of the vWF gene was placed upstream of the −2182 and +246 vWF promoter in the correct orientation (Int-vWFΔInt) or in the reverse direction (RInt-vWFΔInt), with a a pGL3 construct in which the second intron from the human β-globin gene was placed upstream of the −2182 and +246 vWF promoter in the correct orientation (GInt-vWFΔInt) or with a pGL3 construct containing a 2100-bp fragment of the mouse Tie2 promoter linked in the absence or presence of the first intron of human vWF (Tie2P and Tie2P-vWFIn, respectively). (Inset) MEG-01 cells were transiently transfected with a pGL3 construct containing the human vWF promoter spanning the region between −2182 and +246 (which corresponds to the end of the first exon) in the absence or presence of the first intron from the human vWF gene. The results show the mean ± standard deviation of luciferase light units (relative to vWF2) obtained in triplicate from ≥3 independent experiments. *P < .05. n.s., nonsignificant. (B) HUVECs were transfected with vWF2-luc or vWFΔInt-luc constructs. After a 24-hour incubation, total or cytoplasmic (cyto) extract fractions were prepared. RNA was extracted, and luciferase mRNA levels were measured by qPCR. The results show the mean ± standard deviation of luciferase mRNA levels (relative to vWF2) obtained in triplicate from 3 independent experiments. n.s., nonsignificant. (C) HUVECs were transfected with vWF2-luc or vWFΔInt-luc plasmids. After a 24-hour incubation, cells were treated with cyclohexamide (100 μg/mL) and collected at the indicated time points to measure the luciferase activity. Data are presented as percentage relative to 0 hours. n = 3 independent experiments.

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