XB-IMG-138003
Xenbase Image ID: 138003
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FIGURE 5. HIF-1 regulates transcription of nkx2.5. A, effects of various
transcription factors on nkx2.5 transcriptional activity. Luciferase activity was
measured by the lysates from CV1 cells transfected with the Nkx2.5 promoter-
Luc plasmid and the expression plasmid indicated at the bottom (control
vector (pIRES puro3), mouse Gata4, Smad4, or Hif-1 (P577G)). -Fold differences
in relative luciferase activity were calculated as arbitrary luciferase
activity. Transfection efficiency was normalized by Renilla luciferase activity
derived from null-pRL control plasmid. B, RT-PCR analysis using total RNA
from embryos at stage 1 to 25. BothRNAquality and quantity was assessed by
amplification of ornithine decarboxylase (odc). C, visualization of Nkx2.5 promoter
activity in developing embryos. The Nkx2.5 promoter-Venus (100 pg)
was co-injected with 40 ng of CMO or hif-1 MO1. Green fluorescence
reflected activation of the Nkx2.5 promoter. Fluorescence was observed
around the heart primordial region at stage 20 embryos. Nkx2.5 promoter
activity was suppressed with co-injection with hif-1 MO1, but not with CMO.
D, activation of mouse Nkx2.5 promoter (9.0 kbp) in developing Xenopus
embryos. Xenopus embryos were injected with the pGL4.10–9kb Nkx2.5 promoter
(Nkx2.5 promoter-Luc) or promoter-less luciferase reporter gene
(pGL4.10). Dual luciferase assay were performed in the embryos at the neural
stage, stage 19–20 when Nkx2.5 mRNA expression peaked. E, the effect of
injection of either CMO or hif-1 MO on Nkx2.5 promoter activity. The Nkx2.5
promoter-Luc (100 pg) and pRL-null (2 pg) were co-injected with either 40 ng
of CMO or hif-1 MO1. Embryos were harvested at the neural stage, stage
19–20. For each group, 15 pools containing 5 embryos each were used. Luciferase
activities are shown, with error bars representing the mean S.E. (n
15 each group). **, p 0.01 versus CMO). F, RT-PCR analysis for examining
expression of nkx2.5 mRNA using total RNA from embryos at stage 20. Both
RNA quality and quantity was assessed by amplification of ornithine decarboxylase
(odc). Image published in: Nagao K et al. (2008) Copyright © 2008. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |