FIGURE

Fig. 5

ID
ZDB-FIG-080908-21
Publication
Fish et al., 2008 - miR-126 regulates angiogenic signaling and vascular integrity
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Fig. 5

miR-126 Positively Regulates VEGF Signaling in Endothelial Cells by Repressing SPRED1 and PIK3R2

(A) Immunoblot of lysates from HUVECs transfected with control or miR-126 MOs in the presence or absence of VEGF (10 ng/ml, 10 min). VEGF induced phosphorylation of ERK (p-ERK) and AKT (p-AKT), which was blocked by miR-126 inhibition. Total levels of ERK and AKT were not affected. Phosphorylation of SRC was not affected by miR-126 knockdown. Densitometric analysis of normalized protein levels are indicated above immunoblot.

(B) PIK3R2 mRNA was knocked-down by RNAi in HUVECs transfected with control or miR-126 MOs (qRT-PCR). Western analysis indicates a decrease in PIK3R2 protein by introduction of siRNA, even in the presence of miR-126 MOs. Knockdown of PIK3R2 rescued the defect in VEGF-dependent phosphorylation of AKT in miR-126 MO-treated cells.

(C) Western analysis shows reduced SPRED1 levels by transfection of a MO that blocks SPRED1 translation, even in the presence of miR-126 MO. SPRED1 MO rescued the defect in VEGF-induced phorphorylation of ERK in miR-126 MO-transfected cells.

(D) Quantification of percent (%) wound closure of endothelial cells in a “scratch” assay reveals rescue of miR-126 MO effects by knocking down SPRED1. * p < 0.05 compared to control MO.

(E) Treatment of 48 hpf embryos with a VEGF receptor inhibitor (Vatalanib, 5 μM, 18 hr) resulted in reduced circulation of blood cells (gata1:dsRed). ISVs (flk1:GFP) appeared to lack a lumen after VEGF inhibition.

(F) Hemorrhages also occurred in some embryos treated with VEGF receptor inhibitor (arrow).

Expression Data
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Fish:
Condition:
Anatomical Terms:
Stage: Long-pec

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Phenotype Data
Fish:
Condition:
Observed In:
Stage: Long-pec

Phenotype Detail
Acknowledgments
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Reprinted from Developmental Cell, 15(2), Fish, J.E., Santoro, M.M., Morton, S.U., Yu, S., Yeh, R.F., Wythe, J.D., Ivey, K.N., Bruneau, B.G., Stainier, D.Y., and Srivastava, D., miR-126 regulates angiogenic signaling and vascular integrity, 272-284, Copyright (2008) with permission from Elsevier. Full text @ Dev. Cell