DEV029736 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Generation of miR-126 and Egfl7 deletion alleles. (A) The miR-126 targeting construct, engineered to flank 289 bp of Egfl7 intron 7 containing miR-126 with loxP sites, was recombined into the endogenous locus to generate the floxed miR-126 allele (miR-126^flox) in ES cells. The miR-126 deletion allele (miR-126^Δ) was generated by crossing miR126^flox/+ and Hprt-Cre mice to achieve in vivo germline deletion of miR-126 by replacing the 289 bp intronic region containing miR-126 with a single loxP site. (B) The Egfl7 targeting construct with exons 5-7 of Egfl7 flanked by loxP sites was recombined into the endogenous Egfl7 locus, generating the Egfl7^flox allele. Egfl7^flox/+ and CMV-Cre mice were crossed to achieve in vivo germline deletion of exons 5-7, generating the Egfl7^Δ allele. This generated a null allele in which exons 5-7 of Egfl7 were replaced by a single loxP site as opposed to previously described Egfl7 mutant alleles in which a IRES-β-Gal PGK-neo cassette was knocked into exons 5-7, or in which a retroviral gene-trap vector was inserted into intron 2 of Egfl7 (Schmidt et al., 2007). Alignment of Egfl7 message with Egfl7 protein illustrates that the in vivo Cre-mediated deletion removes the C-terminal half of the EMI domain and both EGF repeats of the encoded protein.

• Supplemental Figure S2 -

  Fig. S2. Confirmed targeting of Egfl7 and miR-126. (A) Southern blot strategy to confirm proper targeting of Egfl7 and miR-126. Insertion of the neomycin (Neo) selection cassette into the NheI site in intron 7 of Egfl7 causes a 1.9 kb or 1.8 kb increase in an NdeI or an NheI fragment, respectively. Note that the change in the NheI fragment is slightly smaller than that of the NdeI fragment because cloning the Neo cassette into the NheI site in intron 7 of Egfl7 destroys the initial site but introduces a new NheI site within the Neo cassette. (B) Southern blots of wild-type, Egfl7^flox/+ or mir-126^flox/+ ES cell genomic DNA following NdeI (5′ strategy, left) or NheI (3′ strategy, right) digestion. Probed against regions 5′ of the targeting construct 5′ homology arm (red line in A) or 3′ of the 3′ homology arm (blue line in A), generating bands that were 7.2 kb (wild-type allele) and 9.1 kb (Egfl7 and miR-126 floxed alleles) for the 5′ probe, as well as 4.6 kb (wild-type allele), 6.4 kb (Egfl7^flox allele) and 6.2 kb (miR-126^flox allele) for the 3′ probe.

• Supplemental Figure S3 -

  Fig. S3. Regulation of Spred1 expression by miR-126. (A) Alignment of miR-126 with its binding site in the 3′UTR of Spred1. (B) Spred1 is a direct target of miR-126 as shown by miR-126 repression of luciferase activity of a Renilla reporter construct containing the 3′UTR of Spred1. This activity was specific to the miR-126 binding site as shown by the failure of miR-126 to repress luciferase activity of a Renilla reporter construct containing the 3′UTR of Spred1 with the miR-126 binding site mutated. (C) Western blot analysis of Spred1 expression. Spred1 is upregulated in HUVEC transfected with a miR-126 hairpin inhibitor, as compared with transfection with the control scrambled inhibitor.

• Supplemental Figure S4 -

  Fig. S4. Modulation of VEGF signaling by miR-126. A proposed scheme is depicted in which miR-126 represses VEGF signaling by downregulation of p85β and Spred1, such that miR-126 deletion leads to increased levels of these inhibitors and VEGF signaling antagonism.