Figure 1.
Real-time PCR analysis of SOX18 target genes.
RNA was isolated from non-transduced (mock), control virus (AdvEGFP) and SOX18 adenovirus (AdvSOX18) transduced HUVEC and analyzed for the expression of matrix metalloproteinase 7 (MMP7), ephrinB2 (EFNB2), EPHA7, semaphorin 3G (SEMA3G), interleukin 7 receptor (IL-7R), and cannabinoid receptor 1 (CNR1). Values were normalized for ß2 microglobulin expression, and are expressed as fold induction compared to control cells.
Table 1.
Top SOX18 regulated genes.
Figure 2.
Knock-down of SOX18 diminishes the expression of target genes.
HUVEC were transfected with siRNAs directed against SOX17 or SOX18, or with a scrambled control (Scr), and expression of target genes analyzed by real-time PCR. Values were normalized for ß2 microglobulin levels, and are expressed as fold induction compared to control cells.
Figure 3.
A, Schematic representation of the two MMP7 promoter-luciferase reporter constructs containing either only the proximal or both potential SOX18 binding sites (black diamonds). B, the wild type (wt) constructs were cotransfected together with increasing amounts of SOX18, SOX7 or SOX17 expression vectors into HEK293 cells as indicated. Luciferase values were normalized for ß-Gal expression. C, in the shorter construct, the single potential SOX18 binding site was mutated. D, The short MMP7 promoter construct containing the mutated SOX18 binding site was analyzed as in (B).
Figure 4.
SOX18 binds to the proximal site in the MMP7 promoter in vitro and in vivo.
A, Electrophoretic mobility shift assay (EMSA). Binding of SOX18 to its proximal site in the MMP7 promoter was analyzed using nuclear extracts from HUVEC. Specificity of binding was demonstrated by competition experiments using the same (wt), a mutated (mut) or an unrelated (unrel) oligonucleotide as indicated, the specific complex is indicated by an arrow. B, The same site was analyzed by chromatin immunoprecipitation in HUVEC. Lane 1–3: different α-Sox18 antibodies (SantaCruz, Thermo, Chemicon, respectively) as described in Materials and Methods, Ig: IgG control antibody; inp: input, M: 100 bp marker. The 120 bp PCR fragments obtained after amplification ofDNA precipitated by the anti-SOX18 antibodies and input DNA are marked by an arrow.
Figure 5.
SOX18 and MMP7 are co-expressed in arteries and veins, but not in lymphatic vessels.
Serial sections from human skin were analyzed by immunohistochemical staining with the indicated antibodies. Endothelial cells were revealed by von Willebrand factor (vWF) staining, lymphatic vessels by LYVE-1.