(A) Sanger sequencing validation of SOX7 mutations. Sequencing chromatograms of four heterozygous missense variants. (B) Diagram of SOX7 protein with location of variants identified in this study. (C) ClustalX alignment of the amino acids of the SOX7 mutant proteins among different species. Stars indicate identical residues and dots indicate similar residues. (D) Analysis of mutant SOX7 protein expression. Con means transfection of pcDNA3.1 empty vector, WT means transfection of wild-type SOX7 plasmid, L14V, R153Q, P172R, G187S means transfection of different mutant SOX7 plasmids respectively. Use GAPDH as the internal control (left panel). All protein expression was quantitated with ImageJ software. Protein expression levels in mutant samples were normalized to WT protein (right panel). Data are shown as mean ± SEM, statistical significance was calculated by one-way ANOVA with Dunnett’s post hoc test, n=3 independent protein samples, **P<0.01 vs WT. (E) Effects of SOX7 mutations on transcriptional activation of downstream target gene VE-cadherin. Cotransfection of VE-cadherin promoter luciferase reporter with the WT and mutant SOX7 protein in 293T cells. The luciferase activities were reported as fold increase relative to the activity of the reporter in the presence of an empty expression plasmid (Con). Data are shown as the mean ± SEM, n=3 independent experiments. Statistical significance was calculated by one-way ANOVA with Dunnett’s post hoc test, *P<0.05 vs WT, ***P<0.001 vs WT. Two-tailed unpaired t test was used for statistical calculation, ^###P<0.001 WT vs. Con. (F) EMSA analysis with wild SOX7 and mutant SOX7 protein. SOX7 EMSA analysis using indicated VE-cadherin probes and SOX7 wild and mutant proteins. Bottom arrows indicated unbounded probe, and above arrows indicated shifted complex. The experiments were repeated at least three times independently.