VEGFD modulates SOXF activity in vitro. (A) Luciferase assay to evaluate the effect of VEGFD on the induction of VCAM1, a SOX18 direct gene target. Transfection of lymphatic endothelial cells (LECs) with VC1889 vector in combination with VEGFD treatment leads to induction of luciferase reporter expression, whereas co-transfection of LECs with VC1889 and RaOp (a pSG5 constructs driving the expression of a dominant negative Sox18 mutant) leads to suppression of luciferase activity. (B-D) Expression levels of SOX18 target genes (Prox1, Mmp7, Tie1) (qRT-PCR) in LECs were upregulated by VEGFD treatment (200 ng/mL), and induction was suppressed by transfection with RaOp expression vector. (E) Western blot using nuclear and cytoplasmic extracts from LECs. SOX18 protein levels were elevated in the nucleus after 1 hour of VEGFC or VEGFD stimulation. (F) qRT-PCR and (G) luciferase assay using Sox18 promoter cloned into pGL2B luciferase reporter construct showed that Sox18 transcription was not altered after treatment of LECs with VEGFs. (H) Immunofluorescence staining on LECs for endogenous SOX18 (green), PECAM (red), and DAPI (blue) indicated that VEGFs triggers nuclear condensation of SOX18 (asterisk). Insets show magnifications of area in dashed lines. (I) Immunofluorescence staining on human umbilical vein endothelial cells (HUVECs) for endogenous SOX18 (red), Phalloidin (green), and DAPI (blue). VEGFD treatment triggers nuclear condensation of SOX18 (asterisk). Scale bar represents 50 μm. (J) Intensity scan profile showing intensity values for SOX18 (red) and DAPI (blue), measured across a cell nucleus. The position of the scan line is shown (I, dashed line). (K) Percentage of cells with increased SOX18 in nucleus (approximately 100 cells counted per field of view over at least 3 different fields of view). Cyto., cytoplasm; Nuc., nucleus. The data are shown as the mean ± standard error of the mean of 3 independent experiments. **P < .01, *P < .05 by 1-way ANOVA test.