Synectin regulates Vegfc-driven lymphatic sprouting and Sox18-mediated lymphatic reprogramming in vitro. (A-D) CD31 immunostaining of untreated (A-B) or VEGFC-stimulated (C-D; 200 ng/mL) LEC spheroids in fibrin gels, revealing reduced VEGFC sprouting response by LECs transfected with synectin-specific (B,D; synectin^KD) compared with control siRNA (A,C). Scale bar represents 100 μm. (E) Quantification of the sprout number per LEC spheroid, revealing that synectin knockdown impaired lymphatic sprouting in response to VEGFC (N = 17 for control; N = 26 for synectin^KD), while baseline sprouting of control (N = 17) and synectin^KD LECs (N = 35) was comparable. *P < .001 by univariate analysis. (F) RT-PCR of control and synectin^KD H5V blood vascular endothelial cells (BECs), transfected with Sox18-virus (BECs^Sox18) or GFP-virus (BECs^GFP), revealing that knockdown of synectin impaired up-regulation of Prox-1 in BECs^Sox18 without affecting Nrp1 expression. Results are represented as fold change vs control BECs^GFP. Dashed line indicates baseline expression levels in BECs^GFP. N = 3; *P < .01 by Student t test. Error bars represent SEM.