Tbx1 activates prolymphangiogenic genes in ECs. (A) Fold change histogram representation of qRT-PCR results of RNA from HUVECs transfected with increasing amounts of TBX1 expression vector. (B–E) Anti-IGF1 (green) and anti-VEGFD (red) staining of TBX1-transfected HUVECs (D and E) and control cultures (B and C). Arrows indicate IGF1⁺ and VEGFD⁺ cells. Mock transfections (B, C, and F [white columns]) were performed by transfecting HUVECs with empty vector. TOPRO3 identifies cell nuclei. (F) More IGF1⁺ and VEGFD⁺ cells were present in TBX1-transfected cultures compared with control cultures. 500 cells were counted in three independent experiments for each antibody. (G) Cell proliferation in TBX1-transfected HUVECs increased >1.8-fold above that of control cultures. (H) Western blotting reveals increased TBX1 protein expression after TBX1 transfection of HUVECs. (I and J) TBX1 and VEGFR3 mRNA levels in TBX1-siRNA– treated or control siRNA–treated HUVECs (I) and HMLECs (J) were quantified by qRT-PCR and normalized to GAPDH. Control expression levels were set to 1. In both cell types, TBX1 and VEGFR3 mRNA levels were greatly reduced after TBX1 knockdown. Values are mean ± SEM. (K) Western blotting with antibodies to TBX1 and β-actin showed that TBX1 protein expression was reduced in HMLECs treated with TBX1-siRNA. Bars, 100 µm.