Fig 3. Effect of netrin-4 on HUVECs tube formation.

Tube formation was assessed in tubulogenesis assay in vitro model, HUVECs were serum-starved with EBM2 medium (0.1% FBS, no growth factor) (Lonza) for 12 hours. HUVECs (1.5×10⁴) were then cultured in a 24-well plate (Gibco) coated with 150 mL Matrigel Basement Membrane Matrix GFR (BD Biosciences). Different concentrations of netrin-4 (100, 500, 1000, and 5000 ng/mL) or PBS (control) were added, and the results were quantified 6 h later using Image J. (B) Representative images out of three independent experiments are shown. * p < 0.05; ** p < 0.01. Each experiment was performed three times and representative pictures are shown. Data are expressed as mean ± SD.