BAZF regulates EC network formation in culture. (A) Expression pattern of BAZF mRNA after VEGF-A (50 ng/mL) stimulation in HUVECs. Quantitative RT-PCR of BAZF mRNA was performed using a TaqMan-gene expression system. The results represent mean values from 3 independent experiments. Error bars represent ± SD. (B) Analysis of BAZF protein level in HUVECs after VEGF-A stimulation by Western blotting using an anti-BAZF antibody. Relative intensity of each band was measured by Lane Analyzer 3.0 software (ATTO) and normalized relative to β-actin (loading control). (C) Angiogenic sprouting from AdBAZF-infected or AdGFP-infected HUVECs. Arrows show sprouting cells. Left panels represent microscopic images, and right panel shows total number of sprouting cells in 50 beads. Scale bar: 100 μm (***P < .001). (D) Protein level of BAZF in HUVEC transfected with BAZF or control siRNA with or without VEGF-A stimulation. BAZF protein was detected by Western blotting using an anti-BAZF antibody. (E) BAZF knockdown disrupts VEGF-A–induced network formation of HUVECs on the Matrigel. The cells were stained with Calcein-AM and the images were acquired. Scale bar: 500 μm. (F) Quantification of the network formation of HUVEC transfected with BAZF or control siRNA. The total length of the network per field (mm²) was measured by ImagePro Plus software 4.5. (Roper). The results represent mean values from 3 experiments. Error bars represent ± SD (***P < .001). (G) Time-lapse imaging of the network formation in HUVEC treated with BAZF or control siRNA. Scale bar: 10 μm.