Figure 7.

Bmal1 promotes angiogenesis by transcriptionally regulating the vascular endothelial growth factor (VEGF) expression. (A) Relative mRNA expression of genes involved in angiogenesis in human umbilical vein endothelial cells (HUVECs) which had been transfected with adenoviruses coding for AdGFP or AdBmal1. Data are presented as mean ± SEM (unpaired t-test). ^**p < 0.01 Bmal1 vs. GFP; ^*P < 0.05 Bmal1 vs. GFP. (B,C) Relative mRNA and protein expression of Bmal1 and VEGF in HUVECs which had been transfected with Bmal1 siRNA or si-NC. Data are presented as mean ± SEM (unpaired t-test). ^**p < 0.01 si-Bmal1 vs. si-NC; ^*p < 0.05 si-Bmal1 vs. si-NC. (D) Luciferase reporter constructs were created containing the truncated (−2,000, −1,216, −971, −785, −736, −540, and −43) versions of the VEGF promoter. The luciferase reporter constructs were co-transfected with Bmal1 overexpression vector or with the control vector into HEK293T cells, and luciferase activity was evaluated 24 h later. Data are presented as mean ± SEM (unpaired t-test). ^**p < 0.01 and ^*p < 0.05 Bmal1 vs. PCMV. (E) ChIP assay conducted in HUVECs with anti-BMAL1 or IgG antibody. A qRT-PCR analysis was performed with primer sequences around Bmal1-binding E-box elements in the VEGF promoter. Data are presented as mean ± SEM (unpaired t-test). ^*p < 0.05 BMAL1 vs. IgG. (F) Relative mRNA expression of Bmal1 and VEGF in HUVECs which had been transfected with/without VEGF siRNA and Bmal1/GFP. Data are presented as mean ± SEM (one-way ANOVA with post-hoc Tukey test). ^**p < 0.01 si-NC Bmal1 vs. si-NC GFP; ^##p < 0.01 si-VEGF Bmal1 vs. si-VEGF GFP; ^$$p < 0.01 si-VEGF GFP vs. si-NC GFP. (G) Endothelial tube formation in matrigel in HUVECs. Data are presented as mean ± SEM (one-way ANOVA with post-hoc Tukey test). ^**p < 0.01 si-NC Bmal1 vs. si-NC GFP; ^#p < 0.05 si-VEGF Bmal1 vs. si-VEGF GFP; ^$$p < 0.01 si-VEGF Bmal1 vs. si-NC Bmal1. Each expreiment was repeated 3 independent times.