Effects of BBP on angiogenesis
in vivo. Effect of Huh7 cell-conditioned medium on HUVEC tube formation. Huh7 cells were pretreated with or without Pd98059 (A) or transfected with scrambled siRNA or AhR siRNA (B) followed by BBP treatment, and the conditioned medium was then collected from each culture dish. HUVEC were seeded in BioCoated angiogenesis plates, treated with 20% Huh7-conditioned medium, and incubated for 16 hours. HUVEC were imaged after staining with Calcein-AM. Total tube lengths were analyzed by MetaMorph software. Scale bars: 200 μm. VEGF levels in the conditioned media were measured by ELISA. (C) Huh7 cells were treated with BBP for the indicated times. p-ERK and ERK levels were measured by immunoblotting. β-actin used as an internal control. (D) Huh7 cells were transfected with two different Huh7 shRNAs and then treated with BBP (1 μM) for 15 minutes. p-ERK, ERK levels were evaluated by immunoblotting. (E) Huh7 cells were mixed with Matrigel with or without BBP (1 μM), injected into the flanks of nude mice, and allowed to grow for 21 days. Hemoglobin levels in Matrigel plugs, which indicated blood vessel formation, were measured by Drabkin reagent kit 525. Each value is the mean ± SD of three independent experiments. The asterisks indicate a significant difference between control and test groups, as analyzed by Student’s t-test (*p < 0.05; **p < 0.01).