LCN7 Promotes Angiogenesis Figure 3. LCN7 sensitizes endothelial cells to TGF-b. A. Control and LCN7-overexpressing MB114 endothelial cells were serum starved then stimulated with 5 ng/ml of TGF-b1 for 0, 20, 40, or 80 minutes. Whole cell lysates were fractionated in SDS-PAGE and western blotted with antibodies to detect phosphorylated ERK1/2 MAPK (P-ERK1/2) or b-actin as a loading control. Western blot results from three independent experiments were quantified by densitometry, normalized to b-actin to control for loading differences, and are presented as the fold increase +/2 SE of unstimulated control cells. B. Control and LCN7-overexpressing MB114 endothelial cells were stimulated as in A. Whole cell lysates were fractionated and western blotted with antibodies against phosphorylated SMAD-2 (P-SMAD2), phosphorylated SMAD5 (P-SMAD5), or b-actin. Western blot results from three independent experiments were quantified by densitometry, normalized to b-actin to control for loading differences, and are presented as the fold increase +/2 SE of unstimulated control cells. C. Control or LCN7 overexpressing MB114 endothelial cells were induced to invade through matrigel coated transwells in the presence of 5 ng/ml TGF-b (TGF) or 10 mM TGF-b antagonist SB431542 (SB) and compared to unstimulated control (C) cells. Invading cells were stained with crystal violet and quantified by densitometry of stained invasion membranes. The experiment was performed three times and the resulting data is presented as the fold invasion of control cells +/2 SE. D. Quiescent cultures of control or LCN7 overexpressing MB114 endothelial cells were overlayed with rat-tail collagen containing 5 ng/ml TGF-b (TGF) or 10 uM TGF-b antagonist SB431542 (SB) and compared to unstimulated control (C) cells. Data was collected by manually counting the number of sprouting cells in 10 high power fields and is presented as the average fold increase +/2 SE of three independent experiments. * indicates p,.05, student’s t-test. doi:10.1371/journal.pone.0013905.g003 compared to aortic rings in PBS supplemented collagen. Collec- tively, these data confirmed that LCN7 promotes angiogenic cell behaviors in a variety of cell and culture systems. Based on these in vitro results, we set out to determine if LCN7 performs similar pro- angiogenic functions in vivo. Knockdown of LCN7 in zebrafish causes vascular defects Zebrafish models of angiogenesis have become important tools to examine the function of angiogenic genes in in vivo settings. In particular, zebrafish strains expressing GFP from the endothelial specific Fli1 promoter facilitate microangiogram analysis of even the earliest vascular beds in zebrafish embryos [19,20]. Moreover, the ability to use anti-sense morpholinos (MO) to temporarily knockdown gene expression allows for the rapid analysis of gene function in the absence of targeted genes [21]. In order to determine if LCN7 is important for angiogenesis in developing zebrafish, we first performed a blast search of the full length murine LCN7 protein sequence against the zebrafish PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e13905