LCN7 Promotes Angiogenesis Figure 4. Recombinant LCN7 increases pro-angiogenic activities. A. Murine His-6 and FLAG-tagged LCN7 cDNA was expressed in E. coli and purified by sequential Ni+-agarose (left panel) and anti-FLAG M2 (right panel) affinity columns. Representative coomassie stained gels of the purification process are shown. LCN7 was purified to apparent homogeneity. B. Tissue culture wells were coated overnight with dilute Matrigel, 1 mg/ ml of recombinant LCN7 (rLCN7), or 10 mg/ml of BSA, then all wells were blocked with 10 mg/ml BSA. Adherent SVEC and MB114 endothelial cells were stained with crystal violet. Shown are representative images of an experiment that was performed three times. C. MB114 and SVEC cells were cultured on Matrigel coated trans-wells with 1 mg/ml of rLCN7 or an equivalent volume of 1XPBS. Invading cells were stained with crystal violet and quantified by densitometry of stained invasion membranes. Results are depicted as the fold increase +/2 SE compared to 1XPBS treated cells from a total of four independent experiments. D. Quiescent MB114 endothelial cells were overlayed with rat tail collagen containing 1 mg/ml of rLCN7 or an equivalent volume of PBS. After 48 hours, endothelial cell sprouting was quantified by manually counting the number of sprouting cells present in 10 high power microscopic fields. Results are depicted as the fold increase +/2 SE compared to PBS treated cells from a total of three independent experiments. E. Sections of aorta from a single mouse were embedded into rat tail collagen containing either 1 mg/ml of rLCN7 or an equivalent volume of 1XPBS. Shown are representative images of sprouting cells after 4 days of incubation from a single experiment that was performed three times. doi:10.1371/journal.pone.0013905.g004 PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e13905