LCN7 Promotes Angiogenesis Figure 5. LCN7 is conserved between species. LCN7 protein sequences from human, rat, zebrafish, cow, and mouse were compared using PRALINE sequence alignment tool. The degree of conservation for each amino acid is indicated by color according to the key and in the ‘‘conservation’’ line of the alignment where * indicates 100% identity. Prosite analysis predicted the location of somatoamedin B (AA 74 to 95), lipocalin (AA 272 to 285), and cathepsin B (AA 402 to 452) and (AA 424 to 443) domains that are outlined with dashed lines. Overall, the alignment showed 79% sequence identity over the entire length of the alignment. doi:10.1371/journal.pone.0013905.g005 Figure 6. Morpholino knockdown in zebrafish embryos. A. LCN7 expression was detected in developing zebrafish embryos by RT-PCR analysis. cDNAs pools were generated from embryos ranging from 2 to 48 hours and subjected to RT-PCR analysis with either LCN7 or GAPDH specific oligonucleotides. B. Freshly fertilized zebrafish embryos between 1 to 8 cells were injected with morpholino vehicle (water +.1% phenol red) or equal volumes containing either 1 ng of LCN7 morpholino, or 1 ng of mismatched LCN7 control morpholino. Microangiogram analysis was performed approximately 28 hours later by detecting endothelial-specific expression of GFP. The accompanying graph depicts average ISV pixel length (converted to mM) as measured along the entire dorsal surface in each of five embryos in each experiment. Results shown are the average +/2 SE of five independent experiments. Bar equals 100 mM. C. Freshly fertilized zebrafish embryos were injected with morpholino vehicle (water +.1% phenol red) or equal volumes containing 1 ng of LCN7 morpholinos or 1 ng of LCN7+1.5 ng of p53 morpholinos. ISV vessel length was measured as described above and results are presented as the average +/2 SE of five independent experiments consisting of at least five individual fish per injection. For all panels, * indicates p,.05, students t-test. Bar equals 100 mM. doi:10.1371/journal.pone.0013905.g006 PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e13905