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Fig. 4
Effects of NARFL-knockdown in HEK293T cells and NARFL-knockout in zebrafish. A. NARFL was highly expressed in HEK293T cells detected using western blot. B, C, D. shRNA-A and shRNA-B of five shRNA duplexes (B) induced declined NARFL mRNA level (C) and reduced aconitase activity (D) in HEK293T cells. E. Targeted indel mutations induced by CRISPR/Cas9 at the NARFL gene. The wild-type sequence was shown at the top with the target sites highlighted as blue and the mutant sequence highlighted as red. F. Control zebrafish (94/126) showed dominant subintestinal vessels in 3 dpfs using blood vessel staining. In NARFL knockout zebrafish (87/109), the subintestinal plexus has ectopic sprouts (p< 0.0001, n= 22/109 versus 94/126). G. VEGF mRNA levels were examined using qRT-PCR assay and VEGF mRNA levels were upregulated in NARFL-knockout zebrafish. H. The mRNA levels of SOD and CAT were declined. (*, p< 0.05; **, p < 0.01)