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Fig. 5 Overexpression of zebrafish hjv fails to increase hepcidin expression in zebrafish embryos, but can cooperate with BMP6 to activate the human hepcidin promoter in vitro.
A–D. Whole mount in situ hybridization for hepcidin (A,B) or foxa3 (C,D) as a marker for the liver (arrowhead) and intestine (arrow) at 55 hpf following injection of zebrafish hjv cRNA at the one cell stage. N = 20–30 embryos per group. E,F. Quantitative real-time RT-PCR at 72 hpf demonstrated no significant change in hepcidin expression relative to β-actin (E) or to LFABP (F) following overexpression of hjv cRNA. N = 2 pools per group. G,H. In vitro luciferase reporter assays in Hep3B cells demonstrate the effect of increasing doses of zebrafish hjv cRNA on the human hepcidin promoter (G) or the BMP response element (H) in the absence (black) or presence (white) of exogenous BMP6 (5 ng/ml). Relative light units were calculated as ratios of Firefly (reporter) and Renilla (transfection control) values. Results from luciferase assay experiments were expressed as the means ± standard error of triplicates from representative experiments. * denotes p<0.05, compared to the previous column.