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Fig. 3 Sulfuretin treatment increases body length and Nrf2 target gene expression in zebrafish. (A) Zebrafish at 1 dpf were treated with sulfuretin (50 or 100 μM) for 4 days. Body lengths were then measured at 5 dpf. (B) Zebrafish at 1 dpf were treated with sulfuretin for 6 days and body lengths were measured at 7 dpf. (C) Total RNA was isolated from zebrafish after treatment with sulfuretin (100 μM) for 4 days and relative expression levels of mmp13a, col10a1a, col10a1b, ctgfa, and ctgfb were determined by real-time PCR. (D, E) Sulfuretin (100 μM) treatments increases the length of the ceratohyal (arrow) but not the parasphenoid (*) in zebrafish. (D) Whole mount in situ hybridization of col10a1 and ctgf. (E) Quantification of WISH staining in center of ceratohyal (col10a1), parasphenoid area (col10a1), and total length of ceratohyal (ctgf). (F) Zebrafish at 1 dpf were treated with sulfuretin for 4 days and expression levels of gclm, nqo1, gclc, and maff were measured by real-time PCR. Data are presented as mean ± SEM (n = 10 per group). Statistical significance was determined by Student’s t-test (ns, not significant; *P < 0.05; **P < 0.01).