Fig. S2

Miconazole suppresses brain hemorrhages in fn40a mutants. (A-D) Live image of embryos without PTU treatment. The homozygous fn40a mutant showed evident brain hemorrhage in the midbrain and hindbrain (C, arrow) compared with heterozygous mutant in the absence (A) or presence (B) of miconazole, and the hemorrhage was repressed in homozygous mutants by miconazole treatment (D). Embryos at 2 dpf were orientated in the lateral view. (E-H) O-dianisidine staining of embryos without PTU treatment revealed that erythrocytes accumulated in the brain region of homozygous fn40a mutant (G, arrow) compared with heterozygous mutant in the absence (E) or presence (F) of miconazole, and this hemorrhage phenotype in homozygous mutants was rescued by miconazole treatment (H). Embryos at 2 dpf were orientated in the dorsal view. The numbers on lower right show the phenotypical ones out of total scored embryos. (I-L, I'-L') Live images of Tg(flk1:eGFP; gata1:DsRed) double transgenic embryos of heterozygote siblings (I-J, I'-J') and homozygous fn40a mutants (K-L, K'-L') at 2 dpf in the hindbrain region. Note that gata1+ erythrocytes leaked out from Tg(flk1:eGFP)-labeled vessels and formed hematomas in the mutants (K, K') while erythrocytes remained within vessels in heterozygous siblings (I-J, I'-J') and mic-treated homozygous mutants (L, L'). (M) Intensity of the gata1:DsRed signals in the hindbrain region shown in panels A-D, noting a burst of gata1:DsRed signals in homozygous fn40a mutants compared with heterozygous mutants, which was reversed in homozygous mutants after mic treatment. *p <0.05 by one-way ANOVA with Bonferroni's Multiple Comparison Test, n>10. (N) Miconazole suppresses brain hemorrhages in fn40a mutants in a dose-dependent manner. Note that miconazole at 10 µmol/L completely suppressed brain hemorrhage in fn40a mutants. *p <0.05 by one-way ANOVA with Dunnett's Multiple Comparison Test, compared with control, mean±SEM., n >50 for each independent repeat. (O) Application of miconazole from 6 to 24 hpf had more effective suppression on brain hemorrhage in fn40a mutants. At the concentration of 3.3 μmol/L, miconazole effectively suppressed brain hemorrhage (more than 50%) when its treatment from 6 to 24 hpf, but had little effect when its treatment either from 24 to 36 hpf, or 24 to 48 hpf, and had no effect when having treatment from 36 to 48 hpf. Similarly, at the concentration of 5 μmol/L, miconazole inhibited brain hemorrhage of fn40a mutants (better than that at 3.3 μmol/L) when having treatment from 6 to 24 hpf, and had less inhibition when having treatment either from 24 to 36 hpf or from 24 to 48 hpf, but had no effect when having treatment from 36 to 48 hpf. *p <0.05 by two-way ANOVA with Bonferroni's post test compared with control, mean±SEM, n >100.