Fig. 8.

Fig. 8.

Cercosporamide selectively inhibits caBMPR type I in cultured mammalian cells and zebrafish embryos. (A) HEK 293T cells were transfected with empty vector (pcDNA) or expression vectors for caALK1, caALK2, caALK3 or caALK6 each with a haemagglutinin (HA) epitope tag at the carboxy terminus. The cells were treated with vehicle (1% DMSO), LDN193189 (200 nM) or a range of cercosporamide concentrations (1-10 µM as indicated). Subsequently, the cells were lysed and the lysates run on SDS-PAGE gels. The material on the gel was transferred to blots and parallel blots were probed with antibodies specific for HA (epitope tag on receptors), phosphoSMAD1/5/8 (p-SMAD1), SMAD1 or GAPDH (loading control). Dashed lines indicate the borders of different gels. (B) Cercosporamide partially rescued caAlk2-induced developmental defects in zebrafish embryos in vivo. Bar chart shows the phenotype distribution of embryos injected with caAlk2 mRNA and subsequently treated with 1% DMSO, 100 nM or 200 nM cercosporamide from 2 hpf onwards. The severity of the phenotype (examples depicted in the insets) is plotted as red (severe; no head), orange (intermediate; head structures present, no eyes) and green (mild; head structure with eyes detectable). The total number of embryos (n) is indicated. (C) The phenotypes of the rescued embryos are highly variable; therefore, we depicted two representative individuals of ten embryos that were treated for each condition.