Figure 3. GremlinC141A retains its BMP-antagonist activity.

A. HepG2 cells were transiently transfected with a construct harbouring BMP-responsive hepcidin promoter upstream to the luciferase reporter gene. Serum-starved transfected cells were then stimulated with 50 ng/mL of BMP4 in the absence or the presence of increasing amounts of either gremlin^WT (•) or gremlinC141A (▼). After 16 hours incubation, cells were lysed, and luciferase activity was measured. Data are expressed as percent of luciferase activity measured in BMP4 stimulated cells and are the mean ± SEM of 3 independent experiments. Two-Way ANOVA followed by Bonferroni's test revealed that the dose-response curves of the two proteins were not statistically different. B. 96-well plates coated with 250 ng/mL of BMP4 were incubated with increasing concentrations of gremlin^WT or gremlin^C141A. Then, the capacity of gremlin^WT (•) or gremlin^C141A (▼) to bind to immobilized BMP4 was assessed by ELISA assay using an anti-gremlin antibody. C-D. zebrafish embryos were injected (at 1-4 cell stage) with the indicated doses of either gremlin^WT or gremlin^C141A mRNA. Representative dorsalized embryo phenotypes at 24 hours after injection (C). All embryos were viewed laterally. D, percentage of embryos in different categories at 24 hours after injection. Data are the mean ± SEM of 3 independent experiments. *, p<0.05; **, p<0.01, One-Way ANOVA followed by Bonferroni's test versus control.