Fig. 8. Modifying BMP9 signalling specificity by mutagenesis.

A panel of BMP9 mutants were generated, and tested in vitro and in vivo as described in the Methods. a Mutant proteins were subject to endothelial cell signalling assays (at 0.3 ng ml⁻¹ GF-domain concentration) using induction of ID1 gene expression in hPAECs as a readout, and osteogenic signalling assays (at 10 ng ml⁻¹ GF-domain concentration) using ALP induction in C2C12 cells as a readout. Data were normalised to WT BMP9 and shown as fold change relative to WT upon mutation. Each treatment condition was repeated in 3–7 independent experiments alongside untreated and WT controls. The exact N number for each condition is given under the column. Means ± SEM are shown. b Recombinant WT pro-BMP9, pro-BMP9 D366E, pro-BMP10, as well as BMP2 GF-domain were subject to in vivo heterotopic bone-forming assays in the presence and absence of cardiotoxin. Each data point represents the HO result from an independent injection in one leg. N number for each treatment condition is given under each column. Data are presented as % ossification volume relative to the average of BMP2-treated controls. Means ± SEM are shown. c Representative CT images (left) and histological staining (right) of in vivo formed heterotopic bones after stimulation of indicated BMP molecules in the presence and absence of cardiotoxin. B: osteoid matrix; M: muscle cells. Scale bar = 500 µm.