Fig 1: NPL1010 and NPL3008 rarely inhibit BMP1-dependent COL3A1 cleavage. (A) COL3A1 cleavage assay. rhCOL3A1 was incubated with rhBMP1. The effects of NPL1010 and NPL3008 on BMP1-dependent rhCOL3A1 cleavage were analyzed using SDS–PAGE and Oriole staining. Arrows indicate full-length rhCOL3A1 and cleaved rhCOL3A1. (B) Statistical analysis of BMP1-dependent cleavage of COL3A1. The levels of noncleaved and cleaved COL3A1 were measured by performing densitometry of the Oriole-stained gel, and the percentage of cleaved COL3A1 was calculated based on the fragments detected at each molecular weight and background. **: p < 0.01 (Dunnett’s test). The error bar indicates +s.d. with individual data shown (n = 4).
Fig 2: NPL1010 and NPL3008 inhibit BMP1-dependent Chrd cleavage. (A) Classification of ventralization and dorsalization phenotypes with representative phenotypes shown in the photographs. The phenotypes were classified into ventralized (V1−V4), normal, and dorsalized (C1−C5). Lateral view, anterior to the left. Scale bar, 1 mm. (B–D), Proportion of bmp2b (B), bmp1a (C), or tll1 (D) mRNA-injected embryos displaying the phenotype. DM: dorsomorphin. NPL1010 and NPL3008 partially rescue the bmp1a- or tll1-induced ventralization phenotype (n = 25–30). (E) Chrd cleavage assay. Chrd-Myc was incubated with rhBMP1. The effects of NPL1010 and NPL 3008 on BMP1-dependent Chrd cleavage were analyzed using Western blotting. The schematic illustration shows the cleavage patterns of Chrd. (F) Statistical analysis of the amount of cleaved Chrd detected in the Chrd cleavage assay. NPL1010 and NPL3008 dose-dependently inhibited BMP1-dependent Chrd cleavage. *: p < 0.05 and **: p < 0.01 (Dunnett’s test). Error bars indicate +s.d. with individual data shown (n = 5–6).
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