Fig. 2: BMP8B activates both SMAD2/3 and SMAD1/5/9 signalling pathways in BMP8B-KO HSC.

a–d, Primary murine BMP8B-KO HSCs (confluence 35,000 cells per cm²) activated in vitro (day 7) were treated, after 3-h FBS starvation, with or without recombinant human BMP8 protein (30–75 ng mL^–1) and/or ALK1/2/3/6 inhibitor (i) (K02288, 1 µM) or ALK4/5/7 inhibitor (i) (A-8301, 5 µM) to study phosphorylation (p) of SMAD2 (a,c) or SMAD1/5/9 (b,d) phosphorylation after 30-min incubation. TGFβ (5 ng mL^–1) or BMP7 (5 ng mL^–1) was used as positive control. Immunofluorescence (magnification, ×20) was used to study phosphorylated SMAD (green); αSMA (red) was used to stain the cytoplasm and Hoechst 33342 (blue) was used to stain the nucleus. The percentage of positive nuclei was assessed by Harmony High Content Imaging and Analysis Software. e, Gene expression (relative mRNA expression levels) of BMP–TGFβ targets measured by RTqPCR was assessed after 5 h of incubation with ligands and inhibitors. All the results are shown as mean ± s.e.m.; expression data of biological replicates are represented as dot plots. One-way analysis of variance (ANOVA) plus Fisher’s least significant difference multiple-comparison test was used to estimate the statistical significance among treatments (4 biological replicates per group; each biological replicate is a pool of 3 livers). Lowercase letters indicate post hoc analysis significance: a, reference group; groups with different letters are statistically different per post hoc comparison; differences between groups with the same letter are statistically not significant per post hoc comparison.