Fig. 2

5-bp spaced pGC-SBE homocomposite motifs encode BMP responsiveness: a A library of synthetic firefly luciferase construct was cloned with 1 to 6 SMAD motifs positioned 10 bp before a minimal promotor (MLP) with varying spacer length for pGC-SBEs and varying orientation for npGC-SBE and SBE motifs. b SBE-firefly-Luc constructs were co-transfected with TK-renilla luciferase in HEK293t cells; cells were starved and stimulated with BMP6 (5 nM) for 24 h before analysis using a microplate reader (c–h). c–e Dual luciferase reporter assay displaying BMP6 responsiveness towards constructs with differently spaced pGC-SBE homocomposite motifs. BMP6 responsiveness is observed, when at least two pGC-SBEs are separated by a 5-bp spacer. f Dual luciferase reporter assay displaying BMP6 responsiveness towards constructs with different 5-bp spaced homocomposite motifs taking all potential orientations of non-palindromic motifs into account. 5-bp spaced npGC-SBE, pSBE, and SBE homocomposite motifs show no BMP6 responsiveness compared to pGC-SBE homocomposite motifs. c, g–h Dual luciferase reporter assay displaying BMP6 responsiveness towards constructs with one to six 5-bp spaced pGC-SBE homocomposite motifs (5 nM (g) or 0.04 to 10 nM BMP6 (h)). c–h Data are shown as mean fold induction to unstimulated cells (gray line) in relative luciferase units (RLU) ± SD (c–g n = 3–15; h n = 3 independent experiments). Statistical significance was calculated in between samples using one-way ANOVA and Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001