Fig. 3

FKBP12 regulated ALK2 ligand specificity. A INA-6 cells were treated with BMP6 (20 ng/mL) or BMP9 (0.1 ng/mL) in the presence of BMP10 (125 ng/mL) for 72 h. The relative cell viabilities were measured with CellTiter Glo. B INA-6 BRE-luc cells treated with BMP2 (200 ng/mL), BMP4 (100 ng/mL), BMP10 (100 ng/mL) and BMP6 (7.5 ng/mL), with or without FK506 (100 nM) for 18 h. The results are shown as relative luciferase units (RLU) and each bar represents mean ± s.e.m. of n = 3 independent experiments. Two-way ANOVA and Sidak’s multiple comparisons test was used to analyze statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns—nonsignificant). C To assess downstream effect on apoptosis, INA-6 cells were treated with increasing doses of BMP4 with or without FK506 (100 nM) for 48 h before Caspase-Glo 3/7 Reagent was used to determine caspase 3/7 activity. Two-way ANOVA and Tukey’s multiple comparisons test was used to analyze statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns—nonsignificant). Then, INA-6 cells were treated with increasing concentrations of BMP2 (D), BMP4 (E), or BMP10 (F), with or without FK506 (100 nM) for 72 h. The relative cell viabilities were measured with CellTiter Glo. Two-way ANOVA and Tukey’s multiple comparisons test was used to analyze statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns—nonsignificant). Phosphorylated SMAD1/5/8 was measured using western blot on INA-6 (G) and KARPAS-417 cells (H) treated with BMP4 (5 ng/mL) and FK506 (100 nM) for 18 h. Phosphorylated SMAD1/5/8 was measured using western blot on KATO-III (I) and DOHH-2 (J) after 2 h stimulation with BMP4 (50 ng/mL) and FK506 (250 nM). All graphs are plotted relative to medium control and each bar represents mean ± s.e.m. of n = 3 independent experiments. Two-way ANOVA and Sidak’s multiple comparisons test was used to analyze statistical significans significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns—nonsignificant)