Fig. 4

BMP2, BMP4 and BMP10 activated SMAD1/5/8 via ALK2 and BMPR2 when combined with FK506. A INA-6 BRE-luc cells, all in the presence of FK506 (100 nM), were treated for 18 h with BMP4 (100 ng/mL), BMP10 (100 ng/mL), with or without ALK2 kinase inhibitor K02288 (100 nM) or ALK4/5/7 kinase inhibitor SB431542 (2 µM). Luciferase substrate was added, and relative luciferase units (RLU) were measured. B-D INA-6 cells were transfected with the indicated siRNAs and treated for 48 h with BMP2 (100 ng/mL, only C and D), BMP4 (100 ng/mL, only C and D), BMP10 (100 ng/mL), or BMP6 (7.5 ng/mL), in the presence of FK506 (100 nM). The relative cell viabilities were measured with CellTiter Glo. E INA-6 control cells and ALK2 knock out cells were treated for 72 h with BMP2 (200 ng/mL), BMP4 (100 ng/mL), BMP10 (100 ng/mL), or BMP6 (7.5 ng/mL), in the presence of FK506 (100 nM). F INA-6 control cells and ALK2 knock out cells were treated with BMP10 (100 ng/mL), with or without FK506 (100 nM), for 2 h and subjected to western blotting. The blot is representative of n = 3 independent experiments (upper part). The bands from all independent experiments were quantified to determine the expression of phospho-SMAD1/5 and were normalized to GAPDH levels (lower part). G INA-6 cells stably transduced with BMPR2 or CTR shRNAs were treated for 48 h with BMP2 (100 ng/mL), BMP4 (100 ng/mL), BMP10 (100 ng/mL), or BMP6 (4 ng/mL), in the presence of FK506 (100 nM). The relative cell viability was measured with CellTiter Glo. 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 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)