Figure 3

Activin A competes with BMP-6 and BMP-9 in binding to the type II receptors ACVR2A and ACVR2B. (A) Expression of the type II receptors ACVR2A and ACVR2B in INA-6 and IH-1 cells were determined using QRT-PCR. The delta delta Ct method using GAPDH as housekeeping gene was used to determine the relative levels of mRNA compared to the expression of ACVR2A in INA-6 (Ct-value = 33) which was set to 1. (B) INA-6 cells were treated with BMP-6 (50 ng/mL) or BMP-9 (0.25 ng/mL) for three days in the presence of the soluble Fc-receptors ACVR1/ALK2, BMPR2, ACVR2A or ACVR2B (5 μg/mL) and cell growth was determined using the CellTiter Glo assay. Relative luciferase units (RLU) reflected the amount of ATP in each well, represented as mean ± SEM from three independent experiments. P-values were determined by T-test (*P < 0.05, **P < 0.01). (C) INA-6 cells were treated with BMP-6 (25 ng/mL) with or without activin A (10 ng/mL) and soluble Fc-receptors (5 μg/mL) where indicated and cell growth was determined as in (B). (D) Soluble Fc-receptor ACVR2A and ACVR2B (1 μg/mL) was coated in wells and treated with BMP-9 (15 ng/mL) and different concentrations of activin A (1.5-50 ng/mL). Bound BMP-9 was expressed as absorbance (OD) using BMP-9 DuoSet detection reagents. Shown are mean ± SEM values from three independent experiments. P-values were determined by T-test (*P < 0.05, **P < 0.01). (E) Soluble receptors were coated into plates as in (D) and treated with BMP-9 (15 ng/mL), activin A (15 ng/mL) and different concentrations of follistatin (300–1000 ng/mL). Binding of BMP-9 was determined as in (D).