Figure 1 .

A single 2‐day pulse of Activin‐A reduced osteoblast mineralization. (a) Schematic overview of culture conditions. Human SV‐HFO cells were treated with Activin‐A from Day 5 to Day 7 of osteogenic differentiation. ALP activity, mineralization, gene expression, and miRNA profile were checked at the indicated time points. (b) ALP activity of Activin A‐treated and untreated osteoblasts at Day 10, 12, and 14 of culture. ALP activity was corrected for protein content at each time point. (c) Calcium deposition by Activin A‐treated and untreated osteoblasts at Day 10, 12, and 14 of culture. (d) Calcium deposition at Day 14 of culture, in cell extracts of untreated osteoblasts, osteoblasts treated with Activin‐A, SMAD inhibitor (SB‐505124), and Activin‐A and SMAD inhibitor. Bars indicate mean±SD. (**p<.01; ***p<.001). (e) Phosphorylation of SMAD3 followed by flow cytometry at the indicated time points, in Activin‐A treated and untreated osteoblasts. Values: mean±SD. (**p<.01; ***p<.001 relative to untreated cells). ALP, alkaline phosphatase; miRNA, microRNA; SD, standard deviation