ALK2 is necessary for EndMT. (a) Immunoblotting confirming knockdown of ALK2 expression by ALK2 siRNA in HUVEC and HCMEC cultures. (b) Immunoblotting showing increased expression of FSP-1 and STRO-1 in TGF-β2 or BMP4 treated endothelial cells transfected with negative control siRNA, but inhibition of this expression in cells transfected with ALK2 siRNA. β-actin was used as an internal control. (c) Flow cytometry analysis confirming increased numbers of endothelial cells expressing FSP-1 when treated with TGF-β2 or BMP4, and inhibition of such expression in cells treated with ALK2 siRNA. (d) Positive staining of osteoblast (alkaline phosphatase [AP] and alizarin red [AR]), chondrocyte (alcian blue [AB]), or adipocyte (oil red O [OR]) products in cultures transfected with negative control siRNA and treated with TGF-β2 or BMP4 for 48 h, followed by growth in osteogenic, chondrogenic, or adipogenic culture medium. In contrast, expression of ALK2 siRNA prevented the differentiation of these cells. Scale bar, 100 μm.