Figure 8.

miR‐30b‐c knockdown increases expression of Runx2 target proteins and calcification of human coronary artery smooth muscle cells. Human CASMC were transfected with an antagomir to miR‐30b (anti‐miR‐30b), to miR‐30c (anti‐miR‐30c), to both, or with a scrambled miRNA sequence (SS). After 14 days (A) expression of the Runx2 target proteins osteopontin and osteocalcin was examined. Actin expression levels were examined as a loading control and representative blots are shown (n=3). B, Calcium levels were determined (*P<0.01 vs SS, n=4); and, in the presence or absence of BMP‐2 (100 ng/mL) for the 14‐day treatment period, (C) calcification was evaluated by von Kossa staining (n=3) and (D) activity of the Runx2‐independent protein alkaline phosphatase was assessed using BCIP/NCP staining at 10× magnification (n=4). *P<0.01 vs SS. BMP‐2 indicates bone morphogenetic protein‐2; CASMC, coronary artery smooth muscle cell; BCIP, 5‐bromo‐4‐chloro‐3′‐indolyphosphate; miR, microRNA; NBT, nitro‐blue tetrazolium.