FIGURE 8.

Inhibition of miRNA-223 expression causes aberrant osteoclastogenesis. A and B, RAW 264.7 cells were transiently transfected with antisense miRNA-223 or negative control oligonucleotides, and the cells were treated with RANKL (100 ng/ml). After 5 days, the cells were then fixed and stained for TRAP, and the number of TRAP-positive multinucleated cells was scored. Similar findings were obtained in four independent sets of experiments. C and D, NFI-A, M-CSFR, and PU.1 levels in RAW264.7 cells with negative control or antisense miRNA-223 and infected osteoclast precursors were analyzed by immunoblotting (IB) using whole cell extracts. Quantitative image analyses of these protein expression levels were normalized to GAPDH. D, The expression levels of miRNA-223 was measured by miRNA Northern blotting using biotin prelabeled probes specific for miRNA-223. Sno234 was used as a loading control. The sno234 normalized fold increase in the miRNA-223 in osteoclast precursors with pMX-PU.1 retroviral vector is shown.