Impaired osteoclastogenesis by miR-21 silencing. (A) Total RNA was prepared from BMMs harboring pLVX-shRNA or pLVX-antimiR-21. QRT-PCR was performed with primer for miR-21. The PCR products were normalized to U6 level for each reaction. The data represent the means ± SDs of 3 experiments in triplicate. (B) RT-PCR analysis of PDCD4 in primary cultured BMMs without RANKL treatment. antimiR-21 indicates antisense miR-21; shRNA, short hairpin RNA. (C) Immunoblotting analysis of PDCD4 of primary BMMs without RANKL treatment. (D-E) Immunoblotting analysis of phosphorylated c-Fos (D), NFATc1 (E), or cathepsin K (E) of BMMs with antisense miR-21 or control for indicated times. antimiR-21 indicates antisense miR-21. (F-G) ChIP assay was performed to study the association of c-Fos (F) or NFATc1 (G) with TRAP promoter during RANKL-induced osteoclastogenesis for the indicated days. (H) TRAP-positive multinucleated cell count at 3 days after RANKL treatment, cultured in 24-well plates (200×). Similar findings were obtained in 4 independent sets of experiments.