Impaired osteoclastogenesis by forced expression of PDCD4. (A-B) Immunoblotting analysis of phosphorylated c-Fos (A), NFATc1 (B), or cathepsin K (B) of BMMs with overexpressed PDCD4 or control for the indicated times. (C) ChIP assay was performed to study the association of c-Fos with miR-21 promoter during RANKL-induced osteoclastogenesis for the indicated days. (D) 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. (E) TRAP-positive multinucleated cells count at 3 days after RANKL treatment, cultured in 24-well plates (200×). Similar findings were obtained in 4 independent sets of experiments.