Expression of miR-21 in osteoclastogenesis. (A) The heat map was generated with the use of hierarchical cluster analysis to show distinct miR expression patterns in primary cultured BMMs between M-CSF alone and M-CSF + RANKL treatment groups. The color bar was extracted to show the color contrast level of the heat map. Red and green indicate high expression levels and low expression levels, respectively. (B) RT-PCR analysis of genes related to osteoclastogenesis during RANKL-induced osteoclastogenesis for the indicated days. (C) ChIP assay was performed to study the association of c-Fos and PU.1 with miR-21 promoter. (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; *P < .01 compared with day 0. (E) Total RNA was prepared from c-Fos wt and knock-out (KO) splenocytes. 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. (F) Total RNA was prepared from c-Fos KO splenocytes with retrovirus vectors (pMX-IP or pMX-c-Fos-IP). 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.