FIGURE 1.

JMJD5 repressed osteoclastogenesis with reduced expression of osteoclast-specific genes. A, mature osteoclasts were differentiated from RAW264 cells by RANKL treatment for 6 days. Total RNAs were extracted at the indicated time points and analyzed by real time RT-PCR. Relative mRNA levels are represented by values that were normalized to Gapdh transcript levels. The error bars represent S.D. values from at least triplicate experiments. B, stable cell lines that constitutively express JMJD5-specific shRNA were established in RAW264 cells by puromycin selection. Individual colonies were picked up, and the expression levels of the Jmjd5 gene were detected by RT-PCR. Two stable clones, which have shown high knockdown efficiency, were selected for further study. LacZ-specific shRNA was used for a negative control. C, stable clones were treated with RANKL for osteoclast formation. Differentiated cells were fixed at 3.5 days and assessed with an Olympus IX70 light microscope. Black arrowheads indicate multinucleated osteoclasts. D, during RANKL treatment, cells were harvested at the indicated time points, and total RNAs were prepared. Expression levels of osteoclast-specific genes (Ctsk, DC-STAMP, and Acp5) were analyzed by real time RT-PCR.