Overexpression of PIAS3 suppresses the expression and transcriptioan activity of c-Fos during RANKL-mediated osteoclastogenesis. (A,B) The whole-cell extracts from both mock- or PIAS3-transduced cells were stimulated with 50 ng/mL RANKL for 24 hours. Thirty micrograms of each cell lysate was used for the DNA binding ELISA assay. RLU, relative luciferase unit. (C) Mock- or PIAS3-transduced RAW264.7 cells were cultured with 50 ng/mL RANKL for 2 days. Total RNA was collected from cultured cells and real-time PCR was performed with primers specific for the indicated genes. (D) Mock- or PIAS3-transduced RAW264.7 cells were cultured with 50 ng/mL RANKL for 24 hours. Cells were harvested and lysates were analyzed by Western blot analysis for the indicated genes. Error bars in all panels represent SD. #P < .05; ##P < .01.