PIAS3 negatively regulates RANKL expression in primary osteoblasts and inhibits osteoclast formation in vitro coculture system. (A) Expression of PIAS3 and β-actin mRNA in mock- or PIAS3-transduced mouse POBs was measured by RT-PCR. (B) Expression of RANKL mRNA in POBs that were stimulated with 100 ng/mL IL-6 and 100 ng/mL sIL-6R, or 20 ng/mL IL-11, was determined by real-time PCR. (C) POBs were transfected with negative control and PIAS3 siRNA. Expression of RANKL and PIAS3 mRNA in POBs that were stimulated by adding graded concentrations of IL-6 and sIL-6R. (D) Densitometric values for each band of panel C were quantified and normalized using ImageJ software. (E) Expression of PIAS3 mRNA in POBs that were stimulated with 1,25(OH)₂D3 + PGE2, IL-6 + sIL6R, and IL-11, was determined by real-time PCR. (F) Analysis of osteoclast differentiation by TRAP staining. POBs transduced with mock or PIAS3 were cocultured with WT BMMs for 7 days in the presence of indicated concentration of OSM. Cultured cells were fixed and stained for TRAP. (G) TRAP-positive MNCs having more than 3 nuclei were counted as osteoclast. (H) Expression of RANKL mRNA in mock- or PIAS3-transduced POBs was measured by real-time PCR. (I) Mock- and PIAS3-transduced POBs were incubated with 50 ng/mL OSM for the indicated times. Cells lysates were analyzed by Western blot analysis for the pSTAT3, STAT3, and β-actin. (J) Mock- and PIAS3-transduced POBs were stimulated with 50 ng/mL OSM for 0, 10, and 30 minutes. A total of 60 μg of each cell lysate was used for the STAT3 DNA binding ELISA kit assay. Error bars in all panels represent SD. #P < .05; ##P < .01; n.s, not significant.