ZAS3 promotes RANKL-mediated osteoclastogenesis and expression of osteoclast-associated genes.

<p>RAW264.7 cells (from 500 to 2500 cells) were stably transfected with the empty vector (EV) or the ZAS3 expression construct. (A) Indicated cells were plated onto wells of a 24-well plate and cultured in complete medium supplemented with sRANKL (50 ng/ml) for 6 days, and stained for TRAP. TRAP+ cells with 3 or more nucleic were considered as OLC and counted. Bar charts show the numbers of OLC. (B) Indicated cells were incubated with sRANKL (50 ng/ml) for 0, 2, 4, or 8 days. The induction of ZAS3, TRAP and CTSK (osteogenic markers), and HSP90 (loading control) was examined in total cell lysates by Western blot analysis. (C) Indicated cells were incubated with sRANKL (50 ng/ml) for 6 days and the induction of TRAF-6, RelB, c-Fos, and c-Jun was analyzed by Western blot analysis. (D) EMSA was performed with ³²P-GTTCT<u>GGGGAAGTCC</u>AGTGCTCACATGACC DNA probe corresponding to the mouse TRAP gene enhancer (ZAS3 binding site is shown in underline) and nuclear extracts prepared from indicated cells. The major DNA-protein complexes are designed C1 to C4. Complex C4 (indicated with a black arrow) was observed only in the ZAS3 transfected samples (lane 3). The formation of C2 (indicated with a grey arrow) and C4 was abolished by the addition of ZAS3 antibodies to the binding reactions (lanes 4 and 5).