Fig. 3.

Support of OCL formation by marrow stromal cells from WT, p62KI, TIL-6, p62KI/TIL-6, and MVNP mice. (A) RANKL and OPG expression. Stromal cells from WT, p62KI, TIL-6, p62KI/TIL-6, and MVNP mice were cultured with 1,25-(OH)₂D₃ (1 × 10⁻⁸ M) for 2 days, the cell lysates were collected, and the levels of RANKL and OPG were determined by Western blot analysis using anti-RANKL and anti-OPG antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The ratio of RANKL/OPG expression levels from Western blots were quantitated by densitometry using WT cultures as 1.0. (B) RANKL production by mouse marrow stromal cells. Mouse marrow stromal cells were cultured with 1,25-(OH)₂D₃ for 7 days. Conditioned media from these cultures were harvested at the end of the culture period and the concentration of RANKL present was determined. The data is shown as mean ± SD (n = 4); *p < 0.01 compared with WT cells cultured with the same concentration of 1,25-(OH)₂D₃. (C) TAF12 expression in marrow stromal cells. Stromal cells were cultured with 10% FCS in IMDM for 3 days and then cell lysates were collected. TAF12 expression was analyzed by immunoblot using a polyclonal antibody recognizing TAF12. TFIIB was used as a loading control. (D) Support of OCL formation by marrow stromal cells. Stromal cells from WT, p62KI, TIL-6, p62KI/TIL-6, and MVNP mice were cocultured with CFU-GM derived from WT mouse bone marrow in the presence of 1 × 10⁻⁸ M 1,25-(OH)₂D₃ for 7 days. The cells were then fixed and stained for TRAP, and the TRAP-positive OCLs were counted. Results are expressed as the mean ± SD (n = 4); *p < 0.01 compared with results in WT cultures. OCL = osteoclast; WT = wild-type; RANKL = receptor activator of NF-κB ligand; OPG = osteoprotegerin; 1,25-(OH)₂D₃ = 1,25-dihydroxyvitamin D₃; FCS = fetal calf serum; IMDM = Iscove’s Modi fied Dulbecco’s Media; TFIIB = transcription factor IIB; CFU-GM = colony-forming unit–granulocytemacrophage; TRAP = tartrate-resistant acid phosphatase.