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. 2013 May 6;210(5):969–985. doi: 10.1084/jem.20121187

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© 2013 Kiviranta et al.

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Figure 4. — Zfp521 acts in OC progenitor cells to control OC-genesis. (A) Control and Zfp521^−/− spleen cells were stimulated with 20 ng/ml M-CSF and then with increasing doses of RANKL for 3 d, stained for TRAP activity, and quantified. Bar, 64 μm. (B) BMM-derived OCs were cultured with 20 ng/ml M-CSF and then M-CSF + 100 ng/ml RANKL for the indicated times, and expression of Nfatc1, Ctsk, and Rank was measured by qRT-PCR. (C) Ctsk-Luciferase reporter construct was transfected to RAW264.7 cells together with Zfp521, constitutively active NFATc1, or both. Luciferase activity was normalized to cotransfected Renilla activity. (D) Nonadherent BM cells were cultured for 2 d with 20 ng/ml M-CSF and then stimulated for 4 h with 100 ng/ml RANKL and 20 ng/ml M-CSF, and expression of Ccl9 was measured by qRT-PCR. (E) Control and Zfp521^−/− spleen cells were cultured with 20 ng/ml M-CSF and 50 ng/ml RANKL for 5 d in the presence of blocking anti-Ccl9 antibody, IgG, or vehicle. Cells were fixed and stained for TRAP activity and quantified. All data are means ± SD. Similar cell number and mRNA data were obtained from three independent experiments with three to six replicates per condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001.