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. Author manuscript; available in PMC: 2010 Sep 1.
Published in final edited form as: Nat Med. 2009 Aug 30;15(9):1066–1071. doi: 10.1038/nm.2007

Figure 4.

Figure 4

IRF8 inhibits NFATc1 transcriptional activity and expression, and reduced IRF8 expression may contribute to pathological bone destruction. (a) Expression of Nfatc1 and Acp5 mRNAs induced by 24 h stimulation of BMMs derived from wild-type and Irf8−/− mice with 50 ng/ml M-CSF and the indicated doses of RANKL (Northern blotting). (b) Expression of Nfatc1 and Acp5 mRNAs in retrovirus-infected BMMs (Control, pMX-puro; Irf8, pMX-Irf8-puro) in the absence or presence of 150 ng/ml RANKL with 50 ng/ml M-CSF for 24 h (Northern blotting). Gapdh was used as an internal control. (c) A luciferase activity assay to examine the effect of IRF8 on the transcriptional activity of NFATc1. **P<0.01. (d) The interaction between NFATc1 and IRF8. IP, immunoprecipitation; IB, immunoblotting. (e,f) Analysis of the inhibition of NFATc1 binding to its target DNA sequences using EMSAs (see supplementary methods on line). The arrow heads indicate a specific binding complex, which included the NFATc1–DNA complex; this was confirmed in a supershift assay (lane 1–3) and a cold competition experiment (lane 4). The intensity of NFATc1–DNA bands decreased with increasing amounts of GST-IRF8 (lanes 7–9), or when nuclear extracts from HEK293 cells cotransfected with pcDNA3-Nfatc1 and pcDNA3-Irf8 were used (f), but not with GST alone (lanes 5–6). (g) TRAP staining of mouse whole calvaria (left) and histological sections (right) obtained from wild-type and Irf8−/− mice with or without administration of LPS. (h) Osteoclast formation in BMMs stimulated with various doses of LPS in the presence of M-CSF (50 ng/ml) and RANKL (150 ng/ml) for 5 days. Data are expressed as the mean+SD of 4 cultures. (i) Left, BMM cultures in the presence of 50 ng/ml M-CSF and 0 or 25 ng/ml TNFα on day 3 shown by TRAP staining. Right, TNFα dose-dependent induction of osteoclast formation examined using TRAP activity assays. (j) Expression of Nfatc1 and Acp5 mRNAs in BMMs derived from wild-type and Irf8−/− mice stimulated with 50 ng/ml M-CSF and the indicated doses of TNFα for 24 h (Northern blotting).

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