Figure 8.
Figure 8. Ebf1 and Zfp521 regulate OB-dependent and hematopoietic lineage–dependent OC-genesis. (A) Total BM cells from Ebf1−/− and control mice were plated on 48-well plates, stimulated with 10 nM hPTH(1–34), and stained for TRAP. Number of TRAP+ multinucleated cells (MNCs) per well is shown. (B) Calvarial cells from control and Ebf1−/− mice were co-cultured with nonadherent BM cells as indicated in 24-well plates, stimulated with vitD3 and PGE2, and stained for TRAP. Bar graphs indicate the number of TRAP+ multinucleated cells per well. (C) Rankl and Opg mRNA expression and Rankl/Opg ratio measured by qRT-PCR in the co-culture experiment in B. (D) RANKL protein levels were measured with RANKL ELISA in medium samples from control and Ebf1−/− calvarial cells cultured in 12-well plates and stimulated with vitD3 and PGE2 as in co-culture experiments (n = 3). (E) ChIP with anti-V5 antibody for V5-Zfp521 and anti-Flag antibody for Flag-Ebf1 in MC3T3-E1 cells overexpressing the tagged proteins. IgG was used as control. The PCR-amplified promoter area contained a putative Ebf1 consensus site in the active distal promoter region. (F) The time courses of PTH-induced Rankl mRNA expression and displacement and rebinding of endogenous Zfp521 from the Rankl distal promoter region after stimulation by 10 nM PTH were compared. Zfp521 binding to the promoter was analyzed in a ChIP assay using the anti-Zfp521 antibody. IgG was used as control. (G) Expression of Zfp521 mRNA by qRT-PCR in OC progenitors cultured with 20 ng/ml M-CSF for 2 d and then stimulated with 100 ng/ml RANKL for the indicated times. (H) Expression of Ebf1 mRNA by qRT-PCR in OC progenitors cultured as in G. (I) Control and Ebf1−/− spleen cells were stimulated with 20 ng/ml M-CSF and then with increasing doses of RANKL for 5 d, stained for TRAP activity, and quantified. Bar, 100 μm. (J) Expression of Nfatc1, Ctsk, and Rank mRNAs by qRT-PCR in Ebf1−/− spleen cell–derived OCs cultured with 20 ng/ml M-CSF and then M-CSF + 100 ng/ml RANKL for the indicated times. All data are mean ± SD. Similar cell number and mRNA data were obtained from three independent experiments with four to six replicates per condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Ebf1 and Zfp521 regulate OB-dependent and hematopoietic lineage–dependent OC-genesis. (A) Total BM cells from Ebf1−/− and control mice were plated on 48-well plates, stimulated with 10 nM hPTH(1–34), and stained for TRAP. Number of TRAP+ multinucleated cells (MNCs) per well is shown. (B) Calvarial cells from control and Ebf1−/− mice were co-cultured with nonadherent BM cells as indicated in 24-well plates, stimulated with vitD3 and PGE2, and stained for TRAP. Bar graphs indicate the number of TRAP+ multinucleated cells per well. (C) Rankl and Opg mRNA expression and Rankl/Opg ratio measured by qRT-PCR in the co-culture experiment in B. (D) RANKL protein levels were measured with RANKL ELISA in medium samples from control and Ebf1−/− calvarial cells cultured in 12-well plates and stimulated with vitD3 and PGE2 as in co-culture experiments (n = 3). (E) ChIP with anti-V5 antibody for V5-Zfp521 and anti-Flag antibody for Flag-Ebf1 in MC3T3-E1 cells overexpressing the tagged proteins. IgG was used as control. The PCR-amplified promoter area contained a putative Ebf1 consensus site in the active distal promoter region. (F) The time courses of PTH-induced Rankl mRNA expression and displacement and rebinding of endogenous Zfp521 from the Rankl distal promoter region after stimulation by 10 nM PTH were compared. Zfp521 binding to the promoter was analyzed in a ChIP assay using the anti-Zfp521 antibody. IgG was used as control. (G) Expression of Zfp521 mRNA by qRT-PCR in OC progenitors cultured with 20 ng/ml M-CSF for 2 d and then stimulated with 100 ng/ml RANKL for the indicated times. (H) Expression of Ebf1 mRNA by qRT-PCR in OC progenitors cultured as in G. (I) Control and Ebf1−/− spleen cells were stimulated with 20 ng/ml M-CSF and then with increasing doses of RANKL for 5 d, stained for TRAP activity, and quantified. Bar, 100 μm. (J) Expression of Nfatc1, Ctsk, and Rank mRNAs by qRT-PCR in Ebf1−/− spleen cell–derived OCs cultured with 20 ng/ml M-CSF and then M-CSF + 100 ng/ml RANKL for the indicated times. All data are mean ± SD. Similar cell number and mRNA data were obtained from three independent experiments with four to six replicates per condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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