Figure 1.

Deletion of Tet2 impairs osteoclast differentiation and function

A. Quantitative RT-PCR analysis of Tet1, Tet2, and Tet3 expression (relative to Actb) in the bone marrow-derived osteoclast precursor cells (macrophages, CD11b⁺ gated) of WT mice. B. Flow cytometric analysis for the GFP (Tet2) expression levels in bone marrow derived macrophages from a representative 8-week-old heterozygous Tet2:GFP knock-in mouse. C. Representative photomicrographs showing TRACP-staining of bone marrow-derived osteoclasts cultured in the presence of M-CSF and RANKL in WT and Tet2^−/− mice, respectively. Scale bars, 250 μm. D. Quantitation of the number of TRACP⁺ osteoclasts derived from 5 × 10⁴ BMMNCs. Data are presented as mean ± SEM, n = 5. E. Osteoclast cultures on dentine slices. Representative photomicrographs showing resorptive “pits” generated by osteoclast bone lytic activity. Scale bars, 50 μm. F. Quantitation of the area of the resorbed regions, referred to as ‘pits’. Data are presented as mean ± SEM, n = 3. G. Histological analysis using TRACP stain of the femur from 12-week-old WT and Tet2^−/− male mice. Representative photographs (100×) of the trabecular bone following TRACP staining are shown. The red stained area indicates TRACP⁺ osteoclasts. Scale bars, 100 μm. H. Quantitation of the number of TRACP⁺ osteoclasts. Data are presented as mean ± SEM, n = 3. I. μCT analysis of the femur of 12-week-old WT and Tet2^−/− male mice, respectively. Representative μCT imaging demonstrating the reconstructed 3D microstructure of femoral trabecular bones is shown. The cortical portion of each femur was removed to allow visualization of the metaphyseal architecture. J. Quantitation of the femoral bone volume/total volume assessed using μCT. K. Quantitation of the trabecular number assessed using μCT. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Student’s t test. TRACP, tartrate-resistant acid phosphatase; μCT, microcomputed tomography.