Fig. 5

Atoh8-deficient bone exhibits a higher Rankl/Opg expression ratio, and marrow stromal cells are more potent than WT cells in inducing osteoclastogenesis of bone marrow cells. a–c Primary bone marrow cells of 8-week-old mice were stimulated with 50 ng·mL⁻¹ M-CSF with or without 100 ng·mL⁻¹ RANKL for osteoclastogenesis. Representative images of TRAP staining-positive osteoclasts are presented (a). Scale bar = 50 μm. The number of multinucleated (>3 nuclei) osteoclasts was counted in six fields for each sample (b). Cell lysates were subjected to RT-qPCR for Trap and Ctsk (c) (n = 3). d RNA was extracted from femurs of 8-week-old male mice and subjected to RT-qPCR to measure the Rankl/Opg expression ratio (n = 5 WT males, 4 KO males, 4 WT females, and 3 KO females). IHC of tibial samples from 8-week-old mice using anti-Rankl (e) and anti-Opg (f) antibodies. Scale bar = 50 μm. g, h Coculture of primary bone marrow stromal cells and bone marrow cells of the indicated genotype to induce osteoclastogenesis. TRAP staining-positive osteoclasts are indicated (arrowheads) (g). Scale bar = 500 μm. The number of multinucleated (>3 nuclei) osteoclasts was counted in nine fields for each sample (h). The data shown are the mean ± SD. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001