Expression of ephrinA2 and EphA2 during osteoclast and osteoblast differentiation. A, RT-PCR analysis. MDMs were treated with RANKL, and calvarial osteoblasts were treated with ascorbic acid and β-glycerophosphate for the indicated days. con, control adult mouse brain. B, qRT-PCR analysis. MDMs were treated with RANKL for 0, 0.25, 0.5, 1, 3, 6, 9, 12, 24, 36, 48, and 60 h. Error bars represent means ± S.E. (n = 3). *, p < 0.05; **, p < 0.01 versus 0 h. C, immunoblot analysis during osteoclast differentiation. Upper panel, ephrinA2. Negative and positive controls were MDMs infected with empty (emp) and ephrinA2-expressing (eA2) retroviruses, respectively. Lower panel, EphA2. con, control adult mouse brain. day, after RANKL addition. D-O, expression of ephrinA2 and EphA2 in bone. Hematoxylin and eosin (HE) staining of mouse femurs (D, G, J, and M), and immunofluorescence detection of ephrinA2 (red, E, F, H, and I) and EphA2 (red, K, L, N, and O). Osteoclasts (OC) were identified as multinucleated cells expressing cathepsin K (green, E, F, K, and L). Osteoblasts (OB) were cells expressing osteocalcin (green, H, I, N, and O). Higher magnification of osteoclasts or osteoblasts in E, H, K, and N are shown in F, I, L, and O, respectively. Nuclei are shown in blue (4′,6-diamidino-2-phenylindole). Scale bars, 20 μm.