Fig. 3

SIRT7 positively regulates osteoblast differentiation. a Primary calvarial osteoblasts derived from Sirt7 KO mice and WT mice were cultured for 10 days in differentiation medium. Representative Alizarin Red S-stained images (left) and the Alizarin Red S staining area ratio (right). b Proliferation assay using primary calvarial osteoblasts from Sirt7 KO mice and WT mice. Cell proliferation was measured by direct counting (left) and by the WST-1 colorimetric assay (right). c Expression of Sirt1, Sirt6, Sirt7, and Ocn in MC3T3-E1 cells analyzed by qRT-PCR. d–g MC3T3-E1 cells with Sirt7 knockdown and control cells were cultured for 30 days in differentiation medium. Confirmation of knockdown by western blotting (d), representative Alizarin Red S-stained images (e), Alizarin Red S staining area ratio (f), and expression of osteoblastic marker genes analyzed by qRT-PCR (g). Undiff.: cells cultured in growth medium for 3 days. Diff.: cells cultured in differentiation medium for 30 days. Data are shown as the mean ± SEM. Statistical significance was determined by Student’s t-test. *p < 0.05 vs. WT (a, b), Undiff. (c), or shControl (f, g). Scale bar, 400 μm