miR-34b and -c regulate osteoblast proliferation. (A) Proliferation of osteoblasts in miR-34bc^Osb−/− and α1(I) miR-34c transgenic mice at p14, measured by BrdU incorporation (n = 3–5). Representative images of a BrdU incorporation assay in calvaria of miR-34bc^Osb−/− mice and α1(I) miR-34c transgenic mice at p14. The first two layers of cells lining the cortical bone surface were typed as osteoblasts. Proliferating osteoblasts (arrowheads) were BrdU-positive cells (green), and nuclei were stained as blue (DAPI). BM, bone marrow. Bar, 20 µm. (B) Alignment of miR-34s showing complementary pairing to the 3′ UTRs of Cyclin D1. (C) Effect of miR-34 expression on luciferase reporter plasmids carrying either WT or mutant Cyclin D1 3′ UTRs (n = 3). #, P < 0.001. (D–G) Effect of knockdown (D and E) and overexpressing miR-34s (F) on Cyclin D1 protein level, as well as Cyclin D1 protein level in bones of miR-34bc^Osb−/− and α1(I) miR-34c mice (G; n = 3–5 of each genotype), determined by Western blot and quantified using ImageJ. Quantification of Western blots was summarized from at least three independent experiments. (H) Alignment of miR-34s showing complementary pairing to the 3′ UTRs of CDK4 and CDK6. (I) CDK4 and CDk6 protein levels in miR-34bc^−/− osteoblasts. (J) CDK4 and CDk6 protein levels in bones of miR-34bc^Osb−/− and α1(I) miR-34c mice (n = 3–5 of each genotype). Error bars indicate means ± standard errors of the mean. *, P ≤ 0.05.