Fig. 3

Effects of everolimus (EV) on human and murine osteoblastogenesis in vitro. Human mesenchymal stem cells were differentiated along the osteoblast lineage in the presence of increasing EV concentrations (0, 1, 10, and 100 nM). a Osteoblast marker genes ALP, OPG, RUNX2, and OCN were assessed by quantitative real-time polymerase chain reaction (RT-PCR) at 7 days of differentiation. b The mineralizing ability of these cells was quantified using alizarin red S staining on day 21. Murine mesenchymal stem cells were isolated from the bone marrow of C57BL/6 mice and differentiated along the osteoblast lineage in the presence of increasing EV concentrations (1–100 nM). c Osteoblast marker genes Alp, Opg, Runx2, and Ocn were assessed by qRT-PCR at 7 days of differentiation. d The mineralizing ability of these cells was quantified using alizarin red S staining on day 21. Data are shown as mean ± SD of at least three independent experiments. Data were analyzed using one-way analysis of variance and the Bonferroni posttest, and significance between the control and EV concentrations is denoted (* p < 0.05, ** p < 0.01, *** p < 0.001). Equal volumes of DMSO used to prepare and administer EV concentrations were used in all control conditions. mRNA Messenger RNA