Fig. 2

Effects of everolimus (EV) on osteoclastogenesis of osteoclast progenitor RAW 264.7 cells and murine bone marrow-derived mononuclear cells in vitro. a RAW 264.7 osteoclast precursors were treated with receptor activator of nuclear factor κB ligand (RANKL) in the presence of increasing EV concentrations (0, 1, 10, and 100 nM) for 5 days. Differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) staining and counting. b Osteoclast marker genes Cstk, Oscar, and Trap were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) following differentiation with RANKL and EV for 5 days. c Murine bone marrow-derived mononuclear cells were isolated from the bone marrow of C57BL/6 mice and treated with macrophage colony-stimulating factor (M-CSF) for 2 days prior to further M-CSF plus the addition of RANKL in the presence of increasing EV concentrations (1–100 nM) for 5 days. Differentiation was assessed by TRAP staining and counting. d Osteoclast marker genes Cstk, Oscar, and Trap were also assessed for osteoclasts differentiated from bone marrow-derived mononuclear cells by qRT-PCR following differentiation with RANKL and EV for 5 days. 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 dimethyl sulfoxide used to prepare and administer EV concentrations were used in all control conditions. mRNA Messenger RNA