Additional file 4: Figure S4. of Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer

Standard histological sections of TRAP staining in the femur. Representative images of an OVX control-treated femur and an everolimus-treated femur stained for TRAP (a, ×2.5 magnification, scale bar 200 μm; b, ×20 original magnification, scale bar 50 μm). (PDF 3572 kb

Additional file 1: Figure S1. of Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer

Everolimus inhibits mTOR signaling in cancer cell lines. Quantification of Western blots shown in Fig. 1. Indicated cell lines were treated with everolimus for 24 h, and total and phosphorylated proteins were detected by Western blot analysis. The signals of phosphorylated mTOR (p-mTOR) and phosphorylated p70 S6 kinase (p-p70) were quantified and normalized to corresponding signals of GAPDH for a total of three experiments. Data were analyzed using one-way ANOVA and the Bonferroni posttest and are shown as mean ± SD (* p < 0.05; ** p < 0.01, *** p < 0.001). Equal volumes of DMSO used to prepare and administer everolimus treatments were used in the control conditions. (PDF 14 kb

Additional file 3: Figure S3. of Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer

Everolimus inhibits the bone-resorbing activity of osteoclasts. Murine bone marrow-derived mononuclear cells were differentiated to osteoclasts on bone slices in vitro before being treated with everolimus at concentrations of 1, 10, and 100 nM for 5 days in total. On day 5, supernatants were collected and analyzed for the levels of the bone resorption marker collagen type I cross-linked C-telopeptide (CTx). Data were analyzed using one-way ANOVA and the Bonferroni posttest, and significance between the control and everolimus concentrations is denoted (** p < 0.01). (PDF 9 kb