Fig. 5.

GANT58-NP treatment does not inhibit osteoblast function but reduces tumor-mediated osteoclastogenesis in vitro. (A) hMSCs treated with varying concentrations of free GANT58 (red), Empty-NP (blue), or GANT58-NP (green) were cultured for 1, 3, and 5 days (D1, D3, D5) and proliferation assessed by MTS assay. Gradient colors indicate varying doses: 1 μM (transparent), 5 μM, 10 μM, to 20 μM (opaque). (B) hMSCs were cultured in osteogenic medium treated with same dosage scheme described in (A). Deposition of mineralized matrix by osteoblasts was determined by absorbance of extracted Alizarin Red stain after 14 d of culture in osteogenic media. (C) Representative images of Alizarin Red staining of treated osteoblasts after 14 d culture in osteogenic media. Inset image shows hMSCs cultured in non-osteogenic media. Scale bar: 200 μm. (D) Number of osteoclasts (OC)/well in coculture treated with varying doses of GANT58, Empty-NP, and GANT58-NP after 6 days culture with MDA-MB-231 cells. Three technical replicates are shown in (A), (B), and (D) from single experiments. (E) Multinucleated (3 or more nuclei) and TRAP positive cells were counted as OCs. Red arrows: OCs. (F) Representative images of negative control group (no tumor, -vitD), no treatment group, and 20 μM GANT58-NP treatment group. Red arrows: OCs.