Figure 2. RANK, RANK-L and OPG protein expression levels and RANK-L released concentration after treatment with Den and Doxo in OS cell lines.

(A) RANK-L, OPG and RANK protein expression levels in MG63 and U-2 OS cells determined by WB, starting from 15 μg of total lysates, after 48 h of exposure to Den [30 μg/mL] and Doxo [0,2/0,5 μM] alone or in combination. The most representative WB images are displayed. The proteins were detected using Image Studio Digits software and the intensity ratios of immunoblots compared to MG63 untreated (NT), taken as 1 (arbitrary unit), were quantified after normalizing with respective loading controls for the housekeeping protein β-Tubulin. Histograms show the relative quantification for RANK-L, OPG, RANK expression as mean ± SD of three independent experiments on both cell lines. A One-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, has been used to evaluate differences in RANK-L and OPG expression among groups. ^*indicates p ≤ 0.05 compared to the untreated control (NT). (B) The release of RANK-L from MG63 cells and from U-2 OS (C) cells determined by ELISA assay, after 48 h of exposure to Den [30 μg/mL] and Doxo [0,2/0,5 μM] alone or in combination. RANK-L concentration was determined on a standard concentration curve according to the manufacturer’s instructions. For each cell line the assay was conducted three times. Data are expressed as mean ± SD (pg/ml). A One-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, has been used to evaluate differences in RANK-L release among groups. ^*indicates p ≤ 0.05 compared to the untreated control (NT).