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. 2020 Aug 24;14:3435–3447. doi: 10.2147/DDDT.S256867

Figure 2.

Figure 2

Sarsasapogenin inhibits F-actin ring and bone resorption in vitro. (A) Effect of sarsasapogenin on the fusogenic potential of osteoclast precursor cells. Representative immunofluorescence images of F-actin rings (Acti-stain™ 488 Fluorescent Phalloidin; green) from BMM-derived osteoclasts stimulated with 100 ng/mL RANKL with or without the indicated concentrations of sarsasapogenin. Nuclei were counterstained with DAPI (blue) (magnification = 100×; scale bar = 100 μm). (B) Equal numbers of pre-osteoclasts were seeded onto bone slices and stimulated with M-CSF and RANKL in the presence of different concentrations of sarsasapogenin. After 3 days, bone resorption lacunae were observed by SEM (magnification = 200 ×; scale bar = 100 μm). (C) F-actin rings were quantified using ImageJ (n = 3). (D) The resorption pit area (%) was quantified using ImageJ (n = 3). (E) Dose-dependent mRNA expression profile of osteoclast marker genes. RT-qPCR was conducted using RNA extracted from cells stimulated with 100 ng/mL RANKL with or without the indicated concentrations of sarsasapogenin for 5 days. mRNA levels of c-FOS, NFATc1, DC-STAMP, ATP6V0d2, TRAP, and CTSK were normalized to that of β-actin and then compared to RANKL-only control (0) to obtain the relative fold change (n = 3). Bar graph presents the mean ± SD. **p < 0.01, and ***p < 0.001 versus RANKL-treated only control.