Fig. 3: SP blocks the progression of bone erosion by suppressing osteoclast activation.

A Tartrate-resistant acid phosphatase (TRAP) staining and fluorescent F-actin staining of bone marrow-derived macrophages (BMMs) stimulated with macrophage colony-stimulating factor (M-CSF, 30 ng/mL) and receptor activator of nuclear factor-κ B ligand (RANKL, 50 ng/mL), followed by treatment with different doses of SP for 5 days. B Quantification of number and area of osteoclasts in TRAP stain and area of F-actin belt shown in panel A. C RT-qPCR analysis of Nfatc1, C-fos, and Ctsk in BMMs stimulated with M-CSF and RANKL, followed by treatment with SP for 5 days. D Western-blot (WB) analysis of NFATc1, c-Fos, and Ctsk in BMMs stimulated with M-CSF and RANKL, followed by treatment with SP. (For c-Fos, the samples derive from the same experiment and that gels/blots were processed in parallel, and image was cropped at the dotted line only for the purpose of this figure.) E Semi-quantification of greyscale value in NFATc1, c-Fos, and Ctsk in panel D. F WB analysis of phospho-P38, P38, p-ERK, ERK, IκBα, p-P65, and P65 in BMMs stimulated with M-CSF and RANKL, followed by treatment with SP. G Semi-quantification of greyscale value in p-P38/P38, p-ERK/ERK, IκBα, and p-P65/P65 in panel F. H Immunofluorescence analysis of p-P38 and p-P65 in BMMs stimulated with M-CSF and RANKL, followed by treatment with SP. I Quantification of the integrated optical density (IOD)/DAPI of p-P38 and p-P65 as shown in panel H. All data presented as mean (SD) are from three replicates. Statistical analysis was performed using the Kruskal–Wallis test with Dunn’s post hoc test for ordinal data such as osteoclasts number and the one-way ANOVA followed by Tukey’s post-hoc test for continuous data. The exact p-values were labeled.