Fig. 6. Selective sRANKL inhibitor (S3-15) inhibits the RANKL biological functions of osteoclasts and osteoblasts.

a, b Western blot experimental results show that S3-15 suppresses RANKL mediated NF-κB and MAPK signaling pathway. Images represent three independent experiments. c, d Luciferase reporter assay results show that NF-κB and NFATC-luciferase expression are reduced in doge manage when treated with S3-15. e RT-qPCR experimental results show that S3-15 suppresses osteoclast markers (DC-stamp, Ctsk, MMP9, Tracp, Oscar, and Calcr) expression in BMMs cells stimulated with 100 ng/mL sRANKL. f Cell apoptosis assay results show that S3-15 induces osteoclast apoptosis. The apoptosis (Annexin V stained showing green fluorescence), death (PI stained showing green fluorescence), and nucleic acid breaks (DAPI stained showing blue fluorescence aggregation) of osteoclasts were observed. TRAP stained also observed osteoclast death. Representative images (n = 3 images taken in total with technical triplicate repeat) (left) and dead osteoclast (yellow arrowheads) quantification (right). g Bone resorption experimental results show that S3-15 suppresses RANKL-induced resorption pits in dose-dependent manner. Representative resorption pits images (n = 3 images taken in total with technical triplicate repeat) (left) and resorption pits area quantification (right). h Human primary osteoblasts cell culture experiments show that S3-15 promotes osteoblast mineralization. Image was taken after 12 days cell culture with S3-15 and Alizarin Red S staining. Data are presented as the mean ± s.d. from three biological replicate per group. Statistical difference was determined by unpaired two-tailed Student’s t test. Significant difference p value is < 0.05.