Fig. 7: Interference with DCSTAMP function results in the disruption of RAP1–RAC1 signaling pathways during osteoclastogenesis.

a A principal component analysis conducted on mature osteoclasts derived from primary mBMMs obtained from WT, Dcstamp tail^−/− and WT mice treated with E8431. b Volcano plots illustrating the DEGs between the Dcstamp tail^−/− and WT groups, as well as the WT + E8431 and WT groups. Each group consisted of N = 3 samples. Key osteoclast marker genes, such as Ctsk, Dcstamp, Car2, Mmp9, Acp5, Atp6v1b2, Oscar, Mmp14, Ocstamp and Nfatc1, are highlighted. c, d Venn diagram representations delineating the shared downregulated genes (c) and upregulated genes (d) in the Dcstamp tail^−/− and WT + E8431 groups compared with the WT group. e The GO analysis performed on the commonly downregulated genes in the Dcstamp tail^−/− and WT + E8431 groups compared with the WT group. f A KEGG analysis conducted on the commonly downregulated genes in the Dcstamp tail^−/− and WT + E8431 groups compared with the WT group. g A GSEA examining the Rap1 signaling pathway in the comparison between the Dcstamp tail^−/− and WT groups, as well as the WT + E8431 and WT groups. h A western blot analysis measuring the protein levels of RAP1-GTP, RAP1, phosphorylated Syk (p-Syk), Syk, phosphorylated Src (p-Src), Src, RAC1-GTP, RAC1 and GAPDH. i The quantification of the western blot results, with N = 4 samples analyzed. j The TRAP staining mature osteoclasts differentiated from primary mBMMs of different groups including WT, Dcstamp-tail^−/−, Dcstamp-tail^−/−+ 8-CPT-2′-O-Me-cAMP, WT + E8431 and WT + E8431 + 8-CPT-2′-O-Me-cAMP. k The visualization of F-actin cytoskeleton via the FITC-phalloidin staining of different groups. l The quantification of osteoclast numbers and average size per well from TRAP staining of in vitro differentiated osteoclasts. N = 6 per group. m The quantification of F-actin rings from FITC-phalloidin staining. N = 6 per group. n The transcriptional expression levels of osteoclastic genes including Dcstamp and Ctsk. N = 4 per group. The data are presented as mean ± s.d. with individual data points depicted as dots. Statistical significance levels are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.