Fig. 7

MS-BMDM‑derived exosomal UCHL3 promotes BMSCs osteogenesis by targeting SMAD1. a Western blotting of SMAD1 and UCHL3 protein levels in BMSCs after treatment with the UCHL3 inhibitor TCID or MS-BMDM_siUchl3-EXOs. b, c Relative intensity analysis of a (n = 3). d, e qRT‒PCR of the mRNA expression of osteogenesis genes and Western blotting of RUNX2, UCHL3 and SMAD1 protein levels in BMSCs after overexpression of Smad1 with lentiviruses and treatment with MS-BMDM_siUchl3-EXOs (n = 3). f Relative intensity analysis of e (n = 3). g, h ALP staining and Alizarin red staining in BMSCs after overexpression of Smad1 with lentiviruses and treatment with MS-BMDM_siUchl3-EXOs. Scale bar: 100 μm. i 3D modelling of the interaction between UCHL3 (blue) and SMAD1 (grey) proteins predicted by protein‒protein docking from Alphafold 2. The stick structure represents amino acid residues, and the yellow dashed lines represent hydrogen bonds. j Immunofluorescence images of the colocalization of SMAD1 and UCHL3 in BMSCs (green UCHL3, red SMAD1, blue DAPI). Scale bar: 10 μm. k Endogenous UCHL3 and SMAD1 proteins interacted in BMSCs. UCHL3 protein was immunoprecipitated with anti-UCHL3 antibody. IgG served as a negative control, and endogenous SMAD1 was measured by Western blotting. l Endogenous UCHL3 and SMAD1 proteins interacted in BMSCs. SMAD1 protein was immunoprecipitated with anti-SMAD1 antibody. IgG served as a negative control, and endogenous UCHL3 was measured by Western blotting. m Western blotting of the polyubiquitinated level of SMAD1 in BMSCs after treatment with TCID. SMAD1 protein was immunoprecipitated, and polyubiquitinated SMAD1 protein was measured by Western blotting using anti-Ub antibody. Data are shown as the mean ± SD. Two-tailed unpaired Student’s t test or one-way ANOVA followed by Tukey’s post hoc multiple comparisons were performed. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant