Fig. 4

FXR stabilizes RUNX2 by inhibiting ubiquitination. a Western blot analyses of FXR, RUNX2, Sp7 in BMSCs after 4 days of osteoblastic differentiation transfection with or without FXR shRNA. b-f Quantitative RT-PCR analysis of RUNX2, Sp7, Alp, Col1a, FXR expression in BMSCs after 4 days of osteoblastic differentiation transfection with or without FXR shRNA (n = 3). g, h 6XRUNX2 and OSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2 cDNA or FXR shRNA (n = 3). i, j Gene Ontology (GO) enrichment analysis of differentially expressed genes in (i) Biological Process and (j) Molecular Function. k Western blot analyses of RUNX2 expression in C3H10T1/2 cells, which were transfected with or without FXR shRNA for 48 h, and were treated with cycloheximide (CHX) for the indicated time. l Quantitative analysis of RUNX2 protein level (n = 3). m, n Western blot analyses of RUNX2 expression in C3H10T1/2 cells, which were treated with cycloheximide (CHX) for the indicated time in the transfection of FXR shRNA, and were treated with MG132 (10 μmol/L) for 6 h. n Quantitative analysis of RUNX2 protein level (n = 3). o The embryonic forelimb morphogenesis pathway related genes were obtained via Gene Set Enrichment Analysis (GSEA). p After C3H10T1/2 cells were transfected with Vector or FXR-FLAG for 48 h, lysed, immunoprecipitated with anti-RUNX2 conjugated protein A/G magnetic beads, and immunoblotted with anti-ubiquitin antibody. q After C3H10T1/2 cells were transfected with control shRNA or FXR shRNA for 48 h, lysed, immunoprecipitated with anti-RUNX2 conjugated protein A/G magnetic beads, and immunoblotted with anti-ubiquitin antibody. *P < 0.05, **P < 0.01, ***P < 0.001, ns no significant difference. Data are represented as mean ± SEM