Fig. 5
Thoc6 inhibits osteoblast differentiation by interacting with RUNX2. a Schematic graph of the strategy to identify RUNX2 interacting protein which were regulated by FXR. b Venn diagram representing the intersection gene between Predicted E3 ligases, RNA-seq up-regulated E3 ligases and IP-MS up-regulated proteins. c Western blot analyses of RUNX2 expression in C3H10T1/2 cells, which were transfected with control siRNA or Thoc6 siRNA for 48 h. d Western blot analyses of RUNX2 expression in C3H10T1/2 cells which were transfected with Vector or Thoc6 cDNA for 48 h. e, f 6XRUNX2 and OSE2-luciferase activity was determined in C3H10T1/2 cells transfected with RUNX2 or Thoc6 cDNA (n = 3). g Representative images of Alp staining and Alizarin red staining of BMSCs which were infected with Thoc6 lentivirus. Scale bar = 50 μm. h, i BMSCs were lysed after 4 days of osteoblastic differentiation, immunoprecipitated with anti-IgG control, anti-Thoc6 (h) or anti-RUNX2 (i) antibody conjugated protein A/G magnetic beads, and immunoblotted with the indicated antibodies. WCL means whole cell lysate. j Representative immunofluorescent staining of RUNX2 and Thoc6, which was used to evaluate the subcellular location in BMSCs. Scale bar = 5 μm. k Optimized binding modes of RUNX2 and Thoc6 with the lowest binding energy generated by the HDOCK protein-docking algorithm. l Schematic diagram of the domain structure of Runx2 and its N-terminal deletion mutants. m Interaction between THOC6 and the N-terminal amino acid deletion of RUNX2 in C3H10T1/2 cells. Whole cell extracts from C3H10T1/2 cells transfected with expression vectors for Thoc6 and WT FLAG-Runx2 or various FLAG-Runx2 N-terminal deletion mutants (FLAG-ΔAD, FLAG-ΔRUNT, and FLAG-ΔNLS) were immunoprecipitated FLAG or Thoc6 antibody, followed by Western blot using Thoc6 or FLAG antibody. *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± SEM
