Fig. 3

TMCO1 regualtes RUNX2 stability via promoting RUNX2 acetylation. a The stability of RUNX2 in TMCO1-deficient MC3T3-E1 cells was measured by treating the cells with CHX for the indicated times, followed by western blot analysis. Relative amounts of RUNX2 were calculated and shown in the graphs. Representative results of three independent experiments are shown. b Deficiency of TMCO1 mediates the proteasome-dependent degradation of RUNX2. Control-siRNA and TMCO1-siRNA transfected MC3T3-E1 cells were treated with 20 μM MG132 for 6 h. Protein levels of RUNX2 were measured by western blotting. Representative results of three independent experiments are shown. c Effect of TMCO1 on the ubiquitination of endogenous RUNX2. Control-siRNA and TMCO1-siRNA were transfected into MC3T3-E1 cells for 48 h. The cells were treated with 20 μM MG132 for 6 h, and RUNX2 ubiquitination was analyzed by western blotting using an anti-RUNX2 antibody. Experiments were successfully repeated three times. d Western blot analysis of the E3 ubiquitin ligase for RUNX2 in TMCO1-siRNA-transfected MC3T3-E1 cells. Representative results of three independent experiments are shown. e The E3 ubiquitin ligase Smurf1 is responsible for RUNX2 degradation caused by the loss of TMCO1. Control and TMCO1-deficient MC3T3-E1 cells were transfected with Wwp1 or Smurf1-siRNA for 24 h. Western blotting was used to analyze RUNX2 expression in cell lysates. Representative results of three independent experiments are shown. f Knockdown of TMCO1 using siRNA suppresses RUNX2 acetylation. MC3T3-E1 cells were transfected with control-siRNA and TMCO1-siRNA. The acetylation of endogenous RUNX2 was determined by IP using an anti-Ac-K antibody, followed by immunoblotting with an anti-RUNX2 antibody. The levels of endogenous RUNX2, TMCO1, and GAPDH were determined by western blotting. Experiments were successfully repeated two times. g Representative images of ALP staining in Tmco1^−/− osteoblasts transfected with RUNX2-4KR or not. Scale bars, 6 mm. Western blot analysis of RUNX2-4KR overexpression in Tmco1^−/− primary osteoblasts. h Effect of RUNX2-4KR overexpression on osteoblast-specific genes caused by TMCO1 deficiency. WT and Tmco1^−/− primary osteoblasts were transfected with RUNX2-4KR. Representative results of three independent experiments are shown. Data are presented as the mean ± s.e.m. unpaired Student’s t test, n = 3, **P < 0.01 and ***P < 0.001