Fig. 6
Activation of FXR suppresses Thoc6 expression. a, b Western blot and Quantitative RT-PCR analysis of Thoc6 expression in osteoblast cells from FXRfl/fl and FXROCN mice after 4 days of osteoblastic differentiation (n = 4). c Immunofluorescence assay for Thoc6 expression in osteoblast (OCN-a marker of osteoblasts) of FXRfl/fl and FXROCN mice (n = 3). Scale bars, 50 μm. BM means bone marrow. TB means trabecular bone. d Immunofluorescence assay for expression of RUNX2 and Thoc6 in osteoblast transfection with FXR cDNA or FXR shRNA. Scale bar = 10 μm. e After C3H10T1/2 cells were transfected with Vector orThoc6-myc for 48 h, lysed, immunoprecipitated with anti-RUNX2 conjugated protein A/G magnetic beads, and immunoblotted with anti-ubiquitin antibody. f After C3H10T1/2 cells were transfected with control siRNA or Thoc6 siRNA for 48 h, lysed, immunoprecipitated with anti-RUNX2 conjugated protein A/G magnetic beads, and immunoblotted with anti-ubiquitin antibody. g A schematic representation of the Thoc6 promoter region showing potential FXR (NR1H4) binding sites. h, i C3H10T1/2 cells were co-transfected with one of these designated constructs (constructs 2 066, construct 574, 2 066 Mut with mutated one FXR binding sites) and with FXR cDNA construct for 48 h, and then luciferase activity was measured and normalized (n = 3). j Schematic diagram depicting ChIP assays. k C3H10T1/2 cells were transfected with FLAG-tagged FXR cDNA for 72 h. ChIP assays were performed using anti-FLAG antibody. The Standard PCR products were run and scanned (left panel). The histogram was presented as quantification of the PCR results (right panel) (n = 3). **P < 0.01, ***P < 0.001, ns no significant difference. Data are represented as mean ± SEM
