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. 2016 Jun 29;10:174–184. doi: 10.1016/j.ebiom.2016.06.040

Fig. 1.

Fig. 1

Effect of pS112 and pS273 of PPARγ on adipocyte, osteoblast and osteoclast differentiation.

(a) The effect of S112A and S273A mutations on osteoblast differentiation. U-33 cells were transfected with either WT or mutated PPARγ expression constructs and grown in pro-osteoblastic conditions followed by measurement of extracellular calcium or RNA isolation. (b) Effect of PPARγ S112A and S273A mutations on osteoclast differentiation. RAW264.7 was transfected with either WT or mutated PPARγ expression constructs followed by culture in medium supplemented with 50 ng/ml M-CSF and 50 ng/ml RANKL for 4 days, then either stained for TRAP expression (left panel) or RNA was isolated for analysis of gene expression (right panel). (c) Chemical structure of SR10171 ((S)-2-(3-((5-((1-(4-(tert-butyl)phenyl)ethyl)carbamoyl)-2,3-dimethyl-1H-indol-1-yl)methyl)phenoxy)-2-methylpropanoic acid) and rosiglitazone ((RS)-5-[4-(2-[methyl(pyridin-2-yl)amino]ethoxy)benzyl]thiazolidine-2,4-dione). (d) The effect of SR10171 and rosiglitazone on blocking of S273 phosphorylation in AD2 cells. (e) The effect of ligands on blocking of S112 phosphorylation in AD2 cells. Protein levels quantification was performed using Image J software and displayed as graphs below Western blot renderings. (f)–(j) cell treatment consisted of 10 μM SR10171 (71), or 1 μM rosiglitazone (R), or DMSO as vehicle control (V). (f) Left and middle panels — the effect on adipocyte-specific gene marker expression in marrow MSCs treated for 6 days, as above. Right panel — the effect on transcriptional activity of PPARγ measured in U33/γ2 cells transfected with PPRE promoter construct and treated for 24 h. (g) Osteoblast differentiation as measured in CFU-OB assay. (h) Expression of osteoblast-specific gene markers in marrow MSCs. (i) The effect on osteoclast differentiation. Non-adherant bone marrow cells were cultured as described in experimental procedures. TRAP + multinucleated cells (> 4 nuclei) were enumerated and average calculated from triplicates. (j) Expression of osteoclasts gene markers in cultures described in (i). Data represent the mean ± SD (n = 3–6 experiments). *P < 0.05; **P < 0.01; ***P < 0.001 One Way Anova.