Figure 3.

BMSC differentiation assays. BMSCs were obtained from the tibias and femurs of HFD as well as NC mice. (A and B) BMSCs were cultured in basal culture medium, which was then replaced with an osteogenic-inducing culture medium. On Day 21, the cultures were stained with alizarin red (A), and the number of positive colonies (CFU-osteo) was counted (B). The number of CFU-osteo colonies formed from the BMSCs of HFD mice was significantly higher than that formed from the control BMSCs (n=3, **P<0.01). (C and D) mRNA levels of adipogenic and osteogenic genes in BMSCs from NC and HFD mice. BMSCs were harvested after a 8-day culture, then RNA was extracted and gene expression was determined by real-time PCR. mRNA transcripts of PPARγ and Pref-1 (C) were significantly lower in BMSCs from HFD compared to NC mice. The expression level of Runx2 (D) was significantly higher. The relative expression level of each gene was normalized to β-actin. Data the means of three independent experiments conducted in triplicate (*P<0.05; **P<0.01). (E) Protein levels of PPARγ in BMSCs from NC and HFD mice. BMSCs were harvested after an 8-day culture. Total proteins were collected and PPARγ was detected by Western blotting. GAPDH was used as a loading control. PPARγ production in BMSCs from NC mice was higher than production in BMSCs from HFD mice.