Figure 5.

MLN4924 blocks adipocyte differentiation by destabilizing PPARγ. (a) MLN4924 downregulates PPARγ. 3T3-L1 cells or human adipose tissue-derived stem cells were treated with 0.1 or 0.5 μM MLN4924 for 2 days, and then stimulated with DMI for 8 days. Cell lysates were subjected to western blotting. (b) MLN4924 destabilizes PPARγ protein. 3T3-L1 cells were treated by DMI to stimulate differentiation and treated with MLN4924 for 24 h, and then cells were further treated with cycloheximide for the indicated times. PPARγ protein levels were determined by immunoblot analysis (upper). Band intensities were quantified using ImageJ. Half-lives (t_1/2) were calculated from the slopes of first-order decay curves (lower). (c) MLN4924 blocks adipogenesis. 3T3-L1 cells and four human adipose tissue-derived stem cells were treated with 0.1 or 0.5 μM MLN4924 for 2 days, and then stimulated with DMI for 8 days. Cells were subjected to Oil Red O staining. (d) MLN4924 inhibits the expression of adipogenic genes. 3T3-L1 cells were differentiated by DMI with vehicle or 0.5 μM MLN4924 for the indicated time, and then RT-qPCRs were performed to evaluate the expression of Pparg, Cebpa, Cebpb, Cebpd, Fabp4, Cd36, adiponectin, resistin, Fasn, and Scd1 mRNAs. The mRNA levels (mean+S.D., n=3) are shown as relative values to 18S RNA levels. *P<0.05