Figure 6.

MLN4924 blocks lipid storage in adipocytes. (a) MLN4924 reduces lipid accumulation in mid-differentiated adipocytes. After 3T3-L1 and H-ADSC cells were stimulated with DMI for 5 days, mid-differentiated cells were treated with MLN4924 and further differentiated until day 21 (3T3-L1 cells) or day 30 (H-ADSCs). On the last day, cell morphology was analyzed by phase contrast microscopy, and cells were then subjected to immunofluorescence staining with anti-perilipin antibody. Nuclei were stained with DAPI. (b) Cell lysates from 3T3-L1 and H-ADSCs were subjected to western blotting using anti-perilipin antibody. (c) MLN4924 reduces the expressions of lipid-storing genes. 3T3-L1 and H-ADSC cells were stimulated with DMI, and then MLN4924 (0.5 μM) were administered into culture media from differentiation day 3 (d3) or day 6 (d6). After differentiation of 20-days, RNAs were prepared from the adipocytes, and the expressions of Perilipin, Acs, Cd36, Lpl, Scd1 and Fabp4 were quantified by RT-qPCR. The mRNA levels (mean+S.D., n=3) are presented as relative values to 18S RNA levels. *P<0.05. (d) MLN4924 reduces lipid accumulation in differentiated adipocytes. After 3T3-L1 cells were differentiated by DMI treatment for 8 days, the adipocytes were treated with 0.1 or 0.5 μM MLN4924 for 8 more days. On the last day, cells were subjected to immunofluorescence analysis using anti-perilipin antibody. (e) RNAs were prepared from the adipocytes, and the cellular levels of Perilipin, Lpl, Acs, Scd1, Cd36 and Fabp4 mRNAs were quantified by RT-qPCR. The mRNA levels (mean+S.D., n=3) are presented as relative values to 18S RNA levels. *P<0.05