Figure 5.

MKRN1 reduces PPARγ protein levels and inhibits adipocyte differentiation in 3T3-L1 cells. (a) Effects of MKRN1 overexpression on 3T3-L1 adipocyte differentiation. Stable 3T3-L1 cell lines overexpressing MKRN1 (pBP-MKRN1) or its H307E mutant (pBP-MKRN1 H307E) using retroviral vectors were treated with DMI for 6 days and their adipocyte differentiation was detected using Oil-Red-O staining. 3T3-L1 cells retrovirally transduced with pBabe-puro empty vector (pBP) were used as control cells. (b) Effects of MKRN1 overexpression on the levels of PPARγ and its target proteins. The WCE described above were analyzed using antibodies against the proteins indicated. (c) Levels of adipocyte differentiation markers in 3T3-L1 cells expressing MKRN1. mRNA levels of cells indicated above were measured by qRT-PCR analysis using primers targeted for MKRN1, PPARγ, aP2 and CD36 mRNAs. Data are presented as mean±S.D.; n=3 with **P<0.01 and ***P<0.001 compared with each lane. (d) Effects of MKRN1 depletion on adipogenesis of 3T3-L1. Stable cell lines constitutively depleted of MKNR1 were constructed using a lentivirus expressing shRNA for GFP or mouse MKRN1 (shGFP, shMKRN1 #1, shMKRN1 #3, respectively). The adipogeneses of these three cell lines were induced using DI (dexamethasone+insulin) for 6 days. Cells were stained and detected as described above. (e) MKRN1 knockdown increases the levels of adipogenic proteins. WCE from above were analyzed as above. (f) Effects of MKRN1 depletion on mRNA levels of the adipogenic markers. The mRNA of adipogenic markers were analyzed as described above. Data are presented mean±S.D.; n=3 with *P<0.05, **P<0.01 and ***P<0.001 compared with each lane