E6AP negatively regulates Adipogenesis.

<p>3T3L1 cells were transfected with E6AP (2.0 µg) and E6AP-C843A (2.0 µg). Post 48 h of transfection, cells were treated with MDI for next 8 days followed by Oil red O staining.</p

E6AP downregulates expression of proadipogenic factor C/EBPα.

<p>(<b>a,b</b>) 3T3-L1 preadipocytes were transfected with increasing amounts of E6AP (0.5 µg–2.0 µg) and E6AP-C843A (0.5 µg–2.0 µg). Post 48 h transfection, WCEs were prepared and resolved on 10% SDS-PAGE followed by immunoblotting with C/EBPα, E6AP and β actin antibodies; lysates of 293T alone and transfected with C/EBPα were used as positive and negative control, note that there is no endogenous expression of C/EBPα in 293T. <b>E6AP promotes C/EBPα degradation through ubiquitin-proteasome pathway:</b> (<b>c</b>) 3T3-L1 preadipocytes pre-treated with MDI for 48 h were transiently transfected with expression plasmids for E6AP, E6AP-C843A and His-ubiquitin. Post 48 h Transfection, WCEs were prepared and C/EBPα was co-immunoprecipitated. IgG was used as a control. Co-immunoprecipitates were resolved on 8% SDS-PAGE and blots were probed with anti-His and anti-C/EBPα antibody respectively. <b>E6AP inhibits C/EBPα transactivation potential to activate PPRE-Luc:</b> (<b>d</b>) E6AP mediated downregulation of C/EBPα curtails its transactivation potential: 3T3-L1 preadipocytess were transiently transfected with pPPRE-luc reporter and expression plasmids for C/EBPα, E6AP and E6AP-C843A. 24 h post transfection, luciferase activity was measured. MG132 and lactacytin (LCN) treatment was given 3 h prior to cell harvesting for luciferase activity measurement. Data are representative of three independent experiments. Results are given as standard error of mean (± s.e.m.); *p<0.05; **p<0.001, ***<0.0001.</p

E6AP over expression inhibits while E6AP-C843A promotes adipocyte differentiation of MDI treated mesenchymal stem cells isolated from murine bone marrow.

<p>Mesenchymal progenitor cells were transfected with E6AP (2.0 µg) and E6AP-C843A (2.0 µg). Post 48 h of transfection, cells were grown in presence or absence of MDII for next 8 days followed by Oil red O staining.</p

List of Primers.

<p>List of Primers.</p

siE6AP mediated induction of adipogenesis is marked by increase in expression of proadipogenic factors.

<p>(<b>a</b>) 3T3L1 cells were transfected with siE6AP or scrambled siRNA. Post 48 h transfection, cells were treated with MDI for until ten days; subsequently mRNA expression levels of indicated adipogenic genes was determined with qRT-PCR. <b>Over expression of wild type E6AP down regulates while catalytically inactive E6AP-C843A up regulates proadipogneic factors:</b> (<b>b</b>) 3T3L1 cells were transfected with E6AP and E6AP-C843A. After transfection cells were treated with MDI for 10 days and mRNA expression level of various adipogenic genes was determined with real time PCR. The primers used for C/EBPα, PPARγ, Leptin, SREBP1c and Adipsin listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065330#pone-0065330-t001" target="_blank">table 1</a>.</p

E6AP knock down by siE6AP promotes adipogenesis.

<p>(<b>a</b>) 3T3L1 preadipocytes pre-treated with MDI were transfected with siE6AP or scrambled siRNA, 48 h post transfection, cells were harvested, resolved on 10% SDS-PAGE and probed C/EBPα, E6AP and β-actin antibody (lysates of 293T transfected with C/EBPα were used as control) (<b>b</b>) 3T3L1 preadipocytes were transfected with siE6AP or scrambled siRNA. Cells were grown in the presence or absence of MDI for 10 days followed by Oil red O staining.</p