Figure 1
From: WDR76 mediates obesity and hepatic steatosis via HRas destabilization
Effects of WDR76 on adipogenesis in 3T3-L1 cells. (a–d) 3T3-L1 cells infected with shCon or shWDR76 lentivirus were grown under MDI-induced differentiation condition. (e–h) The 3T3-L1 cells infected with control or WDR76 lentivirus were grown under MDI-induced differentiation conditions. (a,e) Immunoblot (IB) analyses were performed to detect WDR76, PPARγ, C/EBPα, HRas, pERKs, ERKs, and β-actin. (b,f) Quantification of IB analyses as determined by Image J. (c,g) The mRNA levels of WDR76, PPARγ, C/EBPα, and HRas were measured by qRT-PCR using β-actin as an internal control. (d,h) Lipid droplets were stained using Oil Red O staining (ORO). Scale bars, 100 µm. Right panels show graphs presenting the relative area of ORO staining as determined by Image J. (i) The levels of HRas at indicated time points after CHX treatment were determined by IB, and were quantified with β-actin as a loading control. Results plotted on the lower are the amounts of HRas at each time point relative to the level at time 0. (j) The 3T3-L1 cells were infected with control or WDR76 lentivirus and then treated with N-acetyl-leucyl-leucyl-norleucinal (ALLN; 25 µg/mL) where indicated. (k) The 3T3-L1 cells were transfected with pLVX-IRES-Hyg-Myc-H-Ras, pCS4-3xFlag-Ub, and/or pLVX-IRES-Hyg-WDR76 and then treated with ALLN for 12 h. Whole-cell lysates (WCLs) were then immunoprecipitated with an anti-Myc antibody. The ubiquitin-conjugated proteins were detected by IB analyses. Lower panels show graphs for quantification of IB analyses. (l,m) The 3T3-L1 cells were infected with control or WDR76 with or without HRas lentivirus and grown under MDI-induced differentiation condition. WCLs were subjected to IB analyses using PPARγ, C/EBPα, WDR76, HRas, pERKs, ERKs or β-actin antibody. Lower panels show graphs for quantification of IB analyses (l). Lipid droplets were stained using ORO staining. Lower panels show graphs depicting the relative area of ORO staining as determined by Image J. Scale bars, 100 µm (m). All data are presented as the mean ± SD and representative results of at least three experiments are shown. *p < 0.05; **p < 0.01; ***p < 0.005, Student’s t-test.
