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Fig. 3. Silencing of CREB by using specific siRNA prevents triglyceride accumulation in antimycin-A-treated 3T3-L1 cells. (A) Visualization of siRNA transfection efficiency in 3T3-L1 preadipocytes. Cells were transfected for 4 hours with fluorescein-labeled siRNA (20 nM) and processed for confocal microscopy after (a) 24 hours or (b) 48 hours. (B) Effect of a CREB-specific siRNA on CREB protein expression. 3T3-L1 preadipocytes were transfected for 4 hours with different concentrations of siRNA or incubated for 4 hours with the transfection reagent (JetSI). CREB abundance was determined by western blotting of 40 µg of clear cell lysate proteins prepared 48 hours after transfection. TBP served as a loading control. (C) Effect of a CREB-specific siRNA on AA-induced triglyceride accumulation. Cells were transfected for 4 hours with 100 nM siRNA and then incubated for 8 days without (controls, CTL) or with 10 nM AA. The absorbance of cell monolayers was spectrophotometrically determined at 490 nm after Oil Red O staining. Results are expressed in optical density (O.D.) as the mean ± s.d. for n=3 or the mean of n=2 experiments. *P<0.05, significantly different from control cells. (D) Photomicrographs of 3T3-L1 cells transfected or not for 4 hours with 100 nM siRNA and incubated or not (CTL) for 8 days with 10 nM AA before Oil Red O staining. Magnification, 75x.
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