Research Article

Implication of Ceramide Kinase in Adipogenesis

Figure 2

CerK downregulation leads to decreased lipid droplet formation and depletion of TG content in 3T3-L1 cells. The preadipocytes were seeded in 100 mm diameter dishes at 5 × 105 cells/plate and incubated for 48 h as indicated in Section 2.2. They were then electroporated in the absence of siRNA (ctrl) or in the presence of negative (scrambled) siRNA (Neg siRNA) or Cerk siRNA, as indicated in Materials and Methods, and were then differentiated up to day 4. (a) CerK knockdown using specific siRNA was confirmed by immunoblotting. Equal loading of protein was assessed with an antibody against GAPDH. Similar results were obtained in each of 3 replicate experiments. (b) Results of the scanning densitometry of exposed film. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of 3 independent experiments (). (c) Light micrographs of adipocytes stained with Oil Red O at day 4 after differentiation are shown. Pictures were taken with a motorized Nikon Eclipse TS100 microscope at 20x magnification. (d) Cells were stained with Oil Red O, and lipid droplets were quantified at day 4 after differentiation as indicated in Materials and Methods. Data are expressed as the mean ± SEM of 5 independent experiments performed in triplicate (). (e) Triacylglycerol (TG) content of cells was measured at day 4 after differentiation as described in Materials and Methods. Results are the mean ± SEM of 3 independent experiments performed in triplicate ().
(a)
(b)
(c)
(d)
(e)