Implication of Ceramide Kinase in Adipogenesis

Figure 3

CERK downregulation leads to inhibition of leptin secretion and inhibition of PPAR-γ expression. 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. The cells 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. (a) Leptin concentration in supernatants was measured at day 4 after differentiation using an ELISA kit, as described in Materials and Methods. Results are the mean ± SEM of 4 independent experiments performed in duplicate (). (b) PPARγ expression was detected by Western blotting using a specific antibody. Equal loading of protein was assessed with an antibody against GAPDH. Similar results were obtained in each of 5 replicate experiments. (c) Results of scanning densitometry of the exposed film. Data are expressed as arbitrary units of intensity relative to GAPDH and are the mean ± SEM of 5 independent experiments ().

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