Journal: Journal of Lipid Research

Article Title: Sphingosine kinase is induced in mouse 3T3-L1 cells and promotes adipogenesis s⃞

doi: 10.1194/jlr.M800206-JLR200

Figure Lengend Snippet: Abundance of sphingosine kinase (SphK)-1/SphK-2 mRNA/protein and sphingosine 1-phosphate (S1P) content of 3T3-L1 cells during adipogenesis. Shown are the results of experiments in which mouse 3T3-L1 cells underwent a treatment protocol with a hormonal stimulation to induce adipogenesis. Cells were treated with the adipogenic stimuli for the times indicated in each panel as described in detail under Materials and Methods. They were then harvested and subjected to either RNA isolation followed by real-time quantitative RT-PCR (qRT-PCR) (A), protein isolation followed by immunoblot analyses (B), or lipid isolation followed by HPLC analyses (C). In panel A, reverse-transcribed template cDNA samples were subjected to qRT-PCR using the primers specific to mouse SphK-1, SphK-2, as well as 18S. Each data point represents the fold increase in transcript expression levels of SphK-1 (closed circles) and SphK-2 (opened circles) relative to those of 18S, normalized to the values obtained from the cells before hormonal stimulation. n = 5. Panel B shows representative images of immunoblots probed for SphK-1, SphK-2, and Rac1 proteins, derived from five independent experiments that yielded equivalent results. Panel C shows the results of S1P measurement using HPLC. Each data point represents mean ± SEM of pooled data derived from three to eight independent experiments. * P < 0.01, ** P < 0.001 vs. values obtained at the time point 0 in each panel.

Article Snippet: S1P, D- erythro - N,N ,-dimethylsphingosine (DMS) and DL- threo -dihydrosphingosine (DHS) were from BioMol (Plymouth Meeting, PA).

Techniques: Isolation, Quantitative RT-PCR, Western Blot, Reverse Transcription, Expressing, Derivative Assay