Fig 5. Δ4-DA restores the activity of charcoal stripped mouse sera on DimDAF-12.

(A) Dose-response curves of Δ4-DA (25R) on the activation of Bma and DimDAF-12. NIH3T3 cells were co-transfected with Gal4-DAF-12_LBD from Bma or Dim and the luciferase gene reporter construct. Recombinant cells were incubated with increasing concentrations of Δ4-DA (25R). Data representing normalized luciferase activities are plotted with nonlinear regression fit using sigmoidal dose-response with variable slope (Prism 6.0, Graph Pad Software, Inc.). The figure shows one representative experiment out of two. Values are means of technical triplicates ± standard deviation. (B) NIH3T3 cells were co-transfected with Gal4-DimDAF-12_LBD and luciferase gene reporter construct and then incubated with regular or charcoal-stripped mouse serum for 24 hours. Charcoal stripped mouse serum has been supplemented or not with the indicated DAF-12 ligands at 10% of the average concentration found in mouse serum and reported in Table 3 (0.75nM for cholestenoic acid (Chol. ac.) and 0.38nM for Δ4-DA). DAF-12 activity was normalized to the empty vector-transfected cells and to the charcoal stripped serum conditions and is expressed as relative light units (RLU). Data represent the average of normalized luciferase activity and the error bars correspond to the standard deviation of three independent experiments. The significance of the effects was analyzed by unpaired Student’s t-tests using Prism 6.0 (Graph Pad Software, Inc.). ***p < 0.001.