Characterization of IGF-IR, IRS-1 and PPAR-α in human Glioma cell lines. Western blot analysis showing IGF-IR, IRS-1 (Panel A), and PPAR-α protein levels (Panel B) in exponentially growing five human Glioma cell lines in comparison to primary human fetal astrocytes, and R600 mouse embryo fibroblasts, which express 30,000 IGF-IR molecules/cell and high levels of IRS-1. Note that U87MG and U-118MG, which do not express PTEN , demonstrate very low levels of the IGF-IR and its major signaling molecule, IRS-1. In T98G, in which activity of PTEN is compromised by point mutation , IGF-IR is also very low; however IRS-1 is not affected. Equivalent loading was demonstrated by re-probing membranes with anti-Grb-2 antibody. Panel C: Quantification of PPARα nuclear localization (co-localization with DAPI) in exponentially growing LN-229 cells in the presence (FBS + FF) and absence (FBS) of fenofibrate treatment. The nuclear co-localization was calculated from the entire volume of the nucleus by utilizing Mask Operation included in SlideBook4 software, according to manufacturer instructions (Intelligent Imaging Innovations, Denver Co.). The data represent average number of voxels per nucleus +/- SD, (n = 25). * indicates values significantly different from FBS (p≤0.05). Images below the histogram represent examples of PPARα subcellular distribution. Panel D: PPARα transcriptional activity was evaluated in LN-229 cells by utilizing a dual-Firefly/Renilla luciferase reporter system and Femtomaster FB12 chemiluminometer. Data are presented as mean ± SD calculated from two experiments in triplicates (n = 6). * indicates value statistically significantly different (p≤0.05) from control (FBS; cells treated with vehicle only). Statistical significance between two measurements was determined with the two-tailed Student's t-test.