Figure 3

Effects of fenofibrate on intracellular ROS accumulation . Panel A: Exponentially growing LN229 and T98G cells (in 10%FBS) were treated with 50 μM fenofibrate (FF), in the presence or absence of ROS scavenger, N-acetyl-cysteine (NAC). The cells were loaded with Redox Sensor Red CC-1, and MitoTracker Green FM as previously described [41]. A series of three-dimensional images of each individual picture were deconvoluted to one two-dimensional picture and resolved by adjusting the signal cut-off to near maximal intensity to increase resolution. Note strong increase in cytosolic (red fluorescence) and mitochondria associated (yellow fluorescence) ROS accumulation following fenofibrate treatment (FF), which was effectively prevented by NAC (FF + NAC). Panel B: The quantification of intracellular ROS (voxels per cell) in LN-229 and T98G glioma cell lines, respectively. The results were collected from the entire volume of the cell and calculated 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 cell +/- SD, (n = 3). * indicates value significantly different from FBS; ** indicates value significantly different from FF (p ≤ 0.05). Panel C: Cell motility evaluated by cell displacement (upper image) and scratch assay (lower image). Upper image: Trajectories of 50 migrating LN-229 and T98G cells in 10%FBS supplemented with 50 μM fenofibrate in the absence (FBS + FF) and in the presence of NAC (FBS + FF + NAC). Quantification of multiple cell motility parameters is given in Table 1. Lower image: Live cell time-lapse imaging of LN-229 and T98G cells at 10 hrs after scratching the monolayer culture with the pipette tip. The numbers below phase-contrast images indicate % decrease of the scratched areas (+/- SD, n = 3), calculated from the cell-free area measured at time zero and following 10 hrs of continuous cell migration.