Figure 4
Effects of fenofibrate and NAC on mitochondrial function. Panels A and B: Mitochondrial potential was evaluated in LN-229 and T98G cells, respectively, by utilizing flowcytometry based MitoPotential Kit according to manufacturer's protocol (Guava EasyCyte). Loss of mitochondrial inner transmembrane potential (ΔΨm) was evaluated by a cationic dye JC-1 that gives either green or orange fluorescence depending upon mitochondrial membrane depolarization. The cells growing in 10% FBS were treated either with vehicle (DMSO) or with 50 μM fenofibrate in the absence (FBS + FF50) or in the presence of NAC (FBS + FF50 + NAC). Following 24 hrs incubation, the cells were loaded with JC-1 for 30 minutes and analyzed by Guava EastCyte flowcytometer using Mito-Potential software. Note that fenofibrate treatment increases percentage of cells with compromised mitochondrial potential. Quantification of the mitochondrial potential is shown in the last panel. Data represent average percentage of cells showing polarized or depolarized mitochondria +/- SD, (n = 3). Panel C: ATP levels were evaluated by ApoSENSOR ADP/ATP Ratio Assay Kit (BioVision). The luminometric measurement was performed using EnVision multi-plate reader (PerkinElmer). Data are presented as mean ± SD calculated from two experiments in triplicates (n = 6). * indicates values statistically different from FBS. ** indicates values statistically different from FF, (p ≤ 0.05). Note a strong inhibition of ATP production following 48 hrs cell exposure to 50 μM fenofibrate (FF), which was effectively prevented by the ROS scavenger, NAC. Panels D: Effects of IGF-I, fenofibrate and NAC on LN-229 cell migration evaluated in Transwell Chambers. The cells were seeded at the density of 5 × 104/chamber in 200 μl of 10%FBS containing culture medium (control). The cells were additionally treated with 50 μM fenofibrate either in the absence (FF) or in the presence of NAC (FF + NAC). In addition, we have evaluated cell migration in serum-free medium (SFM) and in SFM supplemented with IGF-I (50 ng/ml). Data are presented as mean ± SD from three independent experiments in duplicates (n = 6). Statistical significance was tested between control and FF (*), between FF and FF + NAC (**), and between SFM and SFM + IGF (***); p≤0.05.