Figure 2

Fenofibrate inhibits IGF-induced LN-229 cellular responses. Panels A: Western blot analyses showing phosphorylated (active) forms of IRS-1 (pY612), Akt (pS473), ERKs (pT202/pY204) and GSK-3β (pS21/9) in LN-229 human Glioma cells following 48 hrs serum starvation (SFM); IGF-I stimulation (50 ng/ml) for 30 min (IGF), and when IGF-I stimulation was preceded by 24 hrs cell pre-incubation with 50 μM fenofibrate (IGF + FF). Antibodies against non-phosphorylated forms of IRS-1, Akt, ERKs, as well as anti-Grb-2 antibody, were used to demonstrate equal protein loading. Panel B: Upper image: Trajectories of 50 migrating LN-229 cells in SFM, and following IGF-I stimulation in the absence (SFM + IGF) and in the presence (SFM + IGF + FF) of 50 μM fenofibrate. Data are displayed in circular diagrams drawn in the initial point of each trajectory placed at the origin of the plot. The positions of the "cell centroid" from the consecutive frames were used for generation of cell tracks. Quantification of multiple cell motility parameters is given in Table 1. Lower image: Live cell time-lapse imaging of LN-229 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. Panel C: Upper image: Trajectories of 50 migrating LN-229 and T98G cells the presence of 10% FBS (FBS); 10% FBS + 50 μM fenofibrate (FBS + FF); and 10%FBS + 1 μM IGF-IR inhibitor, NVP-AEW541, (FBS + NVP). Quantification of the cell migration parameters are presented in Table 1. Lower image: Cell migration into the scratched cell-free areas were evaluated at time zero (T0, just after the scratch) and after 10 hrs of continuous cell migration into the cell-free area in the presence of 10%FBS (FBS); 10% FBS + 50 μM fenofibrate (FBS + FF); and 10%FBS + 1 μM IGF-IR inhibitor NVP- AEW541 (NVP). The numbers below phase-contrast images of the scratched monolayer indicate average percentage of the scratched area re-occupied by the migrating cells for 10 hrs (+/-SD, n = 3).