Figure 5

Effects of PPARα siRNA on fenofibrate-mediated ROS accumulation and cell motility. Panel A: Western blot analysis showing PPARα protein levels in LN-229 cells incubated with 200 μM of irrelevant siRNA against nuclear lamins (NL: 200 μM), and with 100 and 200 μM of ON-TARGRT plus SMARTpool siRNA against human PPARα (Thermo Scientific). The histogram below indicates densitometric analysis of the blot analyzed by EZQuant-Gel 2.17 (EZQuant Biology Software Solutions, Tel Aviv, Israel). Panel B: ROS accumulation evaluated in exponentially growing LN229 cells (FBS). The cells were treated with 50 μM fenofibrate (FF), in the presence or absence of 200 μM PPARα siRNA (siPPARα). The cells were loaded with Redox Sensor Red CC-1, and MitoTracker Green FM as previously described [41]. The quantification of intracellular ROS (voxels per cell) is illustrated below. 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: LN-229 cell migration evaluated in Transwell Chambers. Experimental conditions are similar to those described in the legend to Fig. 4D. Exponentially growing LN-229 cells (in 10% FBS) were treated with 50 μM fenofibrate (FF) in the presence or in the absence of 200 μM siRNA against PPARα. After 48 hrs the cells, which did not migrate through the pores, were removed and cells on the bottom surface of the filters were fixed, stained and counted. Data are presented as mean ± SD from three independent experiments in duplicates (n = 3). Statistical significance was tested between FBS (control) and FF (*), and between FF and FF + PPAR siRNA (**); p≤0.05.