FIG 1.
The mitochondrial membrane fraction contains unprocessed FF. HPLC was used to detect FF and its PPARα-active metabolite FA in the mitochondrial membrane fraction (A to C) and the cytosolic fraction (D) isolated from the FF-treated human glioblastoma cell line LN-229. Under the HPLC conditions described in Materials and Methods, FF was eluted at 10.4 min and FA was eluted at 3.9 min. (A) Mitochondrial membranes isolated from LN-229 cells treated with 50 μM FF (FF50) for 24 h. (B) Mitochondrial membranes isolated from control DMSO-treated cells. (C) Mitochondrial membranes isolated from FF-treated cells additionally treated (spiked) with the FF standard prior to HPLC analysis. The insets in panels A and D represent the unique UV-Vis absorbance spectra of the peaks obtained corresponding to FF and FA, respectively (the three different traces in the insets demonstrate three different values for the diode array detector [DAD] to obtain FF and FA spectral analyses). (E) Quantitative analysis of FF and FA in mitochondrial membrane and cytosolic fractions isolated from LN-229 cells exposed to 50 μM FF for 24 h. Concentrations of FF and FA were calculated from the corresponding calibration curves and are presented in μM FF or amounts of FA per 1 × 106 cells. The data are average values and standard deviations from three separate measurements performed in triplicate (n = 9). (Inset) Western blot analysis demonstrating the purity of cytosolic (C) and mitochondrial membrane (M) fractions in which α-tubulin and Cox IV were use as cytosolic and mitochondrial markers, respectively. (F) Quantification of unprocessed FF in mitochondrial fractions isolated from LN-229 cells and NHA. The data are average values and standard deviations from two measurements performed in triplicate (n = 6). An asterisk indicates a statistically significant difference between LN-229 and NHA.