Fig. 7.

Intracellular ROS production inhibits migration by sustained FAK phosphorylation. a Atgl−/− macrophages were pretreated with N-acetyl-cystein (NAC) (20 mM) and diphenyleneidonium (DPI) (2 μM) for 30 min. Intracellular ROS production was determined by hydrolysis of DCFDA (50 μM) to fluorescent DCF in a FlexStation. Data represent mean values (n = 5) ±SEM of two independent experiments measured in quadruplicate repeats. b Atgl−/− macrophages were pretreated with NAC (20 mM) and DPI (2 μM) for 30 min and then stimulated with LPA for 5 and 10 min. Cells were lysed and subjected to Western blotting to detect total and phosphorylated FAK. Representative blots from two independent experiments are shown. Band intensities were quantified using Image J software and ratios of activated to total protein expression were determined. Fold changes were calculated relative to the ratios in unstimulated cells. c Atgl−/− macrophages were pretreated with NAC (20 mM) and DPI (2 μM) for 30 min, and then chemotaxis toward LPA (9 ng/ml) was determined. Data represent mean values (n = 4) ±SEM of two independent experiments performed in duplicate repeats. ***p ≤ 0.001; ^## p ≤ 0.01