Up-regulation of ATX induced by TNF-α is associated with increased lysophospholipase D (lyso-PLD) activity by conversion of LPC into LPA in Hep3B and Huh7 cells. Serum starved Hep3B or Huh7 cells were treated with TNF-α (10 ng/ml) or vehicle (0.1%BSA/PBS) for 20 hours. A. Conditioned media (CM) or control media (DMEM or EMEM) were concentrated (40-fold) and assayed for ATX activity using the FS-3 compound. The results are shown as the average of relative fluorescence activity ± SD from three experiments. B. CM were incubated with 15 μM LPC (18:1) for 3 hours at 37°C. Lipids were analyzed by liquid chromatography-mass spectrometry (LC-MS). Results are level of LPA (18:1) from three experiments and presented as mean ± SD. *, P < 0.05.