Figure 5.

Production of LPA and ATX by MDA-BO2 breast cancer cells. Cell culture media were collected for each indicated cell line placed in the absence or presence of doxycycline (100 ng/ml). (A) Detection of LPA. The production of phosphatidic acid (PA) was due to the transfer of [¹⁴C] fatty acyl chain (FA) onto LPA present in the reaction mixture assay. Experiments were carried out in duplicate for each cell line. Purified LPA (50, 100, and 200 pmol) was used as a positive control. DMEM (Cont.) was used as a negative control. (B) Measurement of ATX/lyso-PLD activity. [¹⁴C]-LPC was used as the substrate of ATX/lyso-PLD to produce [¹⁴C]-LPA. Experiments were carried out in duplicate using culture media from the cell lines described above. Note that MDA-MB-231, MDA-BO2, clone no. 3, and clone no. 79 cell lines did not produce LPA or express ATX.