B16 cells were transduced with lentivirus encoding a shRNA to ATX (ShATX) or an Scr construct. mRNA level (A), ATX protein expression (B) and ATX catalytic activity in 48-h conditioned medium measured using FS-3 substrate (C) were all down-regulated in ShATX cells compared with Scr and wild-type (WT) B16-F10 cells. Scr cells showed an LPC dose-dependent increase in invasion of rat mesothelial monolayers, whereas ShATX cells failed to respond to LPC (D). Both cell types responded equally to LPA with increased invasion of the monolayer. When injected into C57BL/6 mice (5×104/cell per mouse in 100 μl) via the tail vein, ShATX cells yielded 50% fewer metastases compared with Scr and WT B16-F10 melanoma cells (E). n.s., not significant; *P < 0.05; **P < 0.01; P < 0.001. Reproduced from [43] Gupte, R., Patil, R., Liu, J., Wang, Y., Lee, S.C., Fujiwara, Y., Fells, J., Bolen, A.L., Emmons-Thompson, K., Yates, C.R., Siddam, A., Panupinthu, N., Pham, T.C., Baker, D.L., Parrill, A.L., Mills, G.B., Tigyi, G. and Miller, D.D. (2011) Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity. ChemMedChem 6, 922–935. Copyright Wiley-VCH Verlag GmbH & Co. KGaA with permission.