Figure 4

Inhibition of LPAR1 and 3 with Ki16425. A-C: Effect on cell migration. Cell migration was measured after scratch wounding in a confluent cell layer. A: Inhibitor, solvent (DMSO) and/or LPA were added to the wells with serum-free medium as indicated. Digital images were obtained immediately after stimulation and after 24 h (E10), 48 h (SCC-9) and 17 h (D2) and wound closure was measured. B: Inhibitor, solvent (DMSO) and/or LPAR3 agonist OMPT at 48 h gave similar results as LPA at 24 h in E10 cells. C: Inhibitor, solvent and/or EGF were added as indicated in E10 and SCC-9 cells. Digital images were obtained immediately after stimulation and after 24 h (E10), 48 h (SCC-9). Bars represent mean ± SEM, n = 3 or 4. The following concentrations were used: LPA 10 μM, OMPT 2.5 μM, EGF 5 nM, Ki16425 1 or 10 μM as indicated. * indicates p < 0.001, **indicates p = 0.03, NS = Not significant. D: Effects on EGFR, Akt, ERK and p38 phosphorylation. Subconfluent cell cultures were pre-treated with Ki16425 for 30 min, and then stimulated with 10 μM LPA for 3, 5, 10 min (E10 cells) or 1, 3, 5 min (SCC-9 cells) as indicated. Cells were lysed for Western blotting. Western blots show partial inhibition of Akt, ERK, p38 and EGFR phosphorylation in E10 cells (D left), while in SCC-9 cells (D right) very little inhibition was detected. No EGFR phosphorylation with LPA was observed in SCC-9 cells. As a positive control, EGFR phosphorylation with EGF stimulation (abbreviated E) is shown. Densitometric analyses of E10 cell at 5 min stimulation are shown below the E10 blots. *indicates p ≤ 0.01.