Fig. 5. Inhibition of LPA-stimulated cell migration. (A) Influence of dominant negative GTPases. PANC-1 cells were incubated overnight in DMEM without supplements and subjected to wound healing experiments. Microinjection of plasmids into the cell nuclei was performed 1 hour after wounding with either 0.1 µg/µl pEGFP-C3, pEGFP-C1/H-Ras(S17N), pEGFP-C3/Rac1(T17N) or pEGFP-C3/RhoA(T19N). Protein expression was allowed to proceed for 3 hours in medium without supplements. Migration was induced by addition of 10 µM LPA. Cells were fixed after 24 hours, and well-spread, green-fluorescent cells were counted. Cells, which had migrated into the cell-free space, were calculated as per cent of injected cells. Means ± s.e.m. of three independent experiments are shown. The inset shows pEGFP-injected cells 3 hours after microinjection. (B,C) Inhibition of LPA-stimulated cell migration by pertussis toxin and PD98059. PANC-1 and BxPC-3 cells were seeded on top of trans-well cell culture inserts (8 µm pore size) in DMEM with 10% FCS, and treated for 2 hours with 10 µg/ml mitomycin C to inhibit cell proliferation. Chemoattractants [10% FCS (open bars in C), 10 µM LPA (solid bars in C) or 20 ng/ml HGF (hatched bars in C) in DMEM with 0.1% BSA] were added to the lower chamber. The upper chamber was filled with DMEM with 0.1% BSA. PTX (25 ng/ml), PD98059 (25 µM), U0126 (10 µM) or SP600125 (25 µM) was added to both chambers; controls were treated with solvent. PANC-1 and BxPC-3 cells were incubated for 40 hours. Afterwards, cells were fixed, stained with hematoxylin and cells migrated to the bottom side of the filter were photographed. For quantification, cells in three independent visual fields were counted. (B) Representative microphotographs of the bottom sides of the filters. (C) Quantitative analysis of one representative assay out of three experiments. Means ± s.d. of three independent visual fields are shown.

Return to article