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. 2001 Jul;12(7):1937–1956. doi: 10.1091/mbc.12.7.1937

Figure 8.

Figure 8

Cell migration requires ERK1/2 and cyclooxygenase activity. (A) NIH-3T3 cell migration was evaluated with Boyden chamber assays as described in MATERIALS AND METHODS. Cells were treated with 50 μM PD98059 to inhibit ERK1/2 (inhib ERK1/2), 50 μM indomethacin to inhibit both cyclooxygenases (inhib COX1 + COX2), 25 μM SC236 to inhibit cyclooxygenase-2 (inhib COX-2), or 25 μM resveratrol to inhibit cyclooxygenase-1 (inhib COX-1). White bars indicate inhibitor alone, black bars indicate addition of the cyclooxygenase oxidation product PGE2 (50 nM) with inhibitor. Dose-dependent reduction in migration was seen with PD98059, indomethacin, and SC236, the figure shows a typical higher concentration of each reversible by exogenous PGE2. Resveratrol inhibition of COX-1 was dose-dependent for a limited range, but did not reduce migration to <70% of control at concentrations >25 μM (means and SEs; n = 3) (B) Inhibitors of COX-2 or PLA2 added after 30 min of cell spreading block most cell migration. Inhibitors were added to cells after 30 min of cell plating on FN after the initial AA release had taken place, to test the requirement for AA generated in the second sustained release as a substrate for cyclooxygenase activity. Addition of 25 μM AACOCF3 (inhib iPLA2 + cPLA2), 50 μM indomethacin (inhib COX1 + COX2), or 25 μM SC236 (inhib COX-2), after 30 min of cell spreading, reduces migration in a dose-dependent manner; a single representative inhibitory concentration is shown herein that is reversible by PGE2 addition. The COX-1 inhibitor resveratrol at 25 μM (inhib COX-1) producesonly ∼25% reduction in migration when added after 30 min of spreading. Inhibition of ERK1/2 with 50 μM PD98059 at 30 min postspreading also reduced migration only to ≥70% of control, compared with the migration of ≤15% of control seen in A at the same concentration added preplating. Post 30 min spreading addition of 10 μM HELSS (inhib iPLA2) produced a 10–15% reduction in migration that was not dose-dependent. Addition of 30 μM NDGA (inhib all LOX) or 25 μM AA861 (inhib 5-LOX) at 30 min postplating reduced migration also by only 15–20%.