In vitro tube formation of HIMEC requires activation of ERK 1/2 (p44/42 MAPK). In vitro endothelial tube formation assays employed Matrigel™ as a three-dimensional extracellular matrix. HIMEC in complete growth medium (5 × 104) were seeded onto 24-well plates containing Matrigel™ (5 mg/ml). Where indicated, the medium was supplemented with CsA (0.1 μM), p38 MAPK inhibitor SB203580 (5 μM), specific inhibitors of MAPK kinase (PD098059, 10 μM), and COX-2 inhibitor NS398 (10 μM), respectively. Cells receiving DMSO served as a vehicle control, and were equivalent to no treatment (data not shown). Naive HIMEC seeded in complete growth medium display robust tube formation. Addition of VEGF to growth medium resulted in enhanced tube formation. All three inhibitors markedly abrogated spontaneous tube formation of HIMEC seeded in complete growth medium. Note the cells remaining adherent in spherical clusters, lacking mature tube-like structures.