Fig. 2. MEKK1 expression is necessary for fibroblast migration. (A) Fibroblasts were seeded into the upper chamber of a Transwell migration plate with 5% FBS in the lower chamber. Cells traversed after 5 h to the lower surface of the membrane were quantitated. The results shown are the mean ± SEM of at least three independent experiments. (B) Fibroblasts were treated as in (A) except that the bottom well of the Transwell contained either 1 nM EGF, 100 µg/ml fibronectin or the combination of EGF and fibronectin. (C and E) Wild-type or MEKK1–/– fibroblasts were seeded onto coverslips and allowed to grow overnight. In addition, MEKK1–/– fibroblasts stably transfected with full-length MEKK1 (Add-back) were analyzed. Each confluent culture was ‘wounded’ with a razor and observed over the course of 5 h for migration into the wound space (in vitro wound healing assay). (C) is a DIC image of migrating cells. (D) The time required for confluent fibroblasts in a tissue culture plate to close a standardized wound (200 µm) is represented by the graph. Results shown are the mean ± SEM of at least three independent experiments, and the statistical significance was determined by Student’s t-test (*P < 0.05). (E) Cells were first stained with the fluorescent vital dyes PKH26 (red; MEKK1+/+) or PKH67 (green; MEKK1–/–) and then mixed in equal numbers before seeding onto coverslips, and treated as described above. The fluorescence image depicts the migration of MEKK1+/+ (wild type) and MEKK1–/– cells in co-culture. The data are representative samples from at least three independent experiments.