Fig. 7. Wounding triggers delayed cell proliferation but inhibition of proliferation does not affect wound closure. (A) Fluorescence and corresponding phase-contrast images showing BrdU incorporation over the course of wound closure in fixed MDCK cell monolayers. The initial width of the rectangular wounds in A-C was 200 µm. Arrows indicate location of wound margin at 0 and 6 hours, edge-edge contact at 12 hours or former wound area at 24 hours. Bar, 50 µm. In other experiments, wider wounds were made that closed by 24-36 hours, and similar results were obtained, with BrdU incorporation peaking 24 hours (data not shown). (B) Percentage of BrdU-positive nuclei for wounded monolayers as a function of time after wounding, as well as parallel unwounded monolayers and subconfluent cultures (mean and s.e.m.; n=15 fields of view from three independent experiments). (C) The DNA-alkylating agent mitomycin C (MMC, 1.5 µM) blocks MDCK cell proliferation based on BrdU incorporation as in B, as well as the tetrazolium salt assay, with no evidence of general toxicity as assessed by Trypan Blue dye exclusion (data not shown). (D) Wound closure in the presence and absence of MMC added 48 hours before wounding (mean and s.e.m. for n=24 wounds). The initial diameter of the circular wounds in this experiment was 500 µm.