Fig. 2. The catalytic activity of PKC is required for its ability to promote cell spreading and migration. (A) Cell spreading assay. An equal number (10⁵) of MDCK cells or those stably expressing GFP, GFP-PKC (wt), or its kd mutant was suspended in serum-free medium and plated onto collagen-coated dishes. At various times after plating, spread cells in several random fields were counted under a phase-contrast microscope. Each data point is the average of three experiments and expressed as the percentage of spread cells in all counted cells (n=200). Representative micrographs were taken from the cells that had been plated for 60 minutes. (B) Trans-well cell migration assay. An equal number (2x10⁴) of the cells were suspended in serum-free medium and subjected to the assay, as described in Materials and Methods. 6 hours later, the migrated cells were fixed, stained and counted using a light microscope. Values (means ± s.d.) are from three independent experiments and expressed as fold increase relative to the level of the control MDCK cells. ^*P<0.05. (C) Wound healing assay. Cells grown as a monolayer on glass were wounded by manual scratching with a pipette tip. Time-lapse micrographs were taken every 5 minutes for 12 hours to record the healing process. The representative micrographs at 0 hour, 6 hours and 12 hours are shown. The average migratory speed of ten cells at the front was measured by Meta Imaging Series^TM software version 4.5.