BioProject

In an acute skin wound, newly released serum growth factors in the wound bed drive lateral migration of human keratinocytes (HKs) to re-epithelialize the wound. More...

In an acute skin wound, newly released serum growth factors in the wound bed drive lateral migration of human keratinocytes (HKs) to re-epithelialize the wound. However, profiling the migration signal-specific genes has long been challenged by pleiotropic effects of a given growth factor, including proliferation, migration and factor-specific responses. To overcome these technical problems, we 1) took advantage of a unique response of HKs to transforming growth factor-beta (TGFbeta) to selectively suppress growth signal-responding genes and identify motility-specific genes and 2) employed dual stimulation of HKs with TGFalpha and insulin to identify the common genes and eliminate factor-specific genes. Under these conditions, DNA microarray analyses were utilized to study the profiles of both TGFalpha-regualted and insulin-regulated immediate early (IE, 30 min), early (E, 60 min) and delayed early (DE, 120 min) genes. Keywords: time course, growth factor response Overall design: HKs were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes. TGFalpha or insulin was added to separate plates (in the continued presence of TGFbeta) for 30 min, 60 min and 120 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation. Less...