Fig. 3. Reversibility of the inhibitory effect of high TGF-ß1 concentrations on keratinocyte cell cycling. (A) Cells of the Adh⁺⁺⁺ population initially grown on slides without TGF-ß1 were cultured for 24 hours with this factor added to the medium at a concentration of 0, 10, 30, 100, 300, 1000 or 3000 pg/ml. Samples were then processed for cell-cycle analysis by Laser Scanning Cytometry (LSC). Data from one typical experiment are shown (image of fluorescent nuclear DNA staining and cell-cycle analyses). (B) Long-term cultures were initiated with keratinocytes of the Adh⁺⁺⁺ population and maintained in the presence of 100 pg/ml exogenous TGF-ß1 up to day 30. Half of the cultures were continued from day 30 without addition of TGF-ß1 and the rest were continued in the presence of 100 pg/ml exogenous TGF-ß1. (C) Long-term cultures were initiated with keratinocytes of the Adh⁺⁺⁺ population and maintained up to day 21 without addition of TGF-ß1. Half of the cultures were continued from day 21 to day 41 in the presence of 300 pg/ml exogenous TGF-ß1, and then from day 41 without addition of TGF-ß1. The other half were continued from day 21 without addition of TGF-ß1. Data represent means±s.d. of four replicate cultures from a typical experiment.