Fig. 4. Ciz1 and ECiz1 stimulate S phase entry in wild-type and p21 null cells. (A) ECiz1 and the full-length variant, Ciz1, were tagged with green fluorescent protein to allow identification of expressing cells (green), within transfected populations. Examples of Ciz1-expressing and -non-expressing cells that have or have not engaged in DNA synthesis (and incorporated BrdU into DNA, red) are shown. Nuclei are counterstained with Hoechst 33258 (blue). (B) Expression of ectopic GFP-Ciz1 or ECiz1 in the presence of BrdU increases the number of NIH 3T3 cells that undergo DNA synthesis in rapidly cycling and newly confluent populations, during the17 hour period following transfection. Cells expressing GFP alone are not stimulated to engage in DNA synthesis. (C) Similar results were obtained with p21^cip1 null mouse embryo fibroblasts. Histograms show GFP-expressing cells as green bars and non-expressing cells from the same populations as grey bars. Representative results from three independent experiments for each of the conditions are shown. All experiments gave the same effect, but the background fraction of labelled cells in the untransfected population varied according to the density and growth rate of the cells.