Fig. 1

Blocker of HCN family channels, ZD7288, reduces ESC proliferation by lengthening the G1 and S phases of the cell cycle. Culture in the presence of 10 μM HCN inhibitor ZD7288 resulted in reduced mESC cell numbers after 4 days in suspension (Student’s t-test; p < 0.01; n = 9) (a) with a minimal reduction in viability (Student’s t-test; p < 0.05; n = 9) (b). Cells cultured adherently at subclonal densities formed smaller colonies (Mann Whitney test; p < 0.05; n = 3) (c). Despite a significantly longer doubling time (sum of squares f-test; p < 0.05; n = 9) (d) the cells did not exit the cell cycle, as indicated by ubiquitous KI-67 expression in both control and treated cultures (e). Flow cytometry for DNA content (f) showed an accumulation of cells in the S phase (Student’s t-test; p < 0.01; n = 3), and a relative reduction in the number of cells in G2/M (Student’s t-test; p < 0.01; n = 3). Doubling time and cell cycle fraction were used to calculate residence time in each cell cycle phase for control cultures and cultures treated with 10 μM ZD7288 (g). Comparison of cell cycle structure in our study and previous study by Lau et al. is summarized in (h)