Figure 1. Effects of partial pre-mitotic Cdk1 inhibition. (A) Synchronized HeLa-H2B-mCherry cells were released from G₁/S and treated at 6 h post-release (early G₂ phase) with increasing doses of the Cdk1 inhibitor RO3306 (RO). Shown are characteristic fields of G₂ and mitotic images captured, along with specific masks generated using the IncuCyte to count total cell number (green) and accurately identify mitotic cells (blue) based on differential H2B-mCherry intensity and shape. Scale bar = 200 µM (10 µM for zoom). (B) Data from (A) presented as a graph showing the percentage of mitotic cells at each time point. Images were taken every hour up to 20 h post-release from thymidine. Graph is representative of 3 independent experiments. (C) Time-lapse images of representative cells from (B), showing the timing of cell division. Scale bar = 20 µM. (D) HeLa cells were synchronized with thymidine, released and treated with RO at 6 h post-release. Samples were then harvested at the indicated times post-release, lysed, and analyzed by western blot with the indicated antibodies. Triangles indicate phosphorylated (black) and non-phosphorylated (white) forms of Greatwall (Gwl) and Cdc25C. All data shown are representative images from 3 independent experiments.