Figure 1. Cells expressing non-degradable cyclin B1 arrest in mitosis with separated sister chromatids. (A) Schematic overview of the cyclin B1 mutant used. The destruction-box (D-box) is indicated; (B) The fluorescence intensity in live U2OS cells expressing wt-cyclin B1-Cherry was plotted against time in mitosis from NEB. The arrow indicates the start of sister chromatid separation; (C) The fluorescence intensity in live U2OS cells expressing R42A-cyclin B1-Cerulean was plotted against time from NEB. These cells arrest in a post-metaphase state; (D) U2OS cells were transfected with R42A-cyclin B1-Cerulean, thymidine synchronized and released for 14 h. After mitotic shake-off, cells were released in fresh medium for the indicated time in hours. APC/C substrates geminin and cyclin B1 were normally degraded during the arrest. APC3 is used as a loading control; (E) U2OS cells were transfected with R42A-cyclin B1-Venus and collected and processed for chromosome spreads 46 h later. Chromosomes were stained with DAPI. Note that cells expressing R42A-cyclin B1-Venus fully separated their sister chromatids, while all the chromosomes are cohesed in control cells.