Spatial distribution of H2B S6ph and SET during mitosis. (A) The HCT116 cells described in Fig. 9 (F) were induced to trigger GFP-SET expression by the addition of 1 µg/ml doxycycline for 24 h. The top and middle panels show representative examples for fixed cells that were analyzed by confocal microscopy for the distribution of GFP-SET and CENP-A in prometaphase and metaphase cells. Line scan analysis was performed for 24 centromere pairs from three different prometaphase and metaphase cells, respectively. Quantification was done using ImageJ and GraphPad Prism; the maximum of fluorescence intensity was set as 1, and the dotted lines indicate standard deviations. The bottom panel shows the comparison of H2B S6ph and GFP-SET distribution in metaphase cells; line scan analysis was done by quantitative evaluation of 48 centromere pairs from 12 different cells. Bars, 2 µm. (B) Schematic summary depicting the possible molecular mechanisms leading to spatial and temporal control of H2B S6ph. Mitotic chromosomes are shown in blue, and the position of phosphorylated H2B is indicated in red, CENP-A chromatin in yellow, and kinetochores in dark green. Black arrows represent kinase activity of CDK1-cyclin B1 and Aurora B as part of the CPC.