Analysis of chromosomal localization of H2B S6ph by 3D-SIM. (A) RPE-1 cells were stained for H2B S6ph and CENP-A localization with specific antibodies, DNA was revealed by Hoechst dye. Different mitotic phases are shown. Bars: 2 µm (main); 1 µm (magnification). (B) The experiment was done as in A, with the difference that cells were stained for H2B S6ph and Aurora B. The boxed areas in A and B are displayed in 10× magnification. Bars: 2 µm (main); 1 µm (magnification). (C) RPE-1 cells were arrested at G₂/M transition with the CDK1 inhibitor RO 3306 for 16 h and released into mitosis for 1 h in the presence of nocodazole. Chromosome spreads were produced for immunostaining against H2B S6ph, Aurora B, H3 K9 me2/3, or CENP-A as shown. Pictures show chromosome spreads of representative cells. Bars, 1 µm.