RNAi of hMis6 diminishes condensin II at human kinetochores. (A and B) Kinetochore localization of hCAP-H2 on the spread metaphase chromosomes is diminished in cells after RNAi knockdown. Metaphase spread chromosomes were prepared in HeLa-transfected cells for 48 h by siRNA of hMis6 (A) or luciferase (B; control). Cells were stained by Hoechst33342 for DNA (blue), anti–CENP-A antibody for the centromere/kinetochore (red), and anti–hCAP-H2 for the condensin (green) antibodies. The enlarged images correspond to the dashed boxes in the left panel. (C) The percent frequencies of metaphase chromosomes showing the hCAP-H2 signals on kinetochores; the control was 98.2% (n = 112), whereas depleted hMis6 was only 18.3% (n = 131). (D) HeLa transfected by siRNA of luciferase or hMis6 were fixed for 48 h and stained by Hoechst33342 (blue), anti-hMis6 (red), and anti–hCAP-H2 (green) antibodies. Bars, 10 μm.