The effect of Nuf2 depletion on the formation and/or stability of the kinetochore outer plate. HeLa cells 48 h after mock-transfection (A-C), or transfection with hNuf2-siRNA (D-F, H, and I), were treated with 20 μM nocodazole for 3 h to disassemble all MTs and promote growth of the kinetochore outer domain. Cells were then fixed and prepared for EM. (A) Low-magnification view of a nocodazole-treated, mock-transfected cell. Several well-formed, unbound kinetochores are visible in the section (arrows). (B) A pair of well-formed sister kinetochores from a control cell. Note the robust outer plates (arrows). (C) Example of a poorly formed kinetochore from a control cell. The outer plate (arrow) is less dense and robust compared with the example in B. (D) Low-magnification view of a nocodazole-treated transfected cell. Unbound kinetochores (arrows) were significantly harder to locate compared with control cells. (E) A pair of well-formed sister kinetochores from a transfected cell. As in B, the outer plates (arrows) are robust. (F) Poorly formed kinetochore from a transfected cell. The outer plate is much less distinct (arrow). (G) Bar graph showing the numbers of kinetochores found in three control and six transfected cells. The light and dark shadings indicate the number of well-formed (light) and poorly formed kinetochores (dark) found per micron of depth in each of the cells. (H and I) Examples of fibrous material at the putative centromere region of chromosomes from transfected cells. Scale bars: (A and D) 1 μm; (B, C, E, F, H, and I) 200 nm.