KNL1 is required for wild-type kinetochore–MT dynamics. (A) Representative images of control, ZM-treated, and KNL1-depleted early mitotic HeLa cells subjected to a cold-induced MT depolymerization assay and immunostained for tubulin and a kinetochore marker (ACA). Total spindle fluorescence intensities are shown for each condition. Error bars in graph represent SD from independent experiments (n = 2). For each experiment n ≥ 8 cells per condition. ***, P < 0.001 (Mann-Whitney rank sum test); NS, not statistically significantly different. (B) Kymographs of sister kinetochore pairs from live-cell time-lapse imaging sequences in control and KNL1-depleted HeLa cells. Kymographs were generated from the kinetochore pairs indicated in the panels below (arrows). (C) Plots of kinetochore tracks over time in control and KNL1-depleted HeLa cells. For each condition, two representative sister kinetochore pairs are shown. (D) Quantification of kinetochore oscillations in control and KNL1-depleted cells. DAP indicates deviation from average position, a measure of oscillation amplitude (Stumpff et al., 2008). Error bars represent SD from independent experiments (n = 3). For each experiment n ≥ 32 kinetochores were tracked from at least 5 cells. ***, P < 0.001 (t test). Bars, 5 µm.