The KNL1 N terminus partially rescues wild-type MT dynamics and kinetochore–MT attachment regulation. (A–D) Flp-In T-REx HeLa cells were depleted of endogenous KNL1, rescued with the indicated GFP-KNL1 fragment upon doxycycline addition and analyzed for oscillatory kinetochore movements and inter-kinetochore distances in metaphase. (A) Kymographs of sister kinetochore pairs from live-cell time-lapse imaging sequences. Bar, 1 µm. (B) Representative plots of kinetochore tracks over time in cells expressing the indicated fragments. (C) Quantification of kinetochore oscillations. DAP indicates deviation from average position; n ≥ 12 kinetochores per cell line. Error bars represent SD between cells. ***, P < 0.001 (t test). (D) Inter-kinetochore distances were measured on bi-oriented sister kinetochore pairs using ACA as a kinetochore marker. Error bars represent SD from independent experiments (n = 3). For each experiment n ≥ 100 kinetochores were measured from at least 5 cells. ***, P < 0.001 (t test). NS, not statistically significantly different (Mann-Whitney rank sum test).