Figure S3.

Depletion of Tpr partially restores Mad1 KT recruitment in human cells lacking Mps1 activity. Immunofluorescence images (A and B) and quantifications (C and D) of Mad1 levels at unattached KTs of HeLa (A and C) or RPE1 cultured cells (B and D) in the indicated conditions. Cells were incubated with nocodazole (3.3 µM) and MG132 (1 µM) for 3 h (HeLa) or 6 h (RPE). When indicated, cells were treated with Tpr siRNA (50 nM) for 48 h and/or reversine (1 µM) for 1 h before nocodazole + MG132 incubation. Mad1 fluorescence intensities were determined relative to CenpC (n ≥ 4,081 KTs for HeLa cells, n ≥ 519 KTs for RPE cells). Data are presented as mean ± SD. ****, P < 0.0001 (Kruskal–Wallis, Dunn’s multiple comparison test). Scale bars, 5 µm (inset, 0.5 µm).