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Figure S1. shRNA-mediated repression of Mps1. (A) Immunoblot showing that the sheep polyclonal anti-Mps1 antibody SMP1.1 detects a single band of ∼95 kD in asynchronous HeLa cell extracts. In nocodazole-treated cells, Mps1 mobility is retarded, which is consistent with mitotic phosphorylation. BubR1 is used as a loading control. (B) Deconvolved immunofluorescence projection of a prometaphase HeLa cell stained with SMP1.1 (red) showing Mps1 in the cytoplasm and at kinetochores, as seen by colocalization with BubR1 (green). The enlargement of the highlighted (boxed) area shows colocalization of Mps1 and BubR1 at the kinetochore. (C) HeLa cells were transfected with shRNA constructs designed to repress Mps1 and were analyzed by immunoblotting, demonstrating that construct F efficiently represses Mps1. Bub3 is used as a loading control. (D) HeLa cells were transfected with construct F and were fixed and stained to detect Mps1 (green), centromeres (ACA; red), and DNA (blue). The image shows a field of view containing one repressed cell (arrows) and one unrepressed cell (arrowheads). Both the cytoplasmic and kinetochore-bound Mps1 signal is absent in the repressed cell. (E) Bar graph quantifying Mps1 pixel intensities relative to the ACA signal and normalized to 100% in the control cell. Values represent the mean and SEM (error bars) derived from at least 128 kinetochores from five cells. (F and G) HeLa cells were transfected with construct F to repress Mps1 and were treated with nocodazole or taxol for 18 h. Phase-contrast images in F show that the majority of control cells are arrested in mitosis, whereas most cells in the Mps1-deficient population are in interphase. The cells were then fixed, and the mitotic index was determined by MPM-2 staining and flow cytometry. The bar graph in G shows that transfection of construct F reduces the mitotic accumulation in response to both nocodazole and taxol. (H) HeLa cells were transfected with construct F or siRNAs to repress BubR1. The cells were then exposed to monastrol and analyzed by time-lapse microscopy. The line graph plots the percentage of cells in mitosis over time, showing that the SAC is compromised in Mps1-defifcient cells. At least 67 cells were analyzed for each condition. Bars: (B and D) 5 µm; (F) 25 µm.