CENP-C–dependent kinetochore expansion supports the SAC signal. (A) Extracts were immunodepleted of CENP-C and mixed with control extract to produce intermediate levels of CENP-C. (B) Mitotic chromosomes assembled in CENP-C titration extracts shown in A and treated with nocodazole. Bars, 1 µm. (C) Quantification of total signal for samples as in B. (D) Extracts described in A were assayed for checkpoint activity by challenging M-phase extract to cycle into interphase upon addition of calcium, which does not occur over 60 min in control extracts but occurred after 30 min in extracts with 10% and more rapidly in extracts with 0% of endogenous CENP-C. Cell cycle state was monitored by the presence of phosphorylated Threonine 3 of Histone H3 (H3T3ph). (E and F) Mitotic chromosomes assembled in nocodazole-treated control (ΔIgG) or CENP-C–depleted (ΔCENP-C) extracts and stained for the indicated checkpoint protein. (G and H) Quantification of total signal from E and F. Mean and standard deviation in black. Mann–Whitney test: ***, P < 0.0005; **, P < 0.01; *, P < 0.05. All images are maximum intensity projections of 3D-SIM datasets. AU, arbitrary unit.