The anaphase checkpoint reactivation threshold is set by PP2A-B55 and MASTL. (A) Control (siControl), MASTL-depleted (siMASTL), and PP2A-B55–depleted (siB55) HeLa MPS1-GFP CCNB1-mCherry cells were imaged every 1 min until metaphase, identified by spindle architecture, defined by CCNB1 and the lack of MPS1-positive kinetochores, was reached. Checkpoint-silenced cells were then challenged with 3 µM nocodazole and imaged every 1 min for 40 min. (B and C) Fluorescence intensity (I_t) for MPS1-GFP at kinetochores (B) and total cellular CCNB1-mCherry (C) are plotted over time for single cells (n = 13 for siControl, 7 for siMASTL, and 8 for siB55). Color-coded solid lines mark cells showing a spindle checkpoint (SAC) response, and dotted lines mark cells where the checkpoint remained silent. For MPS1, the black lines mark the mean intensity as a function of time for checkpoint active or silent cells.