PP2A-B55 counteracts CDK1-dependent MPS1 localization to kinetochores and checkpoint signaling. (A) Spindle checkpoint response assays are explained in the schematic. HeLa GFP-MAD2 cells arrested in mitosis with 0.3 µM nocodazole for 4 h were treated with 20 µM MG132 for 15 min to prevent mitotic exit. Images were taken every minute after treatment with DMSO (−CDK-i control), 5 µM flavopiridol alone (+CDK-i), or 25 nM calyculin A for 1 min before 5 µM flavopridol addition (PP-i +CDK-i). Representative images at +10 min are shown. Mean GFP-MAD2 intensity at kinetochores as a function of time (I_t) is plotted relative to the starting signal (I₀); error bars indicate the SEM (n ≥ 5 independent conditions). (B–D) Checkpoint signaling and endogenous MPS1 localization were followed in control (siControl; B), PP1αγ-depleted (siPP1; C), and PP2A-B55–depleted (siB55) HeLa MPS1-GFP cells (D). Cells were arrested for 2.5 h with 20 µM MG132 and then either fixed immediately (+MG132), treated with 3 µM nocodazole for 5 min (+Noc), or 5 µM flavopiridol for 1 min, followed by addition of 3 µM nocodazole for 5 min (+CDK-i +Noc). MAD1 and kinetochores (CREST) were detected using antibodies, and MPS1 was detected using GFP fluorescence. (E and F) Kinetochore-associated MAD1 (E) and MPS1-GFP normalized to the mean of the nocodazole-treated control (F) are plotted; error bars indicate the SEM (15 cells and 15 kinetochores measured per cell for three independent experiments).