PP2A-B55 opposes CCNB1 recruitment to kinetochores. (A) Checkpoint signaling and CCNB1 localization were followed in control (siControl) and PP2A-B55 (siB55) depleted HeLa CCNB1-GFP cells. Cells were arrested for 2.5 h with 20 µM MG132, and then either fixed immediately (+MG132) or treated with 3 µM nocodazole for 5 min (+Noc) or with 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 CCNB1 using GFP fluorescence. (B) Mean kinetochore intensity ± SEM of CCNB1 and MAD1 in control (siCon) and B55-depleted (siB55) cells are plotted (15 kinetochores per cell for ≥5 cells in each of three independent experiments). (C) A schematic of the checkpoint response assay. (D) Control (siControl) and PP2A-B55 (siB55) depleted HeLa cells arrested in mitosis for 3 h with 100 µM monastrol were washed into fresh growth medium for 25 min to allow spindle formation. At that point, 0 min, or after a further 10 or 20 min, cells were challenged with 3 µM nocodazole for 5 min to test for the checkpoint response, fixed, and then stained for MAD1 and tubulin. (E) Graphs show the fraction of checkpoint silenced cells at different times after monastrol washout, before nocodazole addition. Bars indicate the SEM (for siControl: 0/10/20 min n = 263/293/277 and for siB55: n = 296/294/277). (F) Graphs show the fraction of checkpoint active cells with unseparated sister chromatids (for siControl: 0/10/20min n = 232/302/297 and for siB55: n = 277/257/306) and MAD1 signal at kinetochores (for siControl: 0/10/20min n = 64/48/65 and for siB55: n = 46/62/49); error bars indicate the SEM. (G) CCNB1 and MPS1 localization are shown for the 20-min time point challenged with 3 µm nocodazole (+Noc) in control (siCon) or PP2A-B55 (siB55) depleted HeLa CCNB1-GFP/MPS1-mCherry cells. MPS1 intensity is plotted relative to the cytoplasmic CCNB1 signal for individual kinetochores with the mean and SD (15 kinetochores per cell in 26 [siCon] or 27 [siB55] cells).