Fig 2: PP1/PP2A inhibition cannot prevent MELT dephosphorylation if MPS1 and PLK1 are inhibited together. (A and B) Effects of phosphatase-binding mutants on KNL1-MELT dephosphorylation in nocodazole-arrested cells treated with PLK1 inhibitor BI-2536 (100 nM) alone (A) or in combination with MPS1 inhibitor AZ-3146 (2.5 µM; B), as indicated in the timelines. Intensities are relative to CENP-C in BUBR1 cells or YFP-KNL1 in KNL1 cells, and all BUBR1 and KNL1 intensities are normalized to their respective WT, 0 min time point. (C and D) KNL1-MELT phosphorylation levels following combined siRNA-mediated knockdown of all PP1 and B56 isoforms (C) or all B56 isoforms in KNL1WT/ΔPP1 cells (D). The quantifications are from nocodazole-arrested cells treated with MPS1 inhibitor AZ-3146 (2.5 µM) alone or in combination with PLK1 inhibitor BI-2536 (100 nM), as indicated. Representative images are displayed in Fig. S4, D and G. (E) KNL1-MELT dephosphorylation in Hela FRT cells arrested in nocodazole treated with kinase inhibitors in the presence or absence of the PP1/PP2A phosphatase inhibitor calyculin A (25 nM), as indicated. Representative images are displayed in Fig. S4 H. (F) Effects of PP2A-binding mutants in combination with the PP1/PP2A phosphatase inhibitor calyculin A (25 nM) on pMELT dephosphorylation in nocodazole-arrested cells treated with PLK1 (100 nM BI-2536) and MPS1 (2.5 µM AZ-3146) inhibitors, as indicated in the timeline. Representative images are displayed in Fig. S4 I. In all kinetochore intensity graphs, each dot represents the mean kinetochore intensity of a cell, and the violin plots shows the distribution of mean intensities between cells. The thick vertical lines represent a 95% CI around the median, which can be used for statistical comparison of multiple time point/treatments by eye (see Materials and methods). A–D derived from 30–40 cells per condition, three or four experiments. E and F display 40–50 cells, four or five experiments. Timelines indicate treatment regimen before fixation. MG132 was included in whenever MPS1 was inhibited to prevent mitotic exit. (G) Schematic model to show how kinetochore phosphatases restrain (PP2A) or extinguish (PP1) autonomous PLK1 activity to control the SAC. MCC, mitotic checkpoint complex.

Fig 3: Effect of BUB1/BUBR1 knockdown in PLK1, BUBR1, and BUB1 kinetochore levels (related to Fig. 1). (A–C) Relative kinetochore levels of PKL1 (A), BUBR1 (B), and BUB1 (C) after siRNA-mediated depletion of BUB1, BUBR1, or both BUB1 and BUBR1 combined in HeLa FRT cells. Graphs show the mean kinetochore intensity of 40–60 cells per condition, four experiments. For all kinetochore intensity graphs, each dot represents a cell, and the error bars display the variation between the experimental repeats (displayed as ±SD of the experimental means). Two-tailed, nonparametric Mann-Whitney unpaired t tests were performed to compare the means values between experimental groups. Example immunofluorescence images were chosen that most closely resemble the mean values in the quantifications. The insets show magnifications of the outlined regions. Scale bars, 5 µm. Inset size, 1.5 µm. ****, P < 0.0001.

Fig 4: Example images and kinetochore quantification KNL1 and KNL1-pMELT after calyculin A treatment with 25 nM and 50 nM doses (related to Fig. 4). (A) Quantification of KNL1 levels in cells treated with 25 nM calyculin as in Fig. 4 E. Graph shows 30–40 cells per condition from three or four experiments. (B and C) Kinetochore levels of KNL1 (B) and pMELT (C) after treatment with 50 nM calyculin A. B displays 30 cells from three experiments, and C represents 50–70 cells from five to seven experiments. (D) Effects of PP2A-binding mutants in combination with the PP1/PP2A phosphatase inhibitor calyculin A (50 nM) on pMELT dephosphorylation in nocodazole-arrested cells treated with PLK1 (100 nM BI-2536) and MPS1 (2.5 µM AZ-3146) inhibitors, as indicated in the timeline. Graph displays kinetochore intensities of 30 cells per condition from three experimental repeats. MG132 was included in combination with MPS1 inhibitor in every case to prevent mitotic exit. For all graphs, each dot represents a cell, and vertical bars show 95% CIs. All images were chosen that most closely resemble the mean values in the quantifications. The insets show magnifications of the outlined regions. Scale bars, 5 µm. Inset size, 1.5 µm.

Fig 5: Representative images from kinetochore quantifications in Fig. 4. (A and B) Example immunofluorescence images of the kinetochore quantifications shown in Fig. 4 A (A) and Fig. 4 B (B). (C) Representative images and quantification of KNL1 (BUBR1 cells) or YFP-KNL1 (KNL1 cells) after 30 min pretreatment with PLK1 inhibitor followed by coinhibition with MPS1 and PLK1 inhibitors, as in Fig. 4 B. Graph represents 40 cells from four experiments. (D–F) Immunofluorescence images of pMELT (D) and BUBR1 (E), and kinetochore quantification BUBR1 (F) from cells treated as in Fig. 4 C. Graph shows median of 30–40 cells per condition from three or four experiments. (G–I) Representative immunofluorescence images of the kinetochore quantifications shown in Fig. 4 D (G), Fig. 4 E (H), and Fig. 4 F (I). MG132 was included in combination with MPS1 inhibitor in every case to prevent mitotic exit. For all graphs, each dot represents a cell, and vertical bars show 95% CIs. All images were chosen that most closely resemble the mean values in the quantifications. Scale bars, 5 µm. Inset size, 1.5 µm.