Fig 2: The molecular features of GBM stem cell. (a) Violin plots showing selected marker of GSCL. (b) IHC staining of BRCA1, C16orf59, CASC5, CCNF2, CHAF1A, FBXO5, TIMELESS, MCM2, and NCAPH in GBM samples. (c) Immunofluorescence colocalization of selected markers (red) and CD133 (green).

Fig 3: Kinetochore phosphatases PP1 and PP2A-B56 remove PLK1 from the BUB complex to silence the SAC. (A–D) Effects of phosphatase-binding mutants on KNL1-MELT dephosphorylation (A and C) and duration of mitotic arrest (B and D) in nocodazole-arrested cells treated with MPS1 inhibitor AZ-3146 (2.5 µM) either alone (A and B) or in combination with the PLK1 inhibitor BI-2536 (100 nM; C and D). MG132 was included in all MELT phosphorylation experiments to prevent mitotic exit after addition of the MPS1 inhibitor. A and C display kinetochore intensities from 30 cells per condition, three experiments. 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. B and D show mean (±SD) of 150 cells per condition from three experiments. (E and F) Effect of mutating the PLK1 binding site on BUBR1 (pT620) on MELT dephosphorylation (E) and mitotic exit (F) in nocodazole-arrested BUBR1WT/ΔPP2A cells, treated as in A and B. E displays kinetochore intensities of 30–80 cells per condition from three to seven experimental repeats. F shows the means (±SD) of 200 cells from four experiments. (G and H) Effect of mutating both PLK1 binding sites BUBR1 (pT620) and BUB1 (pT609) on MELT dephosphorylation (G) and mitotic exit (H) in nocodazole-arrested cells, treated with MPS1 inhibitor AZ-3146 (2.5 µM in G and 1.25 µM in H). The 1.25 µM AZ dose was selected because it is then possible to see effects that weaken or strengthen the WT SAC response (2.5 µM AZ-3146 data are displayed in Fig. S3 I). G displays kinetochore intensities of 40 cells per condition from four experimental repeats, and all intensities are normalized to BUBR1-WT+BUB1-WT control. H shows the means (±SEM) of 200–250 cells from four or five experiments. In all kinetochore intensity graphs, each dot represents the mean kinetochore intensity of a cell, and the violin plots shows the distribution of intensities between cells. The thick vertical lines represent a 95% CI around the median, which can be used for statistical comparison of multiple time points/treatments by eye (see Materials and methods). Timelines indicate treatment regimen before fixation.

Fig 4: 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 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.